COAGULATION BEHAVIOUR OF DIFFERENTLY ACIDIFIED AND

RENNETED MILK AND THE EFFECTS OF PRE-TREATMENT OF MILK

A Theab

Pmented to

The Faculty of Graduate Studia

of

The University of Guelph

br
CAROLE CLAUDE TRANCHANT

In partial fullllment of rquirtments

for tbe degree of

Doctor of Philarophy

Mirch, 2000
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 COAGULATION BEHAVIOUR OF DIFFERENTLY ACIDIFIED GND

RENNETED MILK AND TFIE EFFECTS OF PRE-TREATMENT OF MILK

Carole Tranchant Drs. D.G. Dalgleish & A.R. Hill

University of Guelph, 2000 Advisors

The dissertation focuses on the variations in the coagulation behaviour of milk that mise

when renneting and acidification proceed simultaneously. Expiments and literature reviews

were conducted along two major axes:

(1) Estimation of the changes in the surface structure of casein piuticles of milk between

pH 6.7-5.5, with statistical assessrnent of the (interaction) effects of direct pre-

acidification and pre-heating of milk.

(2) Systematic investigation of different modes of coagulation of milk inoculated with

diffennt amounts of acidiQing starter bacteria andor rennet enzymes (and,

occasionally, pre-treated, e.g., pre-heated).

Apparent hydrodynamic diameter of dilutcd casein particles (estimated by photon comlation

spectroscopy at 2YC) decreased by up to CU. 10 nm upon exposure to increasingly acidic

solvent. The decrease in particle size was rclated to-inainly-gradual collapsing of the surface

layer of K-cwin amund the particles, with concomitant rcduction of particle stability.

Estimations of suiface layer apparent thickness from changes in particle diameter upon muicting

at constant pH nnging from 6.7-5.5 supported this interprctation. Pre-heating mik at 900C-1

min appead to d u c e the thickness of the surfaee Iayer.

 Qualitative and quantitative analyses of gel development h m differently cultured and

rcnneted milk by dynamic heometry OJarnetrc and Carri-Med rhcometers. with monitoring of

p H and hydrolysis of K-wein)pointed to the prcdominant influence of rennet concentration on

the evolution of gel viscoelastic pmperties. In addition to the profiles characteristic of

coagulation by strictly acidification or renneting, two distinct types of coagulation profiles were

evidenced. depending on the relative contributions of renneting and bacteriological acidification

to gel formation. Two situations were distinguished:

(i) Conditions of coagulation such that the effects of continuous acidification were

integral-albeit with minimal renneting,

(ii) Conditions such that the cffects of-substmtial-renneting prevailed.

Largely similar patterns of coagulation were observed for differently pre-(heat) treated

milks, with quantitative differences.

A conceptual scheme was proposed to account for the gradations in coagulation khaviout.

with delineation of basic stages of gel development and discussion of the physico-chernical

processes (casein dcmineralization) likely involveû. nie pmnise is that different patterns of gel

y-diffcmit patterns of succession of acidification and

development stems fiom~ssentiall

renneting.
A mes parents qui m'ont entraînée dans le bleu étonnant de l'existence
Avec toute ma tendresse.

And to Didier, exquisite cornpanion and CO-adventurer

Si loin, si proche...
(6,500 plus km across the Atlantic will not get the better of our Love!)
 Preparing this dissertation has b e n a far more stimulating experience than 1 cver expected. It

was my pleasure and good fortune to have crossed the path of Dr. Elisabeth Dumoulin (Ecole

Nationale Supérieure des Industries Agricoles et Alimentaires, France). She whetted my appetite

for venturing abroad and her early influences led me to discover beautifil Canada.

1 am deeply grateful to Dr. Douglas G. Dalgleish (presently with Groupe Danone, France)

for ernbarking me on the milky way and for the fmedom he allowed in developing the project,

which has ultimately stretched my curiosity in surprising new directions. Thank you for your

initial guidance and encouraging, especially at times when the assembling of al1 that we

researched and learned did not seem easily within mach. In bringing this study to completion, 1

also acknowledge the generous commitrnent of Dr. A. R. Hill and the occasional contribution of

the other members of the advisory and examination committees, Drs. A. Clarke (department of

Microbiology), D.H. Goff, J. A. Lucey (University of Wisconsin-Madison), and R. Y. Yada.

Along the way, 1 have enjoyed the help and cheery temperament of many spirited

individuals. Ttianks to fellow members of the colourfiil band of fnends of milk and life,

Jacqueline Brun, Milcna Corredig, Dr. Yuan Fang, Deryck Penaud, home Rabalski. Susan

Tosh, Mark Yoshimasu & Co. Also, and especially, to Edita Verespej for your heartening words

and deeds, and sparkling thought energies. To Juan Amilcar Colindm, thmk you for caring the

ways you do. To Dr. Chandnni Atapmu, Mario Balona, and the people and fiiends with the

department of Animal and Poultry Science (you know who you are :-) for precious technical and

emotional support. And certainly to Dr. Massimo Manone, for your gracious assistance in so

many important ways fiom beginning to completion, including the chairing of the examination

cornmittee.
 1 am th&l too, to Dn. C a d e Butau and Cyril Duitschaever for sdvice in the

micmbiology aspects of the smdy and rehshing causeries; Dis. Elisabeth A. K. Gullett and

Marc Le Maguer for opening talkr and encouragement; Drs. D. H. Goff and R. E. SuMen for
allowing me to use their equipment; and William Matthes-Sears (Ontario Veterinary College) for

helpful hints in canying out the statistical 'magic' or, as some would joke, "torture the data till

they told some mth..."

For administrative assistance, i am particularly obliged to Donna Moytane and Linda

Peteranac. John van Esch, Monika Okoniewsùa, and Hanydath Ramsuen occasionally helped

with canying starter cultures, and 1 thank them. The cooperation of Charlie Fulton and Bill

Lachowsky of the Guelph Central Milk Testing Labotatory in analyses of milk samples was

much appreciated as well.

A special word of thanks goes to the Dairy Fanners of Ontario, the Ontario Agricultural

College, the Ontario Dairy Council, and the Ontario Ministry of Education and Training for

scholarship support and reseuch hnds for this pro-. Additional financial assistance was

received initially h m the Ministhre fiançais de l'Agriculture et de ta Forêt and, most critically

over the last two years, from my farnily and from my cornpanion.

Merci, mes parents et ma famille, for the gifb of reading anci leaming, the unstinting

support, et pour la chaleur a l'affection qui m'ont permis de voir toujours plus loin. Merci de

votre immplaçablc amour qui fleure si bon la Normandie!

Merci, Didier, pour ta complicitd et tes douces folies. Après avoir gofit4 i l'amour & des

kilomhtrcs ailleurs et au bonheur par dkctrons libres interposés ('virtuous redity'?), j'ai bien

hâte de découvrir une autre voie lac& pour prolonger notre histoire jusqu'aux Ctoiles de la vie...

Thank you cach bemroup! And many blcssings to all.

Abatract
Dtdication

Ackaowledgments i
Contents iii
Liat of Tables viii
List of Figum X
Frepuently Used Abbmiations and Notations xix
Terminology xxii

1. Introduction 1

2.1. Casein Micelles in Bovine Milk and the Pmicles Derived from Them by
Changing Their Environment
2.1.1. Molecular Characteristics of the Caseins and Semm Proteins
-
2.1.2. Casein Micelles Structure and Stability
Physical and Chernical Charactetistics
Structural Models and Implications on Micelle Stability
2.1 3. Modification of Casein Micelles by Acidification and Heat
Changes on Lowering the pH Below Physiological Value
Changes on Heating Beyond Pasteurization

2.2. Formation and Properties of Milk Gels

2.2.1. Studies on Gel Formation in Acidified Milk
-
2.2.2. Rennet Coagulation of Milk Enzymatic Proteoiysis and Aggrcgation
of Casein
Effects of Concentration of Rennet
Effects of Low pH
Effcets of Re-Heating
2.2.3. Gel Asscrnbly and Syneresis
Early Gelation Events
Gelation as a Multiphasic Process
The Phenornena of Syneresis
2.2.4. Physical Characteristics of Milk Gels
2.2.5. The Use of M W Concentratcd by Ulbitiîtration
2.2.6. Aggrcgation on Lowcring the pH- Acid Coagulation of Milk
Effects of Re-Hcating
Effbcts of Protein Concentration
2.2.7. Combincd Rcnnct a d Acid Coagulation of Milk
 3.1. Dynamic Light Scattering (DLS) -Photon Comlation Spectrosçopy (PCS) 66
3.1.1. Particle Size by Dynamic Light Scattcting 66
Principks of Mokpuremcnts 66
Experimental Dctermination of Autocomlation Functions and
Difision Coefficient 68
Analysis of Autocomlation Functions for Polydispcne Systems 70
3.1.2. Application to the Study of Particle Surface Stnicture 72

3.2. Fluorirnetry
3.2.1. Protein Hydrophobicity by Fluorescence Probe Methods
3.2.2. Anilino-8-Naphthalene Suiphonaie (ANS)-Fluorimetry

3.3. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 77

3.3.1. Electrophoretic Sepmîtion of Ptoteins 77
3.3.2. Densitometric Scanning and Quantification 78

3-4. Dynamic (Oscillatory) Rheornetcy 79

3.4.1. Rheological Characterination of Viscoelastic Materials 79
Principles of Mawremcnt 79
Dynamic Shear Stress, Shear Strain, Shear Rate, and the Conditions
of Linear Viscoelasticity 80
Sinusoidal Straining 81
Interpretation of Rheological Data and Experimental Difficulties 84
3.42. Dynamic Testing with the Nametre Rhcoliner RheometerTY 86
3.4.3. Dynamic Tcsting with the Carri-Med Controlled Stress RheometerTM 88

4. Hyddynamic Size and Hydrophobicity of Casein (Pseudo) Micelles

and Thcir Possible Relation to Cbanges in the Structure of Particle
Surface Between pH 6.7 and 5.5 91

4.1. Outlook 91

4.2. Experimental Details .

4.2.1. Fresh Milk and Prc-Trcatmcnts
4.2.2. Heating Pmcedure
4.2.3. Casein Micelles of Reduced Size Polydispersity
4.2.4. Pm-Acidification of Milk
4.2.5. Milk Ultrafiltrate (MUF)
4.2.6. Renneting of Resuspcnded Micelles
4.2.7. Photon Comlation S p e c t m s q y
Instrument Sctup and Run Conditions
Data Acquisition and Treatment
4.2.8. ANS-Fluorimetry
4.2.9. Statistical Analyses
 4.3. Results and Discussion for Photon Correlation Spectroscopy
4.3.1. Apparent Hydrodynamic Diameter of Casein Particles Diluted in MUF
at Diffemnt Values of pH
43.2. Apparent Hydrodynamic Diameter of Casein Puticles Isolated fiom
Pte-Hcated Milk and the Effect of Law pH
4.3.3. Effect of Rennet Action on Pmicle Diameter at Different Values of pH

4.4. Results and Discussion for ANS-Fluorimetry

4.4.1 .Pm-Tests
Background Fluorescence
Effbct of Sodium Azidc on Fluorescence Intensity
Sensitivity of ANS Fluorescence to the Chemical Environment
Effect of Dilution Range on the Estimation of Appafent Hydro-
phobicity
4.4.2. Apparent Hydmphobicity o f Casein Particles Diluted in MUF at Diffe-
rent Values of pH
4.4.3. Apparent Hydrophobicity of Casein Parthles from Pre-Heated Milk and
the Effcct of Low pH

4.5. Sumrnary Discussion

5. Quantification of Rennet Hydrolysis of ~-Cascinin Chemically Acidifitd

Skim MW by SDS-Polyacrylamide Gel Electraphomis
5.1. Outlook

5.2. Experimental Details

5-2.1. Fresh Milk and Prc-Treaûncnts
5.2.2. Renneting, Sarnpling, and Prepmtion of Milk
5.2.3. Gel Electrophorcsis, Staining, Densitometric Scanning, and Quantifica-
tion
5.2.4. Statistical Analyses

5.3. Results and Discussion

5.3.1 .Pm-Tests
53.2. Kinetics of U-CaseinHydmlysis in Skim Milk Renneted at Diffcmit
Values of pH
5.3.3. Kinetics of K-CaseinHydmlysis in Pre-Heated Skim Milk and the Effect
of Low pH

5.4. Conclusions on the Uscfiilness of the Mcthod

6. SmaU Strain Dynamic Rheological Analyicr of Gel Development fmm

Cultureà and Rennetcd MUk 1. Pmctical hpccts
6.1. Outlook
6.1.1. Expcrimcntal Plan. and Rcference Systms and Conditions
 6.2. Experimental Details
6.2.1. Milk Samples and Pre-Treatments
6.2.2. Heating Procedures
6.2.3. Protein Concentration by Laboratory-Scale Ulbafiltration
6.2.4. Lactic Acid Bacteria and Propagation Conditions
6.2.5. Bacteriological and Chernical Acidification of Milk
6.2.6. Renneting
6.2.7. Memurement of pH
Data Acquisition and T ~ t m e n t
6.2.8. Rheological Measurements with the Nametre Rheometer
Instrument Setup and Run Conditions
Data Acquisition and Treatment
6.2.9. Rheoiogicai Measurements with the Carri-Med Rheometer
Instrument Setup and Run Conditions
Data Acquisition and Treatment
6.2.10. Complementary Analyses
SDS-Polyacrylamide Gel Electrophoresis
ANS-Fluorimetry
Isothennal Microcalorimetry
6.2.1 1. Statistical Analyses

-
6.3. Pre-Tests Results and Discussion
6.3.1. The Use of Skim Milk Reconstituted fiorn Powder
6.3.2. Dynarnic Testing with the Nametrc Rheometer
Sensitivity and Repmducibility
Temperature Fluctuations Accompanying Gel Development
6.3.3. Dynarnic Testing with the Carri-Med Rheometer
The Approximation of Linear Viscoelasticity of Gels
Sensitivity and Repducibility
Gel Development at Different Frequencies of Oscillation
6.3.4. pH and Calorimetric Measurements, and Activity of Bacterial Cultures
Acidification Kinctics
Effects of Growth (Gclation) Conditions
Heat Production During Renncting and Bacterial Growth
6.3 .S. Microbial Deterioration of Unacidified Renncted Milk

7. SmaU Shrin Dynamic Rheological Analyses of Gel Development fmm

Cultureâ and Renneteci Milk n, Rmults and Dbcussion
7.1. Phenomenology of Gel Devclopment
7.1.1. Examples of Different Typcs of Gelation Profiles Rcsulting h m Varying
the Concentrations of Rennct and Starter Cultures 197
Cornparison of Time-Pmfiles for Nametrc Consistency and Carri-Med
Dynamic Moduli 202
7.1.2. Analysis of Gelation Profiles 209
Uscfiilness of Tirne-Derivative Curves 209
Conversion of K-Casein 213
Variations in ANS-Fluorescence 219
 7.1.3. The Problem of Syneresis 220
7.1.4. Analysis of Gelation Profiles for Reference Milk Systems 223
Evolution Over Time of (Derivative)Consistency, Dynamic Moduli,
and Loss Tangent for Standard Milk Coagulateâ by Rennet at
Constant pH 223
Effects of Concentration of Rennet at Constant pH 228
Effects of Relatively Acidic pH at Renneting at Constant Concentration
of Rennet 228
Cornparison with Milk Coagulated by Lactic Acid 229
Efftcts of Concentration of Starter Cultures 239
7.1 S. Analysis of Gelation Profiles for Cultured and Renneted Milks 242
Effects of Concentration of Starter Cultures at Constant Concentration
of Rennet 242
Effects of Concentration of Rennet at Constant Concentration of
Starter Cultures 247

7.2. Gel Development fiom Cultured and Renncted Milk as Affected by Pre-Treat-
ment of Milk
7.2.1. The Use of Different Milks and the Eflects of Various Additions
Coagulation of Whole Milk vs. Reconsttuted Skim Milk and Effects
of Homogenization
Effects of Various Additions
7.2.2. Effects of Gelation Temperature
7.2.3. E f f of ~ Pm-Heating Milk
Gelation Profiles for (Derivative) Consistency, Dynamic Moduli, and
Loss Tangent
Possible Interpretation of the Coagulation Behaviour of High-Heated
Milk and Cornparison witb that of Ultra-High Heated Milk
7.2.4. Efkcts o f Pre-Concentrating Milk by Ultrafiltration

7.3. General Discussion

7.3.1. Key Parameters in the Progress of Gel Development on Combined
Biological Acidification and Renneting of Milk
7.3.2. Relation of Experimental Rcsults to Plcvious Work
Studies of Gcl.ation of Acidifying and Renneting Milk
Studies of Gelation of Acidifying Milk
7.3.3. Proposed Interpretation of the Processes of Gel Dcvelopment in
Acidifying and Renneting Milk
Concurrent Acidification and Gel Formation
Largcly Concurrent Aciditication and Gel Formation
Largely Squential Gel Fornation and Acidification
7.3.4. Possible Tschnological and Nutritional Relevance
Processing of Dairy Products
Gastnc Digestion of Milk by the Pm-Ruminant Calf

8. Concluding Remarks 342

vii
Table 4.1. Results fiom L e significance testing of the effects of MUF pH (6.7-SS),
milk pre-heat treatment (90°C- i min), and week on the average hydre
dynamic diameter of casein psiticles (dh),overall decrease in particle
hydrodynarnic diameter upon renneting (Mmat 2S°C, and particle hydro-
phobicity (HO)at 20°C. 101
Table 43. Overall apparent hydrophobicity Ho (arbitmy intensity unitsfpercent wlv
of micellar casein) of casein particles isolated fiom unheated and pre-heated
(90°C- 1 min) fresh milk and serially diluted in MUF at different values of
pH at CU. 20°C. 128

Table 5.1. Results fiom the significance testing of the effects of pH, pre-heat treatment
(90°C- 1 min), and week on characteristic parameters of the renneting process
in fresh skim milk [0.006% (vlv) rennet, 2S°C]. 142
Table 53. Effect of pH on some characteristic parameters of the renneting process in
unheated fresh skim milk [0.006% (vlv) rennet, 2S°C]. 142
Table 5.3. Effect of pH on some characteristic parameten of the renneting process in
skim milk pre-heated at 90°C- 1 min [0.006% (vlv) rennet, 2S°C]. 147

Table 6.1. Experimental conditions for heat treatment of milk and approximate extent
of denaturation of whey proteins. 155
Table 6.2. Average composition (in wt. %) of ultrafiltration retentates prepared from
skim milk reconstituted to 9%. 159
Table 6.3. Reproducibility of experimentation with the Nametre rheometer: mean,
standard deviation (SD), and coefficient of variation (CV) for characteristic
parameters of combined enzyrnatic and lactic acid coagulation kinetics.
9% RSM,C14-Rx4, 40°C. 175
Table 6.4. Reproducibility of experimentation with the Carri-Med rheometer: mean,
standard deviation (SD), and coefficient of variation (CV) for characteristic
parameters of combined enzymatic and lactic acid coagulation kinetics.
9% RSM,CI4-Rx4, 40°C, 5% strain, 0.1 Hz. 180
Table 7.1. Percentage hydrolysis of K-casein,as estimated by SDS-polyacrylarnide
gel electrophoresis, at various stages during the coagulation of standard
reconstituted skim milk at 40°C under difkrent conditions of concentration .
of acidifying starter cultures (C/i) and rennet enzymes (Rxj]. 218
 LIST OF FIGURES

Figure 1.1. Typical methods of processing milk leading to defined products.

Figure 2.1. Primary structure of the A genetic variant of bovine K-casein.

Figure 2.2. Essential pathways to destnbilimtion and coagulation of m ilk casein.
Figure 2.3. Network modcl of a 'hairy' casein micelle showing a mon or less spherical,
highly hydrated, and fairly open particle.

Figure 3.1. Block diagram of the Malvem Photon Conelator SpectrometerN.

Figure 33. Decrease in average apparent hydrodynamic diameter dh of casein micelles
as the surface layer of K-caseinmacropeptide is broken down by the action
of rennet enzymes (chymosin).
Figure 3.3. Schematic picture of SDS-polyacrylamidegel electropherograms of bovine
milk proteins from untreated and partly renneted milk on a 20% homoge-
neous ~ h a s t ~ e l @ .
Figure 3.4. Comparison of the idealized sliear stress responses, o(t), of an elastic solid,
a viscous fluid, and a viscoelastic semi-solid under oscillating shear strain,
y(t), when deformation (strain) is within the linear viscoelastic range.
Figure 3.5. Block diagram of the Namctre Rheoliner 2010 RheometerTM.
Figure 3.6. Block diagram of the Carri-Med CLS 100 Controlled Stress RheometerTM.
Figure 3.7. Concentric 'cylinders' with cone and plate end (Mooney-Ewart geometry).

Figure 4.1. Schematic of sample preparation for particle size and hydrophobicity
measurements by photon correlation spectroscopy (PCS) and ANS-
fluorimetry, respectively.
Figure 4.2. Apparent average hydrodynamic diameter dh of casein particles diluted in
MUE: at 25°C as a function of the pH of MUE
Figure 4.3. lntensity distribution of particle sizes for casein particles isolated from
unheated f k h miik and diluted in MUF at pH 6.7 and 5.5 at 2S°C.
Figure 4.4. Apparent average hydrodynamic diarneter dh of casein particles diluted in
MUF at 2S°C with and without ethanol added as a hinction of the pH of
W.
Figure 4.5. Apparent average hydrodynamic diametcr dh of @mu) casein particles
isolated fiom a single sample of unheated fresh milk as a function of time
after adding rennet enzymes under diffennt conditions of pH of MUF at
25°C.
Figure 4.6. Apparent average hydrodynamic diameter dh of @mu)casein particles
isolated fiom a single sample of fiesh milk pre-heated at 90°C for 1 min as
a function of time ûfter adding rennet enzymes under different conditions
of pH of MUF at 25OC.
Figure 4.7. Apparent average hydrodynamic diameter dh of @mu)casein particles
isolated froin a single sample of unheated fresh milk as a function of time
after adding rennet enzymes at pH 6.7 and 2S°C.
Figure 4.8. Overall decrease in hydrodynarnic diameter MH of @ara) casein particles
isolated fiom unheated and pre-heated milks upon the action of rennet as a
function of the pH of MUF in which the particles were diluted at 2S°C.
Figure 4.9. Intrinsic, extrinsic, and net fluorescence intensity FIof casein particles
serially diluted in MUF at pH 6.7 and 5.5 at Ca. 20°C.
Figure 4.10. Effect of sodium azide (NaNi)on the fluorescence intensity FI of casein
particles serially diluted in MUF at pH 6.7 and 5.5 at CU. 20°C with and
without NaN3.
Figure 4.11. Overall apparent hydrophobicity Ho of casein particles isolated from
unheated and pre-heated (90°C-1 min) fresh milk as a function of the pH of
MUF at CU. 20°C. 127

Figure 5.1. Disappearance of K-caseinand appearance ofpura-K-caseinas functions of

time after the addition of rennet enzymes under different conditions of pH
of unheated skim milk at 25°C. 138
Fiyre 5.2. Semi-logarithmic plots of the progress curves of rennet hydrolysis of
=casein shown in Figure 5.1.
Figure 5.3. Contrasted tirne-courses of the disappearance of K-caseinand of the
appearance ofpara-K-caseinunder different conditions of pH of unheated
fipsh skim milk at 25°C. t 40
Figure 5.4. First-order rate constant of rennet hydrolysis k, visual clotting time CT,and
percentage K-caseinhydrolyzed at CT as fiinctions of the pH of renneted
skim milk at 25°C. 141
Figure 5.5. Contrasted evolution of the first-order rate constant of rennet hydrolysis k
and of the hydrodynamic diameter dh of casein particles as functions of the
pH at 25°C. 145

[Note: Complementary illustrations relevant to Cbapten 6 and 7 (331 figures) are bound as
a body of graphical appendices separate fmm the main body of the dissertation, and listed
therein.1

Figure 6;l. Synopsis of gelling systems and gelation conditions for small strain
dynam ic rheotogical testing with the Nametre and Carri-Med rheometers. 1 52
Figure 6.2. Schematic of ultrafiltration system with the ~rniconmspiral-wound
membrane cartridge S1Y 10. (57
Figure 6.3~.Time-courses of consistency development and bacteriological acidification
for standard 9% RSM vs. (pasteurized) fresh whole milk and pasteurized
hornogenized fresh whole milk cultured and renneted at C/4-Rx 1 at 40°C. 172
Figure 6.36. Time-courses of consistency development and bacteriological acidification
for standard 9% RSM vs. (pasteurized) fresh whole milk and pasteurized
homogenized (commercial) whole milk cultured and renneted at C/4-Rx8
at 40°C.
Figure 6.4~.Typical evolution of the pH of milk with time during the incubation of
different amounts (Ch) of a co-culture of 1 :3 Lactococcus lactis subsp.
Iactis with Lactobacillur delbrueckii subsp. bulgari~~~/Streptococm
salivaris subsp. thermophilirs in standard RSM at 40°C.
Figure 6.46. Typical evolution of the pH of milk and its rate of change with time
dpWdt (i.e., rate of acidification) with time during the incubation of a co-
culture of lactic acid bacteria at level C/4 (no rennet) in standard RSM at
40°C.

xii
Figurea 4.Su&b. Contrasted evolution of the pH and consistency C of milk, and their
rate of change with time (Le., dpWdt and dCldt) with time d h n g the
incubation of a CO-cultureof lactic acid bacteria at level Cl4 in differently
renneted standard RSM at 40°C. 187-88
Figure 6.6~.Contrasted time-courses of heat production, (uncontrolled) acidification,
K-caseinhydrolysis, and consistency development for standard RSM
renneted at CO-Rx8 at pH 6.4 and 40°C. 191
Figure 6.66. Contrasted time-courses of heat production, acidification, and consistency
development for standard RSM cultured at Cl8-RxO at 40°C. 192
Figure 6.6~.Contrasted time-courses of heat production, acidification, K-casein
hydrolysis, and consistency development for standard RSM cultured and
renneted at Cl8-Rx8 at 40°C, 193
Figure 4.6d. Contrasted time-courses of rate of heat production AQ for standard RSM
di fferently cultured andor renneted at 40°C. 194

Figure 7.1.1. Set of typical consistency development curves for standard RSM cultured
at level Cl4 and difl'erently renneted at 40°C. 198
Figure 7.1.2. Set of typical consistency development curves for differently cultured
standard RSM renneted at level Rx4 at 40°C. 199
Figure 7.1.3. Set of typical elastic modulus development curves for standard RSM
cultured at level Cl4 and differently renneted at 40°C. 200
Figure 7.1.4. Set of typical elastic modulus development curves for differently cukured
standard RSM renneted at Ievel Rx4 at 40°C. 20 1
Figure 7.l.S~.Contrasted profiles of gel development obtained for non-renneted
standard RSM cultured at level Cl4 at 40°C using the Nametre rheometer
and the Carri-Med rheometer. 203
Figure 7.l.Sb. Contrasted profiles of gel development obtained for standard RSM
cultured at level Cl4 and renneted at level R x l at 40°C using the Nametre
rheometer and the Carri-Med rheometer. 204
Figure 7.1.5~.Contnsted profiles of gel development obtained for standard RSM
cultured at level C/4 and renneted at level Rx4 at 40°C using the Nametre
rheometer and the Carri-Med rheometer. 205

xiii
Figure 7.1.Q. Contrasted profiles of gel development obtained for non-renneted pre-
heated RSM cultured at level Cf4 at 40°C using the Narnetre rheometer
and the Carti-Med rheorneter.
Figure 7.1.6b. Contrasted profiles of gel development obtained for pre-heated RSM
cultured at level Cl4 (CI2) and renneted at level Rx 1 at 40°C using the
Nmetre rheometer and the Carri-Med rheometer.
Contrasted profiles of gel development obtained for pre-heated RSM
Figure 7.1.6~.
cultured at level CI4 and renneted at level Rx4 at 40°C using the Narnetre
rheometer and the Carri-Med rheometer.
Figures 7.1.7a&b. Typical curves of consistency development vs. tirne for standard
RSM cultured at level Cf4 and differently renneted at 40°C, and the use of
time-derivative data [i.e., instantaneous rate of change of consistency C with
time or gradient of consistency curves, Le., dC(t)/dt] for detining characte-
ristic pointdregions along the primery curves. 21 1-12
Figure 7.1.&. Typical profiles of consistency C vs. pH for non-renneted standard RSM
differently cultured at 40°C. 214
Figure 7.1.8b. Typical profiles of consistency C us. pH for differently cultured standard
RSM tenneted at level Rx 1 at 40°C. 215
Figure 7.1.û~.Typicd profiles of consistency C vs. pH for differently cultured standard
RSM renneted at level Rx4 at 40°C. 216
Figure 7.1.9. Contrasted evolution of the percentage hydrolysis of K-casein,
(derivative) consistency C, and pH of milk with time for standard RSM
cultured at level Cf4 and differently renneted at 40°C. 217
Figure 7.1.10. Parallel evolution of consistency C and temperature vs. time for standard
RSM coagulated at 40°C under conditions of acidity and renneting conducive
to conspicuous syneresis of gel. 222
Figure 7.1.11.Overview of elastic modulus developrnent curves for non-cultured
standard RSM treated with 0.02% (wfv) NaN3 and differently renneted at
pH 6.4 at 40°C. 225
Figure 7.1.12. Profiles of elastic modulus G', its rate of change with time dG'ldt, and
loss angle 6 (tan 6 = G"/G') vs. time for non-cultured standard RSM pattially
pre-acidified to different values of pH below 6.4 and renneted at level Rx4
at 40°C. 227

xiv
Figure 7e1.13. Profiles of consistency C and pH of milk. their rate of change with time
(i.e., dC/dt and dptildt), and temperature vs. time for non-culhued standard
RSM partially pre-acidified to different velues of pH below 6.4 and renneted
at level Rx4 at 40°C. 230
Figure 7.l.14~.ûvewiew of consistency development curves for non-renneted standard
RSM differently cultured at 40°C. 23 1
Figure 7.1.146. Overview of time-derivative curves of consistency (i.e., rate of change
in consistency C with tirne, dC/dt) for non-renneted standard RSM differently
cultured at 40°C.
Figure 7.1.1Sa. Overview of elastic modulus development curves for non-nnneted
standard RSM differently cultured at 40°C.
Figure 7.1.lSb. Overview of time-derivative curves of elastic modulus (Le., rate of
change in elastic modulus G' with time, dG'/dt) for non-renneted standard
RSM differently cultured at 40°C.
Figure 7.1.16~.Overview of consistency development curves for non-renneted RSM
pre-heated at 90°C- 1 min and differently cultured at 40°C.
Figure 7.1.166. Overview of time-derivative curves of consistency (i.e., rate of change
in consistency C with time, dC/dt) for non-renneted RSM pre-heated at
90°C-1 min and differently cultured at 40°C.
Figure 7.1.17a. Contrasting of the primary and derivative profiles of consistency C
and pH, and elastic modulus G' and loss angle 6 vs. time for standard RSM
renneted at level Rx4 at pH 6.0 (rennet control) and for standard RSM
cultured at level Cl8 (lactic acid control) at 40°C.
Figure 7.1.17b. Contrasting of the primary and derivative profiles of consistency C
and pH and elastic modulus G' and loss angle 6 vs. time for pre-heated
RSM renneted at level Rx4 at pH 5.8 (rennet control) and for pre-heated
RSM cultured at level C/8 (lactic acid control) at 40°C.
Figure 7.1.1&i. Overview of consistency development cuwes for difierently cultured
standard RSM renneted at level Rx l at 40°C.
Figure 7.1.186. Overview of consistency development curves for differently cultured
standard RSM renneted at level Rx4 at 40°C.
Figure 7.1.19. ûverview of elastic modulus development curves for differently
cultured standard RSM renneted at level Rx4 at 40°C.
Figure 7.1.2ûu. Ovewiew of consistency development curves for standard RSM cultured
at level C/4 and differently renneted at 40°C. 248
Figun 7.1.20b. Ovewiew of consistency development curves for standard RSM cultured
at level C/2 and differently nnneted at 40°C. 249
Figure 7.1.21~.Overview of elastic modulus development curves for standard RSM
cultured at level C/4 and differently renneted at 40°C. 250
Figun 7.1.216. Ovewiew of elastic modulus development curves for standard RSM
cultured at level Cl2 and differently renneted at 40°C. 25 1
Figure 7.1.32. Typical evolution of loss angle 6 (tan S = G"/G') upon the coagulation
of cultured and renneted standard RSM ( ~ / ~ - R Xvs.
J >nnneted (CO-Rxj at
constant pH) and biologically acidified (C/i-RxO) standard RSM at 40°C. 254
Figure 7.1.23~.Average values of maximum rate of consistency development dC/dt
(before point PM, or its deemed equivalent, that is) for standard RSM
diffetently cultured and renneted at 40°C. 257
Figure 7.1336. Average values of consistency C at point PM, or its deemed equiva-
lent for standard RSM differently cultured and renneted at 40°C. 258

Figure 7.2.1. Overview of consistency development curves for (pre-heated) RSM with
various additions or pre-cycling of pH (6.7 to 5.8 to 6.4) prior to culturing
and renneting at Cl4-Rx4 at 40°C. 266
Figure 7.2.2a. Overview of consistency development curves for standard RSM
cultured and renneted at different temperatures ktween 20 and 40°C. 274
Figure 7.2.26. Overview of consistency development curves for RSM pre-heated at
90°C-1 min and cultured and renneted at diffennt temperatures between
20 and 40°C. 275
Figure 7.23. Typical evolution of loss angle 6 (tan 6 = G"1G') upon the coagulation of
cultured and renneted standard RSM (Cl2-Rx8) vs. renneted (CO-Rx8 at pH
6.4) and biologically acidified (C/2-RxO) standard RSM at 25 vs. 40°C. 277
Figure 7.2.40. Ovewiew of consistency development curves for differently cultured
RSM pre-heated at 90°C-1 min and renneted at level Rx 1 at 40°C. 282
Figure 7.2.46. ûvewiew of consistency development curves for differently cultured
RSM pre-heated at 90°C-1 min and renneted at level Rx4 at 40°C. 283

xvi
Figure 7.2.5. Overview of consistency development curves for RSM pre-heated at
90°C-1 min, cultured at level C14 and differently renneted at 40°C. 284
Figure 7.2.. Overview of elastic modulus development curves for RSM pre-heated
at 90°C-1 min, cultured at level C/4 and differently renneted at 40°C. 285
Figure 7 3 . 7 a Typical evolution of loss angle 6 (tan 6 = G"/G1) upon the coagulation of
cultured and renneted RSM (C/4-Rxj') pn-heated at 90°C- 1 min (or 1 15OC-
10 min) vs. renneted (CO-Rxj at constant pH) and biologically acidified
(C/i-RxO) pre-heated RSM at 40°C. 290
Figure 7.2.8. Overview of consistency development curves for differently pre-heated
RSM cultured and renneted at C/4-Rx8 at 40°C. 295
Figure 7.2.9. Overview of elastic modulus developrnent curves for differently pre-
heated RSM cultured and renneted at Cf4-Rx8 at 40°C. 296
Figure 7m2mlO. Overview of consistency development curves for differently (directly)
pre-concentrated RSM cultured and renneted at C/4Rx4 at 40°C. 303
Figure 7m2m11m Overview of elastic modulus development curves for differently (directly)
pre-concentrated RSM cultured and tenneted at C/4-Rx4 at 40°C. 304
Figure 7.2.12. Typical evolution of loss angle 6 (tan S = G"/G') upon the coagulation
of cultured and renneted (C/4-Rx4) differently pre-concentrated RSM vs.
renneted (CO-Rx4 at pH 6.4) and biologically acidified (C/4-RxO) pre-
concentrated RSM at 40°C. 308
Figure 7.2.13. Typical evolution of loss angle S (tan S = GW/G')upon the coagulation
of cultured and renneted (C/4-Rx4) pre-heated and differently concentrated
RSM vs. renneted (CO-Rx4 at pH 6.4) and biologically acidified (C/4-RxO)
pre-heated and concentrated RSM at 40°C. 309
Figure 7.2.14. Overview of typical evolution of loss angle 6 (tan S = G"/G') upon the
coagulation of differently pre-treated (or coagulated) RSM cultured and
renneted at Cf4-Rx4 (or C/2-Rx8) at 40°C. 310

xvii
Figure 7.3.1. Tentative contrasting of the coagulation profiles for cultured and renneted
milk as obtained by dynamic rheometry (i.e., present work with the Carri-
Med rheometer, low heat 9% RSM,C/4-Rx4,4O0C) and difising wave .

spectroscopy (DWS; after the data of Dalgleish & Home [199la&b], fresh
pasteurized skim milk, relatively high (or intemediate) rrnnet and low (or
intermediate) acidifying starter, 30-33OC). 3 16
Figure 73.2~.Schematic representation of the basic patterns of succession of continuous
(bacteriological) acidification and renneting as influenced predominantly by
the effective concentration of rennet enzymes in standard milk. 32 1
Figure 7.3.2b. Basic patterns of coagulation of (standard) acidifying milk as defined by
the (approximate) contrasted evolution of elastic modulus G', first time-
derivative theteof dG'/dt (i.e., instantaneous rate of change in G' with time),
and loss angle tan 6 (= GW/G')over time of incubation (pH) under the condi-
tions of continuous (bacteriological) acidification relative to renneting
illustrated in Figure 7.3.2~. 322
Figun 7.3.3. Schematic representation of the effects of certain treatment and composi-
tional parameters on the succession of continuous (bacteriological) acidifi-
cation and renneting in milk. 323
Figun 7.3.4. Typical curves of consistency development for standard low heat RSM
cultured at level C/8 and renneted at level Rxl and Rx4 at 40°C. 335
Figure 7J.5. Typical curves of consistency development for RSM pre-heated at 9O0C-
1 min, cultured at level C/8 and renneted at level Rxl and Rx4 at 40°C. 337
Figure 73.6. Approximate evolution of the pH of the abomassal contents of pie-ruminant
ruminant calf foilowing the ingestion of non-acidified fiesh milk (afier Roy
[1980]) and putative parallel evolution of the initial consistency of the casein
coagulum (before extensive disintegration, that is). 340

xviii
 FREQUENTLY USED ABBREVATIONS AND NOTATIONS

ANS 1-Anilinonaphthalene-8-sulphonate
Consistency (cP.g.cmJ) as measured with the Nametre rheometer; with dC/dt,
the instantaneous rate of change of consistency with time. i.e., the gradient or
slope of a curve of consistency vs. time at a given time t (cP.g.cm-%)
C/i Different levels of concentration of mixed starter culture
Ca Calcium (Ca2+, calcium ions)
CaC12 Calcium chloride
CCP ColIoidal calcium phosphate (used interchangeably with MCP)
CT Clotting or coagulation time (h or min)
cv Coefficient of variation (= SDImean)
dh Hydrodynamic diameter (nm); with Mf and Adfi the decrease in particle
hydrodynarnic diarneter upon renneting and exposure to acidic environment,
respectively; and dd@ (M&ît), the instantaneous rate of change in dh with
time (ndmin)
dpWdt instantaneous rate of change in pH with time (pH unitdh)
D Translation diffusion coeficient
DLS Dynarnic l ight-scattering
DWS Diffusing wave spectroscopy
Ea Activation energy (kl.mol- 1)
f Frequency of oscillation (= ol2x; Hz)
FI Fluorescence intensity (arbitrary unit)
G* Complex (shear) modulus (Pa) as measured with the Carri-Med rheometer
G' Elastic or storage modulus (in-phase component of complex modulus, Pa; also
refemd to as 'rigidity modulus'); with dGW, the instantaneous rate of change
of elastic modulus with time (Pdh)
G" Viscous or loss rnodulus (out-of-phase component of complex modulus; Pa)
GDL Glucono-64actone
GMP G lycomacropeptide of r-casein
Ho Apparent hydmphobicity (aibitrary unii/concentration of micellar casein)
IR Infrared

xix
LAB Lactic acid bacteria
LVE Linear viscoelasticity
M Molar (mole.1-1)
MCP Micellar calcium phosphate (also CCP)
moi. wt. Mo lecular weight (Da)
MUF Milk ultrafiltrate
n Number of replicates
NaN3 Sodium azide
NMR Nuclear magnetic resonance
Pi Point of inflection
PMU Point or tegion of local maximum (Le.. stationary point of zero slope) on a
graph of consistency or modulus of milk gel as a function of time (also used as
P M and
~ P ~ M to ~denote
~ the regions of local maximum in the rate of
bacteriological acidification of milk)
Point or region of local minimum (Le., stationary point of zero slope) on a
graph of consistency or rnodulus of milk gel as a function of time (also used to
denote the region of local minimum in the rate of bacteriological acidification of
milk)
PAGE Polyacry larnide gel electrophoresis
PCF Protein concentration factor
PCS Photon correlation spectroscopy
Q Heat; with AQ,the rate of heat production (pca1.s-1). and dAQ/dt, the
instantaneous rate of change in A Q with time (pca1.s-h)
Q 1o0c Temperature coefficient
r Sample correlation coefticient (a measure of linear correlation); with r2 (= R2),
the coefficient of detennination
h j Different levels of concentration of rennet enzymes
RSM Reconstituted skim milk
SD Standard deviation of a set of n sample measurements
SDS Sodium dodeçyl sulphate
SDS-PAGE Sodium dodecyl sulphate-polyacrylamidegel electrophoresis
t Time (h or min)
Tan 6 Loss tangent (= G"/Gi) as measured with the Carri-Med rheometet
UF U ltrafi ltration
v (or vol.) Volume
VCF Volumetric concentration factor (= volume of milk initially/volume of UF
retentate)
Volthour
w (or wt.) Weight
a-La a-Lactaibumin
P-LB PLactoglobulin
6 Loss angle (phase angle, phase lag or phase shift; degrees) as measured with the
Carri-Med rheometer
Amplitude of shear strain (dimensionless)
Apparent viscosity (mPa.s or cP)
Dynamic viscosity (mPa.s or cP)
Cornplex viscosity (mPa.s or cP)
Dynarnic viscosity (in-phase viscous cornponent of complex viscosity; mPa.s or
c P)
Out-of-phase elastic component of complex viscosity (mPa.s or cP)
Density (g.ml-1)
Amplitude of shear stress (Pa)
Amplitude ratio (= G*; Pa)
Angular frequency of oscillation (= fx2n; rads-1)
Aging. The process of change in structure and properties of a gelled material afier gelation.

Coagulation. Generally defined as a process of random aggregation. Coagulation of proteins

typically occurs subsequent to denaturational changes mermansson, 19791. As gelation,
coagulation may mult in a continuous network. Most systems arc classified as 'coagula' or
'gels' based on tlieir ability to immobilize a liquid (water-holding capacity) or their tendency to
synerese. Schmidt [1981] pointed out the ambiguities which arise from the use of the terms
'coagulation' vs. 'gelation'. These ambiguities are not easily resolved and so the two terms are
used more or less interchangeably throughout the dissertation.

Consistency. The property of a substance or material by which it resists permanent change of

shape [Reiner & Scott-Blair, 19671. Most ofien in the context of cheese making, this and other
related terms such as gel or curd 'firmness', 'rigidity', 'strength', or 'tension' are measured by
empirical devices and used to describe a composite of elastic and viscous effects (the elastic and
viscous moduli defined hereafter).

Elastic (or storage) modulus. The ratio of a stress to its corresponding elastic strain.

Gel. A substance that contains a continuous solid (e.g., protein) matrix enclosing a
discontinuous liquid phase. The continuity of the solid (polyrneric or particulate) structure gives
elasticity to the gel.

Gelation. The formation of a continuous network (gel) which exhibit. a certain degree of order
[Hennansson, 19791. As coagulation, gelation is generally interpreted as a prwess of random
aggregation and cross-linking.

Linear viscoelastic region. Generally defined as the region in which the responsc of a matenal
(e.g., strain) at any time is directly proportional to the value of the applied force (e.g., stress). A
linear viscoelastic material has properties that depend upon time alone and not on the magnitude
of stress applied. A non-linear viscoelastic material exhibits properties that are a function of time

xxii
und the magnitude of stress Ferry, 19801. Rheological testing within the bounds of linear
viscoelasticity commonly refers to (essentially) non-destructive testing. Beyond the material
L W region, the efastic structure begins to partially break down.

Losa angle. The Iag phase between stress and strain under sinusoidal stress in the absence of
inertial forces [Reiner & Scott-Blair, 19671.

Macrosyneresh. See 'syneresis' .

Microsyneresis. A process of phase separation in which the solid phase in a gelled system
separates from the liquid on a local scale. Microsyneresis may also be envisaged as a local
expansion of the average pore size of the gel, possibly involving swelling of the gel [Brinker &
Scherer, 19901. In casein gels microsyneresis can result in an increase in permeability but no
large-scale changes in the height of the gel, otherwise it would be teferred to as
.
'macrosyneresis' [See Walstra, 1993 for various definitions of syneresis most relevant to casein
gels.]

Rate. The changing with time of a variable.

Rheomalaxia (or rheodestruction). Irreversible thixotropy.

Strain. Relative deformation (changes of shape andor volume).

Stress. The ratio of a force (or system of forces) to the area of a surface element on which the
force(s) act(s).

Syncmis (or macroryueresir). A process of spontaneous contraction of the network of a gel

and concomitant expulsion of pore liquid. Syneresis is generally attributed to the formation of
new bonds through continuing aggregation reactions. The driving force is the greater affinity of
the gelled material for itself than for the liquid in the pores ptinker & Scherer, 19901.

Tbixotropy. A tenn refemng to the time-dependent decrease in viscosity due to shearing (Le.,
time-depndent thinning), and the subsequent recovery of viscosity when shearing is removed
[Barnes, 1997; van Vliet, 19991. van Vliet wams not to confuse this with situations in which
there are some imvenibie stmchupl changes to the product, e.g., acid milk gels that are stirred
in the manufacture of stirred yoghut (see 'rheomalaxis').

Toque. The moment of loads producing torsion [Reiner & Scott-Blair, 19671.

Viscoelmticity. Elasticity accompanied by viscous resistance [Reiiier & Scott-Blair, 19671.

Viscous (or lou) rnodulua. The ratio of the amplitude of that part of the stress which has a
phase lag (loss angle) of 90° with a sinusoidal stmin of angular fiequency CU,to the strain [Reiner
& Scott-Blair, 19671.

World line. The trajectory or trace of a partick in space-time.

xxiv
 Water Ic the soutce of ive, mil& the essence of the d n d
Tuareg proverb

The entire matter of food, and especially that of milk, is not only a nostalgic and persistent

part of our cultural baggage, it is a fescinating study too. Highly nutritious by nature, (cow) milk

also tums out to be a (deceptively) complicated polyphasic system, particularly when it cornes to

rationalizing key aspects of its industrial transfomation. Milk can be modified casily. Mature

bovine milk contains numeious compartmentalized components, including lactose (a 46 g.L-i),

fat (a 40 g.L-i), proteins (m 32 g.L-1;caseins and whey or serum proteins), and minersls (= 7

g.~-l [Jensen, 1995; McDonrld, 19971, that cm react, and many (bio) chemical and other-often
interrelatecCchanges can take place, particululy when milk is subjected to conditions that differ

markedly fiom physiological conditions. In a sense, the craft and technology of dairy practice is

still running ahead of any complete and coherent scientific understanding. Potential

repercussions of the seemingly endless complexities that can d s e when milk is pmcessed not

only sue a challenge to the imagination, they also open the way to on-going tcchnologicaV

product innovation and diversification.

Contiolled development of instability leading to coagulation is central to a number of

(sometimes ancient) methods of processing milk (Figure 1.1). Most cheeses and kindred

products, for instance, depend for their formation on the effects of two destabilization processes,

vu., renneting (wherc selectd protcolytic enzymes am adâcd to the milk and induce coagulation

of the caseins in the prcscnce of calcium (Ca) if the temperature is high mough) and

acidification ( w h m the pH is lowend h m its original value around 6.7 and the caseins are

precipitated around tbcir iso-ekctric region, CU. pH 5.34.6 at 20°C).

 In many cases the two tnatments are combined, selected strains of lactic acid bacteria or a

slowly hydrolyzing acid precursor king used so that gradua1 loweting of the pH (traditionally by

rn icrobial fermentation of lactose tc+inainlHactic acid) occun during the mneting reaction.

Limited proteoiysis, acidificaîion

Milk

Addification, limited pmtsolysis Cottage chasse, quarg

& cream chssse
Yoghurt
L k m n t e d rnilks
High heat, addHication
m ) Ricotta, Panesr

Figure 1.1. Typical methods of pmessing milk leading to defined productç [adapted from
Dalgleish, 1989aI.

Essential features of the rennet-induced clotting of milk are fairly clearly understd

[reviewed in Dalgleish, 1992, 1993~1.Initially, the acid proteinase (chymosin, or rennin, the

main coagulating enzyme active during tenneting) contained in rennet specifically hydrolyzes

the K-caseinwhich appears to k locatcd ptimarily on or near the surface of the so-called 'casein

micelles', the ubiquitous dispersed tiny proteinaceous particles found in milk and most of its

detivatives. This causes the particlcs to k o m e unstable and to flocculate or aggregate. As

aggregation pmceeds, a thm-dimensional macioscopic network of casein 'micelles' eventually

foms throughout the milk, physically converting fluid milk into a semi-fim vis~clasticgel. At

well-defincd fimness, the (mainly w c i n ) gel, or to use a common term, the ~ dis usually
, cut

into small pieces to pmmote the expulsion of the whey (mostly water, lactose, small ions, and

soluble whey proteins), a process called synereslr. Variations in checsc-making procedum relate

in part to differcnt methods of controlling synensis of the curd to obtain the desircd prduct
moisturc, acidity, and texture. [Sec for instance the accounts by Walstra & van Vliet, 1986; Hill,

1995~;Holsinger et al., 1995; Kililb, 199%on electronic medium, and Walstra et al., 1999.1

The versatility of milk and its technological behaviour in ternis of coagulability, syneresis,

and cheese curd (or yoghurt) chamcteristic9--and, ultimately, yield, quality, and consumer

acceptance of the final pmduct-are constrained to a gnat extent by the unique structures and

properties of the casein particles. Depcnding on the precise treatrncnts undergone by the

micelles, a range of distinct products c m k obtained, including countless varieties o f chases.

[Standard practices and products have k e n described in detail by Emmons 8 Tuckey, 1967;

Kosikowski, 1977; Raiiic & Kurmann, 1978; Fox, 19930,6; Kosikowski & Mistry, 1997.1 Fat

globules in unhomogenized whole milk and (denatured) whey proteins do interfere with the

formation of gel structure and are influential in modulating the final pioperties of the coagulum

[van Vliet & Dentener-Kikkert, 1982; Kilaib, 1985; van Vliet, 1988; Aguilera & Kessler, 1989;

Aguilera, 19921, but they are not the 'motor centre' in the aggregatiodgelation processes which

essentiolly affect the stability behaviour of the casein particles.

Why a study about the eRects of acidification on the coagulation behaviour of renneted

miik? Well, many investigations of mcchanism of remet coagulation have becn made around the

physiological pH of the milk, that is, many puzzles still remain about the details of the reaction

at lower, mlistic pH valua. In particulai, existing views about renneting tend to take link

account of the inhercntly dynamic and ephemeral character of the casein micelles. What is oAen

overlooked is that the typicrl composition, properties, and most likcly also, the very nature o f

these pmtein micelles alter considerabiy upm acidifkation [Hewtje et al., 1985; Rafs et ai.,

1985; van Hwydonk et al., 19860; Vrccman et al.. 19891, and that may bring extra twists to the

picturc (i.e., variations of the h i c theme of casein coagulation). The moleeuiar and kinetic

aspects of such 'denaturation' (physico-chernical) changes are largely enigmatic at pmcnt, as

are their consequences for pmtcolysis and ensuing arscmbly of a gel.

 Pm-rcnacting treatmcnts of milk such as high hcating and protein concentration ue other

important steps in the manufacture of a numkr of dairy spccialties, especially because they offer

attractive ways to i n c m cheese yield [Marshall, 1986; Howard et al., 1994; nviews by Luccy

& Kelly, 1994 and Leliévre, 19951. It is still a matter of dsbate. however, how such technological

treatments, either individually or in combination, affect the course of renneting under various

conditions of pH. The main r e m for heating milk to temperature x time conditions exceeding

conventional pasteurkation (> 7S°C for > 30 s) is to induce denaturation and complexation of

part of the rrum protcins with the micellar fnmework so that they get incorporated into the

curd. High pte-heating also remarkably impairs the rennetability of the milk [Momssey, 1969;

van Hwydonk et al,, 1987; Singh et al., 19881. The detrimental effects of temperature can be

(partly) reversed, if thc hcating was not t w scvcre, e.g, by acidifcation, with or without re-

neutralization Panks & Muir, 1985; Singh et al., 1988; Reddy & Kinsella, 19901. This is usually

related to the dectcasc in the repulsive forces among wnneted casein particles and to the increase

in concentration of Ca2+ in the scrum phase, although this may be only one side of the story. The

surfaces of the pseudo-micelles in heatcd milk seem to bear little resemblance to the surfaces of

native casein micelles. It is not impossible that adjusting the pH also induces more substantial

reorganization (mt3trucniring) of the overall micellar architecture, yet how excessive hcating and

subsequent acidification actually modify the average stability and rcactivity of the particla is far

fiom clear.

In the longer tem, prccioe pdictions of the influence of critical operational parameters,

such as acidification and pre-heat tmtment of milk shall assist the manufacturers to optirnize the

production of dairy foods. As well, better comprchension of the intricatc mechanisms involved in

ml rcnnet coagulation of mik, we hope, shall frilitate successful fonnulation and pmcessing

of (alternative) gel-bwd qstems of prcviously illdefined hnctionalities.

 In the p m m t dissertation, we take a closer look at the variations that may k introduced in

the renneting reaction scheme when proteolysis and acidification take place simultancously. We

conducted laboratory research and literatun reviews along two major lines. with a view to define

further L e curd-fonning reactions in non-pre-heated and pre-heated (skim) milks:

(1) Estimation of the changes in the structure of the surface of untreated and heat-treated

casein (pseudo) micelles of milk ktween pH 6.7 and 5.5, with statistical assessrnent of the

(interaction) effects of direct partial pie-acidification and pre-heating of milk.

(2) Systematic rheological study of different modes of coagulation (i.e., enzymatic vs. acid

vs. combined enzymatic and acid coagulation) in differently pre-treatcd milks.

We obtaincd information about average particle dimensions and surface structure through

the use of dynarnic light scattering @LS) in the fonn of photon comlation spectmscopy (ES)

combined with renneting. These and complementary studies of micellar hydrophobicity

(describcd in Chapter 4) were made on simplified (diluted) systems fiom milk; the pH was

adjusted by direct chemical acidification. Fresh milk was used to ensure that the starting material

had undergone as little uncontmlled altentions as possible.

Skim milk teconstitutcd h m powder was generally used in subsequent rheo-kinetic studies

(Chapters 6 through 7) to stmdardize the raw matcrial and minimize natural quantitative

variations in composition. We defined the rheology of gclling milks by pefionning continuous

(essentially) non-destructive dynamic measurements using two commercial oscillatory

rheometcrs with different measuring systems. The attendant strengths and weaknesses of each

instrument were considercd. Gels werc fomed in situ under a number of experimental

conditions; their viscodastic chatacteristics under smalt defonnation were related to the kinetics

of rennet hydrolysis of K-cwin as estimatcâ by polyacrylamide gel electmphorcsis (PAGE).

Milk was acidified by inoculating with lactic starter cultures rather than using acid prccursors
such as glucon~Iactone(GDL), in part because the latter may intcract with milk proteins

[Trop & Kushelevsky, 1985; other limitations to the use of GDL for simulating bacteriological

acidification have k e n discussed by Luccy et al., 199MJ.Bacterial starters are more dilficult to

handle in pmctice, but this appmach reflects common industrial practicc and prcsents interesting

possibilities for probing mechanistic aspects of rennet-lactic acid-induced coagulation: by

varying the relative concentrations of rennd and starters one can creatc a broad spectrum of

gelling situations in which the gels are fomed predominantly by the action of rennet, by

acidification, or by both proctsses.

Investigations revolved around the effects of milk acidification. Variables other than (but

related to) pH (e.g., Ca concentration, ionic stnngth and ion activity of the serum, rennet vs.

starter concentrations, and temperature at which coagulation is carried out) are important in the

clotting of milk but they were not of primary intcmt hem These and additional controllable

variables (e.g., variables relevant to indusaial application, such as incrcascd pmtein

concentration or pertinent combinations of test parameters) have only been looked at insofar as

they cou!d allow us to gain insights into reaction rncchanisms. Factors involved specifically in

the syneresis phase have hudly k e n touched on. It was not our purpose eithu to inquin about

the effects of acidification on the activity or stability of remet enzymes [outlined in van

Hooydonk â Walstta, 19871. In particulu, we did not attmipt to use purified enzyme

preparations. Rathcr, a standard checse remet (i.e., a mixture containing both acid proteinases

chymosin [EC 3.4.23.41, which contributes for about 87% to the spccitic pmtcolytic activity

under standard tcchnological conditions [van Hooydonk & Walstra, 19871, and pcpsin [EC

3.4.23.11) has been used throughout the pmject.

 To give context to the work, Chapter 2 presents a s w c y of the major fcahires of casein

micelles for agpgationlgelation and of determinant phenornena pertaining to gel (CU

rd>sett ing

starting fiom fluid milk. Elabontion o f the review chapter was influenceci by my perception that

the abundance and Pace of (scattemi) research and technical reports in the field have made it

dificult fur newcomers to catch up to the statc of the art, and alm thnaten to oveMmelm

researchen alreidy involved in the subject-a problem that is manifested, e.g., by publications

that (unwittingly) menly replicate previous works and by interpretations that stop short of

integreting previous observations. An important purpose of the dissertation actually was to

colkct and unify available information that could be used to consolidate understanding of milk

coagulation by remet and acid.

Some fkquently used terms and notations are defined more closely at the opening of the

dissertation. The underlying principles and inherent limitations of the techniques central to the

studies discussed in Chaptea 4 through 7 arc highlighted in Chapter 3.

 2. LITERATURE OVERVIEW

Wodd i( c m i r and nion of it t h m m thin&, incowigib3,plurol.. .

Louis McNeice, Snow [1935]

2.1. Casein Micelles in Bovine Mük and the Particlci Derived from Them by
Changing Tbeir Environment

Casein so-calleâ 'micelles' are a charismatic and omnipresent figure (1 always want to say

'enfànt terrible') of dairy chernistry and physics, a field day for scientists fiom many disciplines.

[The tem 'micelle' is used loosely in this context to mean limited aggregates of protein

m o l e c u l e ~ oconventional
t (soap or surfactant) micelles.] Numerous workea have studied the

constitution, ultrastructure, and stability of both native and aitificial bovine casein micelles

under the environment in milk over the last thm decades. In sorne aspects, the research

literature on the subject amounts to an imposing (not to say overwhelrning) pyramid. Sunly, the

intnnsic stn~cturaland functional complexities of the micellar bioassembly are not in thcmselves

a barrier to understanding (if only tentatively) its properties and behaviour at the fundamental

physico-chemical level. [For rcviews, s a Schmidt, 1982; McMahon & Brown, 1984; Walsba &

Jenness, 1984; Walstra, 1990; Holt, 1992; Jensen et al., 1995; Dalgleish, 19976; Home, 1998;

Tarodo de la Fuente et al., 1999; Walstra et al., 1999; and reports in Int. Duiry J., 9 (3/6), 1999.1

Still, should the= k but one certainty with milk micelles and relatcd mattcrs, it ought to k

that the situation is usuaîly more inb'icatc than originally thought. Principdly because the

micelle system pmcnts us with an inescapable and essential changefulness ('denaturability '),

which means that a reductive appioach (behaviour of constant particles with changing

conditions) can hardly k adoptcd. It may even be ugued on scmantic gmunds [Dalgleish,

19890. 199ûuJthat casein micelles as such only exist in the 'natunl' milieu of mature milk, at
the pH (r 6.7), ionic sücngth (a 0.08 M), and temperature (m 37OC) of scention. The casein

particles derived h m micelles in theu original form by changing their sunoundings (processing

imposesjust such conditions) are different h m their native counterparts; they pmbably cease to

khave as typical (intact) casein micelles when subjected to extreme changes. To nflect the

'mutability' of the material, micellar characteristics seem to be ktter pictured as evoiving dong

continuum lines &in to 'world lines' in physics, the path of each particular line retracing the

processing history of milk.

Wc currently have little depth of undentanding of the principles and reactions that govem
the formation of modified (transient) casein particles or 'temnants' fiom indigenous casein

micelles; nor do we have a clear and definite picture of the species of particles that emerge on

such process operations as heating, enzymatic proteolysis, andlor lowering of pH, partly because

their investigation is bset with (methodological)dificulties, and partly (in some cases) because

of insufficient research. Interactions among casein (pscudo) micelles in different States of

dispersion largely determine the formation, (micro) structure, and rheology of milk gels and

derivatives. Clearly, one ought to consida the possibility that nuances in the recictivity

(coagulability) of the pnmary andor secondary (modified) micelles may introdwe subtly

different pathways to destabilization, aggregation, and pst-aggrcgation piocesses.

2.1.1. Molecnlar Ckaracte&tiès ofthe Càseins ond Serum Proteins

The casein micelles in uncoolcd bovine milk contain virhially al1 of the caseins (ca. 80% of

the total nitrogen of milk) clustercd togediet with inorganic matter-prcdominantlycalcium (Ca)

phosphate (CCP or MCPWnto roughly spherical, physically stable aggregates of colloidal size,

for the most part 50-300 nm in diameter and of the order of 107-109 molcculu weight WcGann

et al., 19801, with substantial interstitial moisture or, more prcciscly, a solution similu to milk

scrum. It is the prcsence of these casein particles that gives milk its characteristic white ('milky')

appcarancc. Under conditions as in raw milk, the micelles, togcthr with fat globules, are
disperd in an aqueous solution of lactose, simple salts, and serum or whey proteins [i.e., non-

casein: mainly PlactoglobuIin (PLg, m 0.3 g.L-1). a-lactalbumin (a-La, * 0.1 CL-i), and

vuious y-globulins) callcd serum or whey.

Unlike casein micelles, whey pmteins are soluble, mainly sepamte molecules in their native,

globular fonn (monomeric or oligomeric, depending especially on the pH). They are particululy

heat labile cornpucd to micellar casein and can fonn (stiff) polymer products when milk is

heated above 60-70°C [Lyster, 1970; de Wit 1Yr Klarenbeek, 1981; Dannenberg & Klesser,

198&-,Parris et al., 1991; G M n & Griffin, l993a,b; Gimel et al., 1994; Elofsson et al., 1996;

see Jelen & Rattray, 1995; Wong et al., 1996 for reviews; M i c & Kurrnann, 1978 and

Dannenberg & Kessler, 1988~-cfor references about the relationships between heat treatment of

milk and the extent of whey protein denatuntion]. The PLg fraçtion is uniquely characterized in

milk as the main source of sulphydryl groups (-SH;one per mole) which can be either active

(exposcd) or inactive (buried) depending on the spatial conformation of the protein. Many of the

phenornena related to the efTects of heat on milk are vested in the reactivity of the thiol group of

PLg. [See McKenzie, 1971 for a revicw of thermal denatuntion of PLg; Roefs & de b i f ,

1994; Roefs, 1995; Mandenon et al., 1995; Qi et al., 1995 for ment fundamental studies.] PLg

also contains two intmmolecular disulphides; a-Lahas four such disulphide bonds but no k e

sulphydryl.

Bovine casein consists of four distinct primary proteins (gene products), VU.,a,l-, a@, p,

and K-caseinin the approximate proportions, by weight, 38%, 10%. 36%. and 13%; and several

minor protcins, including y-clseins (pmieolytic fngmcnts of bcasein) and proteose-peptones

[Davies & Law, 1980, 19831. The major caseins exhibit 'micro-hetcmgeneity' because of

variations in the degree of (pst-translational) phosphorylation, glycosylation (i.e., carbohydrate

binding), disulphide-linkcd polymerization, and genetically-controllcd amino-acid substitution

(genetic polyrnorphism) [mviewcd by Holt, 19%; Swaisgood, 1992, 1993, 19951. Extensive

10
studies have suggesteâ that genetic variability (including dcgree and type of K-casein

glycosylation motifs), in particular, hm some technologicd sipifhance, espccially in relation to

cheese-making (revieweâ by Jakob & Puhan, 1992; Jekob, 1994; Dziuba & Minkiewicz; 1996;

also Allmere et al., 1998; Walsh et al., 19983.

Al1 the caseins are phosphorylated, albeit to variable extents, the phosphate king esterified

to the polypeptides as monoesters of serine (or, rarely, of h o n i n e ) [West, 1986 for a review].

The K- (and a*) caseins contain two half-cystine nsidues per mole. These seem to fonn

intemolecular disulphide bonds under physiological conditions and thus bovine r-casein may

well exist as an oligomcr (six molecules on average?) in its native state in milk micelles

[McKenzie & Wake, 1961; Swaisgood & Btunner, 1962; Swaisgood et al., 1964; McKinley &

Wake, 1965; Woychik et al., 1966; Pepper & Farrell, 1982; Carroll & Fanell, 1983; Gmves et

al., 1992; Rasmussen et al., 19921.

The caseins are strongly hydrophobic in the order P > a,i > K > a ~although
, the primiry

structures indicaie that the hydmphobic and polar or charged residues are not unifonly

distributcd throughout the sequences (Payens, 1982; Walstra & lenness, 19841. Clustering is

particularly apparent for the centres o f phosphoryiation and hm a marked influence on the metal

(Ca2+)-binding properties of the pmtcins [reviewed by Farrell & Thompson, 19881. All the

caseins have a remarkably high content and fairly unifonn distribution of proline. Together with

the constellations of chuged groups along the peptide chains, the effect is to give relatively

disordered, open and mobile ('rhcomorphic' rather than 'random coil' in the words of Holt &

Sawyer [1993]) conformations in solution and, demonstrably, also in the (interiot of) casein

particles-ti1though to a Iesser degrec kcause interactions of the phosphocaseins with Ca in the

micelles rcstrict the fmdom of motion of the molecules, as show by nuclear magnetic

resonance spcctroscopy, or NMR, in D$l [Rollema et al., 19881.

 K-Caseinappem to k the rnost highly structured of the caseins [Loucheux-Lefebvre et al.,

1978; Ono et al., 1987; Richardson et al., 1992; Farrell et al., 1993; Plowman et al., 1997;

Crearner et d . 19981. Loucheux-Lefebvre and CO-workersproposed that the two predicted

tums mund the chymosin-susceptible bond may cause the relevant sequence in the vicinity of

Phtlo3-Metl~to protmde h m the sutface of the ic-in molecule, enabling it to fit into the

active-site clefi of acid proteinases.

1 t Flexible glycomacropeptide
H I PHPHLSFi~Mi~IPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVAT
clcrivage + 1 t by chymoain

LEDSP 1soEVIESPPEMTVQVTSTAV169
C-terminal

Figure 2.1. Pdmary struchue of the A genetic variant of bovine K-casein [after Holt & Home,
1996). The molecule divides readily into a faitlv riu hydrophobie N-terminal half and a
flexible, hydrophilic C-terminal half.

A = Ala C = Cys D = Asp E = Glu F = Phe

G = Gly H = His 1= Ilc K = Lys L = Leu
M = Met N = Asn P=Pm Q = Gln R = Arg
S, = Ser T = Thr V = Val W=Trp Y=Tyr
 As illustratcd in Figun 2.1, w-casein can be divided into two distinct regions vu.. a fairly

hydmphobic N-terminal with a small positive charge m n d neutml pH (al= known as para-K-

casein, appently containing most of the rccondary structure) and a flexible, anionic C-terminal

portion containing al1 the hydmphilic glycosidic moieties (thne or four hexose residues with

varying numbers of N-acetyl neuraminic acid or NANA [Walstra & Jenness, 19841).

The prosthaic carbohydnte groups appear to have protective hinctions against proteolysis

and in particulsr retard the action o f rennet enzymes (chposin). They are important for the size-

determining and structure-stabilking rôles of K-casein in micellar casein [Slattery, 1978;

Gahmkrg & Tolvanen, 19961(see Section 2.1.2).

In its native position on the surface of the micelles, K-casein is thought to be Iinked to the

remainder of the proteinaccous hamework via the pma-K-casein part of the molecule. The C-

terminal hgment is released as a short soluble giycomacmpeptide (GMP) upon hydrolysis by

milk-clotting enzymes (rennet), setmingly with little change in the average secondary structure

ofporo-K-casein,ai lest when it is in a non-micellar fom at around neutral pH and 30°C [Ono

et al., 19871. Structural and sequence homologies between K-casein and blood fibrinogen, and

the broad similarities between the coagulation of milk by chyrnosin and the final stages of in vivo

coagulation of blood by thrombin have k e n highlighted by Visser et al. [1981], Jolks &

Henschen [1982], and Jollts & Caen (199 11.

Such prominent features as the prcsence of separate hydrophobie and hydrophilic domains,

phosphorylation, and a distinct Iack of ordereâ secondary and tertiary structure confer on the

caseins properties that are very much in evidencc in the formation and (industrial) khaviour of

the casein particles [reviewed by Wong et al., 1996; Dalgleish, 19976; Home, 19981. They also

ought to be scen as key featurcs contributing to the biological assembling and the great diversity

of biological activities of the cuein syotem, whether they k nutritional and physiological for the
young or physiological for the cow [Hill et al., 1969; Roy, 1980; Holt, 1992, 1995; Yamauchi,

19921.

2.1.2. Casein MiceIIes -Structure and Stubifliiy

(a) Phvsical and Chernical Characteristics. Milk casein, then, occurs as complexes of Ca

cascinate-'colloidal' Ca phosphate [plus magnesium (Mg)and some citrate], in respective

proportions about 94 and 6% by dry weight, and psrticles of this complex are collectively

refemd to as 'casein micelles'. Apparently, the ar and pcaseins combine with Ca-phosphate to

form the interior ('core') of the micelles, while the K-casein,which represents about 13% of total

casein, appears to be located predominantly in. or close to, the outemost regions of the particles,

together with some of the other (a,i-,plus portions of P?)caseins [McGann et al., 1980; Canoll

& Famll, 1983; Donnelly et al., 1984; Mehaia, 1984; Rollema et al., 1988; Dalgleish et al.,

1989; Leaver & Thomson, 1993; Diaz et al., 1996; Dalgleish, 19970, 19981. Native casein

micelles are fairly voluminous, containing approximately 4 mL serum per g of dry casein

(though values greater than 10 mL.g-1 are not uncornmon in individual, Le., not pooled milks)

[Walstra, 19791. They are polydisperse in size and molecular weight, and show considerable

variation in average composition (especially content of K-cwin and Ca phosphate, which is

teflected in average casein particle size and voluminosity [Saito & Igaiashi, 1981; Davies &

Law, 1983; Dalgleish et al., 19891) and structural organization; milk serum also varies.

The micelles are cacher fluctuating arrangements and show numemus changes, from the

pcrpetuil Bmwnian motion of the flexible surface Iayer (demonstrated by proton NMR [Griffin

& Roberts, 1985; Rollema et al., 19881) to the diffusion of ions molecules, or larger entities into

and out of the (loose or porous) casein particles (Ribadeau-Dumas & Garnier, 1970; Tarodo de la

Fuente & Labiée, 19871. Such dynamic equilibria are f u to the micelle side, however, at least in

fmh milk [Walrtrs & Jcnness, 19M].Any change in the physico-chernical environment (e.g.,
temperature, pressure, pH, and-various additions) disturbs the cornparimcntalization in milk,

including the relative distribution of milk salts and cascins (and whey pmteins) betwcen micellu

and bulk senun phases (Figure 2.3). The salt equilibria are particularly intricate, depend on

several conditions, and o h exhibit slow changes [Walstra & Jenness, 1984; review by de la

Fuente, 19981. Once the micellar structure is dismpted, it is unlikely that it can be re-assembled

in its original statc [McGann & Pyne, 1960; McGann & Fox, 1974; Lucey et al., 19961. Still,

many questions m a i n about, e.g., the changing with time (Le., the rates) and reversibility of

exchange with the serum. Surely, the concept uf 'casein micelle' is a multifaceted one because

there is no unique compositional and structural definition of a micelle, and it is perhaps more

useful to think of the particles as functional entities instead.

The crucial rôle of colloidal or micellar (i.e., insoluble or undissociated) Ca phosphate (CCP
and MCP, respectively), rather than of Ca2+ ions alone (though these are closely related

variables), in maintaining the structural integriwence, stability-of the micelle system has

long k e n highlighted malt, 198Sa, 1997, 19981. In fact, the propertics of casein particles and

associated salts can hardly k considercd independently. CCP also seems to play an important

part in buffering d u h g the acidification of milk and checse curd [overview in Lucey & Fox,

1993; Lucey et d 1993~1.The (chemical) nature of the attachmcnt of (indigenous) Ca phosphate

to micellar cuein is not easy to uncover, although many investigators belicve it exists as

amorphous tertiary Ca phosphate [Ca3(PO4)2] intersperscd throughout the micelles. Some

authors have suggested that there are distinct types of micellu Ca and phosphate with

differential exchrngeability [Pierre et al., 1983; Yamauchi et k , 1996; Zhang & Aoki, 19961.

The presence in milk of other ions, particularly Mg2*, and of the cwins. is thought to pmvcnt
tntnsfonnation of amorphous Ca phosphate to more stable fonns, such as hydmxyapatite [Holt &

Sawyer, 1988; Holt & van Kcmcndc, 19891. A somewhat differcnt viewpoint on the rôle of Ca
phosphates in micelle stn~ctureand stability, and on its dependence on pH, has been proposod by

van Dijk [1990a,b, 19921. [See also Walstra, 1999.)

(b) Stn~cturalModels and Imdications on Micelle Stability. Historically, ideas of casein micelle

structure and stability have evolved in tandem. The use of models has k e n helpful for

rationalizing known and conjectund facts and for maturing views on the mechanisms of

(de)stabilization of the micelles. Agreement on the details of micellar structures is by no means

complete, however. Here it should be noted that the term 'stability' is usually defined vis-84s

aggregation and coagulation; another (related) dimension to micelle stability, viz., stability

against intemal (intramicellar) rearrangements may be distinguished. The different States of

association of the caseins in both cases are apparently govemed by a balance of, rnainly,

attractive hydrophobie interactions and electrostatic repulsion (see the dual-bonding model of the

casein micelle explicated by Home (19981; alpo Bringe & Kinsella [1987]).

(0 The 'hairy' casein micelle.model has focused attention on the hydrophilic nature of the
micelles and steric (or polymeric [Napper, 19831) stabilization mechanisms [reviewed by Holt &

Home, 19961, providing a consistent p a d i g m picturc in the study of the particles and related

mattea. According to this unifying niodel, the C-terminal glycomacropeptides (and, possibly,

portions of the p u - c a s e i n moieties [Raap et al., 19831) ofsome of the r-casein protrude fiorn

the surface of the micelles out into the continuous phase and fom a highly hydrated difise laycr

of flexible, hydrophilic, and negatively chuged 'hairs', which prevent close appmach of intact

micelles. Destabilization and suboequent aggregation of the particles may occur if the protective

hain are physically removed on enzyrnatic action (e.g., renneting) or if t h u n t e m l a t e d ~ c r i c

andor charge interactions ktween micellu surfaces are pcmirbed upon reduction of solvent

quality (e.g., acidifcation and ethanol stability), or both (Figure 2.2).

At pnsent, the mcchanisms by which the stcric/elcctrostatic b d s is otherwisc rendereâ

ineffeetive during, rg., acid and hcat mcdiated coagulation rue Iargely unrcsolved. Evcn in the
cases in which the mechanistic bases for (de)stabilization arc understd, dissccting and

quuntifying the relative contributions of stcric vs. electrostatic components m a i n a challenging

task. Modeling of the stability of casein micelles h u been attempted using the adhesive hard-

sphete theory, with or without incorporating the concept of fiactal aggtegation [de K ~ i etf ai..

1995; de Kmif & Rafs, 1996; de k i f & Zhulina, 1996; de Kniif, 1997; Dickinson, 1997;

summarized by de Kniif, 1W].

Milk

I
Disperscd casein micelles

[- -
Ca caseinate colloidal Ca phosphate
94% -6% by dry wt. 1
Acidification

- -
Casein serurn remet - -
Casein semm soluble Ca & phosphate
(Ca para-caseinate matrix) -acid (demineralized caseinate matrix)

Firm, elastic geVcurd Jelly-like gcVcurd

Figure 2.2. Essential pathways to destabilization and coagulation of milk casein. Not only the
mode of formation, but also the composition and poc-treatment of milk and the conditions of
coagulation detemine the typc of teactions and interactions duthg coagulation, and hence the
typc of gekurd obtained.

(li) Of the several accepted models undet the umbrella of the hairy micelle model, two am

broadly in Iine with the extensive phenomenology of milk systems: the sub-~ssernblymode! and

the network mudel. The sub-assembly (or subunit) model of the casein micelle, originating in

the work of a host of authors, portrays the cote of the micelles as king divided into a luge
number of discrete basically spherical structures (the 'sub-micelles'. diameter 5 15-20 nm) with

a distinctly different character h m the hairy layer of K-casein [Shimmin & Hill, 1964; Morr,

1967; Calapiij, 1968; Rose, 1969; Schmidt & Buchheim, 1970; Waugh. 1971; Buchheim &

Welsh. 1973; Slattery, 1973, 1976, 1978; Slattery & Evard, 1973; Schmidt, 1974, 1980, 1982;

Schmidt & Payens, 1976; Payens, 1979; Pepper & Farrell, 1982; Famll & Thompson. 1988;

Ono & Obata, 1989; Kakalis et al., 1990; Walstra, 1990, 1999; Kumosinski et a!., l994a,b; Chu

et al.. 1995; McMahon & McManus, 19981. The putative sub-micelles are heterogeneous in

terms of both size and composition (esp. u-casein content). The forces among the individual

molecules in a sub-micelle are mainly hydrophobic and electrostatic (include intemal salt

bridges). Regions of CCP-and some (covalent) protein-protein bonds?-contribute to the

clustering of sub-micelles in micelles [Walstra, 1990, 19991. (Though we note it may as well be

the other way around.) The simple 'coat-corn' representation of Waugh & Noble [1965], in

which a predominantly hydrophilic 'coat' of K-casein envelopes a predominantly hydmphobic

'core' of rosettes of as and pcwins plus Ca phosphate. may also be seen as a declination of

the sub-assembly model. Based primarily on electron microscopical evidence, classical sub-

micellar models ail1 receive w i d e s p d support. To bc sure, Nature fkquently relies on sub-

assembly systems. But sub-assembly models of the casein micelles are not wholly satisfactory on

at least two counts. Firut, it can be shown that the hyddynamic diameter of the micelles

remains essentially constant during the urly stages of dissociation [induced by removal

(chelation) of Ca24 at neutral pH9 2S°C]. This would imply that cascin micelles are constructed

on a si=-detcrmining moleculu fnmework whose tcmplatc may relax but remsins essmtially

intact until the final stages of disintegration [Lin et al., 1972; Gtiffh et al., 19881. Second, it is

found that Basein (and, to a lesser extent, K- and a6asein) is prcfcrentially lost during the

initial stages of dissociation, especially at low temperature [Roefs et al., 1985; Roefs, 1986;
Dalgleish & Law, 1989; Gastaldi et al., 19961, indicating that either (i) the dissociation pn>ccss

does not involve cornpletc sub-units or (il) subunits rich in (and K-) casein dissemble first.

Network (or framework) model is the gencric namc for a micellar structure in which each

putick is considered to be a large continuous (and variable) chernical compouncl+nade from

btanched [Garnier & Ribadeau-Dumas, 1970; Garnier, 19731 or cross-linked [Holt, 1975. 1992,

1995; Graf & Bauct, 1976; Horne, 1986; Visser et al., 1986; Visser, 1991; Holt & Home, 19961

caseins fonning a loose and inhomogeneous gel-like structure, with at least some of the K-casein

et the outer surface of the particle. The telatively open conformation of the P and ~ecaseinsin
solution [Payens & Vreeman, 1982; Rollema et al., 1988; HoIl 1992; Holt & Sawyer, 19931 has

led to suggestions that the outside of the miceiles may be qualitatively no different in stnicture

from the core, and that a three-dimensional tangled web of polypeptide chains in the core may

only kcome pmgressively more difise towards the periphery of the micellar particle, as

illustrated in Figure 2.3. The structure-stabilizing fùnction of r-casein still is envisaged as an

essential feature. We will proceed largely based on this model.

In Figure 2.3, some 'sub-structure' (zones of relatively closer-packed caseins) is depicted in

a pmtein gel wifhout requiring the existence of sub-micelles. Such sub-structure might build up

dynarnically amund small domains ('nanoclustm') of morphous acidic Ca phosphate and

interact with one another hydrophobically [Holt et al., 1986; Holt, 1997, 19981. It may bc

suficient in practice to conceive the micelles as 'ftactal' association biocolloids rather than seck

to delineate the details of the (ever-vanishing) molecular arrangements.

It was Holt [1975] who initially suggested that cssein micelles bc rcgarded a9 coarse, partly

mineralized, micro-gel particles with regions of variable hydrophobicity. In this variant of the

concept, (partly rcversible) dissociation ptoceeds by loss h m the more hydrophilic regions of B

and r-cwins in prefemnce ta the more strongly associating asi-cueins. The high and variable

voluminosity is then undentood to rcflect the vm'able protein content in the more hydrophilic

19
regions of the micro-gel particles. nie effective hydmdynamic si= of die network particles

depends on their dcgree o f swelling (voluminosity or amount of solvent they contain), as

detenined by the solvent qudity of the medium in which they are d i s p c d . The extent of

aggregation of casein m o l c u l e ~ dconscquently,

, the size of the micelleAs limited by their

interactions with K-cascin.

Acidification ..- - -t - . - iieating

Hairy
layer

Figure 2.3. Network mode1 of a 'hairy' casein micelle (section) showing a more or less
spherical, highly hydratcd, and fairly opm particle [mainly aRer Holt, 19921. Polypeptide chains
in the con are cross-linked (@y) by nanometer-si& clusters of Ca phosphate (a); the intemal
structure gives rise insensibly to an extemal region of lower segment density known as the 'hairy
layer' which confers steric andlor (+/O)charge stability to native casein particles. Some (pseudo)
equilibria betwecn milk micelle and rrum arc depictcd schematically. Sce text for deuils.

This view, representing a continuity in pmtein association and agpcgation, is M e r able to

explain the assacietion behaviour of proteins in geneml [Clark et al., 19811 and of caseins in

particular woll 1992; Rollema, 19921. It would also accommodate the existence of metastable
quilibriurn States of the micelles and a certain nndomness (inherent 'fluctuability') of the

arrangement of the structural elements.

The hairy micelle model accounts for the dominant eff& of the suiface layer of (mostly) K-

casein in determining the stability of the casein particles against aggrcgation [Waugh & von

Hippel, 1956; Holt, 1975; Guthy & Novak, 1977; Walstra, 1979; Home & Parker, 19810.6;

Walstra et al., 1981; Home, 1984a; Griffin & Robeits, 1985; Holt & Dalgleish, 1986; Home.

1986; Home & Davidson, 1986; Dalgleish & Holt, 1988; overviews in Dalgleish, 1990a;

Walstm, 1990; de k i f & May, 19911. In contrast, the other caseins in the micelles likely

contribute more to the stnictum and less to the stability; they may be removed (esp. the

caseins, together with some CCP, e.g., by lowering the temperature to 4OC pose, 1968, 1969;

Downey & Murphy, 1970; Crearner et al., 1977; Ali et al., 1980a,b; Pierre & Brulé, 1981;

Reimerdes, 1982; Davies & Law, 1983; Roefs, et al., 19851). at leest in part, without drastic

deterioration of the stability of the particles. Non-K criseins are of considerable importance,

however, in defining the local forces which hold aggregatcd 'micelles' together.

(iio Somewhat different interpretations of the nature of the hairy layer have been put

forward. Hem it must bc pointed out that the regions identified as 'surface layer' need not be

strictly identical because different types of treatrnents [notably enzymatic (rcnneting) vs. ethanol

matment] probably probe different aspects o f suiface properties, as emphztsized by Dalgleish &

Hallett [1995]. Holt & Dalgleish [1986] and Dalgleish & Holt [1988] describcd the

hydrodynamic properties of the micelles in terms of particles with a partly draining outer layer of

thickness Ca. 12 nm-esscntially independent of micelle size-made up of (only) about 10% of

the total K-caseinin the micelles. The surface appears thinncr cxpcrimentally (about 5 to 7 nm as

shown by difiennt methods [Walstra et al., 1981; Home, 1984a; Home & Davidson, 19936; de

K ~ i&f Zhuiina, 1996; Alexander, 1997; a b Scott-Blair & Oosthuizcn, 1961;Guthy & Novrk,

19771) as a mult of hydration and dmining, and possibly, also, the presence of gaps betwcen
surface molecules. Home & Davidson [1986] envisioncd the micelle surface as an extended

(soft) gel-like structure ('gel-sheath' model), in which the individual molecules am more or less

Jtencally comlated, rather than as a mon fmly draining layer. de Kruif & Zhulina [19%] put

more emphasis on the idea of 'electrosteric' stabilization using the adhesive hard-sphere model.

They regarded the surface layer as a 'polyelectrolyte b ~ s h 'made up of rtasein hairs and

attempted to map out the colloidal stability of the micelles in tenns of a (sharp) stretched

(swo1len)-to-collapsed conformational transition of the salted polymer brush. They derived a

scaling equation to predict transition of the b ~ s hwhen either the charge density along the

polyelectrolyte chain or the chain density is lowered (e.g., by lowering the pH or renneting.

respectively), or both. [See also the work by de Kruif et al., 1995; de Kruif & Roefs, 1996; de

ffiif, 1997, 1999; Dickinson, 1997.1

(lu) Although the (mainly) steric stabilization mechanism of the hairy micelle model

represents an advance on early concepts, the pichire is probably more complicatcd bccause K-

casein molecules are integrated within the micelle h e w o r k rather than simply grafied or

adsorbcd ont0 the smooth and ncutral surface of hard-core spherical particles. Not only does the

prescnce of the surface layer lead to combined steric and electrostatic stabilization, but the core

itxlf is likely subject to variations depending on the way milk has ben tmated-ie., disruption

of the core structure of the casein micelles may be (dircctly) implicated in the loss of stability,

e.g., in acid coagulation of milk (Section 2.2.6). It is also conceivable chat rearrangements of

casein components in the interior of the particks may be felt in the extemal portions (e.g.,

propagating instability), thereby modulating the pmperties of the surfaces. Similady, surface

molecules may 'morb' or migrate to some extent, rcversibly perhaps, to the interior of the

particles.

Additionally, the model encourages the vicw that the topogmphy of the surface molecules is

rather uniform. What if r-casein actually is oligo or polymeric on the native micellar surfaces?
Then it is possible that only a fiaction of the surface molecuks provides the w-casein which is

observai by hyddynarnic or electmphorctic mobility measurrments to be eflkctive in

stabilizing the m i c e l l e d e more so if the surfaces are implar, Le., 'rough' or 'bumpy'

[Dalgleish, 1990~1.From studies of milks of 0 t h species, such as human, whose K-cascin

cannot polymerize (contains only one cystcinc) [Azurna et al.. 1984; Brignon et al., 1985; Kunz

& Wnnerdd, 1989; Wnnerâal & Atkinson, 19951, it seems that less K-caseinis prcsent. But why

and to what extent the casein micelles in bovine milk-and possibly, goat milk (goat r-casein

has three cysteine residues wrcier et of., 1976])-should be over-endowed with r-casein. and

what effect(s) this has on the structurai and functional pmperties of the particles is largely an

unwritten story so far. It may also be ch.1 the (native) surfaces consist of K-casein-richand K-

casein-depletcd arcas in a patchwork fashion because thcre does not seem to be enough I<-casein

to covcr al1 the surfaces [Dalgleish, 1997a, 19981. Clumps of wasein u~venlydistributed over

the surfaces would still provide long-range stabilizing forces among the particles. ksides, this

representation makes it easier to p i c m a mute by which incoming (bulky) molecules. such as

nnnet enzymes and (stiff) polymcrs of whey proteins, could penetrate into the layer and move in

(deeply) towards the vulnctable sites in @ma) r-casein without expcriencing detrimental

geometric or steric hindranccs. Perhaps pmtniding portions of kcasein complement K-cwin at

the suiface of the micclks, rs envisagcd by Dalgleish [1997a, 19981 in light of the rcsults of

Leaver & Thomson [1993] and Diaz et al. [1996].

Presumably, a more exact p i c m of native and partly denatureâ cascin particies cm ôe

obtained by making a synthcsis of a numkr of the aspects prexnted hercin. To be sure, the

remarkable compositional and structural diversity and the configuration d y ~ m i c sof the

'micelles' ought to k borne in mind in dcaling with any working model.

21.3. M'iflcaîit~no/Cosein M&elfts by Acidiflcatlo~)and Hwl

(a) Changes on Lowerino the OHBdow Phvsiolo&al Valuq. It is well documentcd that lowering

the pH of milk hwn Ca. 6.7 onwuds Imds to dissolution of micellar calcium phosphate

Bvenhuis & de Vries, 1959; Davies & White, 1960; Pyne & McGann, 1960; Bru16 et al., 1974;

BnilC & Fauquant, 1981;Pierre & Brulé, 1981;van Hooydonk et al., 19864; Visser et al.. 1986;

Dalgleish & Law, 1989; Gastaldi et al., 1996; Law, 1996; Singh et al., 1996; review by de la

Fuente, 19981. Apparently, this dissolution does not occur sharply as for titration curves in

gened, but rather gradually, the amount of micellar Ca phosphate being roughly proportional to

the pH over the pHsrange of ca. 6.7 to 5.0 at temperatures above 20°C. Althougti micelle-like

pariicles seem to remain, et least initially (pH > 5.4). they have difietent properties. Here the

method of acidification [e.g., direct or dialysis regulated acidification with inorganic acids vs.

glucono-&lactone (GDL) or bacterial acidification]may not be as important as the rate of acid

addition or production, and how long the milk is kept at lower than physiological pH, at least for

acids that do not chelate Ca2+,

The solubilization of MCP can be expccted to weaken the casein h e w o r k , but at

temperatures above 2S°C, then seems to be little pH-induced dissaciation of casein from both

rennet-treated and non-nnnet-treated (heated) micelles, even at values o f pH at which most of

the MCP is in solution [Rose, 1968; van HooyQnk et al., 19860, Dalgkish & Law. 1988; Law.

1996; Singh et al., 19961. Pmbably t h m is concurrent ncutrolization of the phosphoserine charge

by acid, which maintains an attractive interaction balance in favour of hydrophobic interactions.

The progressive loss of MCP and titration of acid groups on the cascins appear to be

primarily responsibk for the changes obscrvcd. [A graphic summary of rcported data is given by

Walstm, 1990.1 This is espccially clcu for the (negativc) ekctrophoretic ('surface' charge or 59)

potential which rises to about zero near pH 5.2-5.4 at mund 20°C [Schmidt & Poll, 1986; atm

Darling & Dickson, 1979; Bmon & Hardy, 1992; Wadc et al., 19961. It should k noted that a
lower pH in milk leads to a higher ionic stnngth and, in puticular, a higher activity of Ca2+ ions

([Cd+]) in the serum (2 to 3 time increase at pH 6.0 [van Hooydonk et al., 198641). which

contributes to lowering the negative charge on the particles palgbish, 19841. No obvious

difference in the mobility of the proteins constituting the micelles could ôe detected by 1H-NMR

in the range of pH 6.7-5.8 at 20°C Folkma & Brinkhuis, 19891. Variation in the voluminosity

(solvat ion) of @ara) casein particles at about the same temperature (or 30°C) in the pH range 6.7

to 5.2-5.4 is mon debatable; besides, some slight shifk in the size distribution of the particles on

reducing the pH [Vreeman et al., 19891 may be a confounding factor. Some findings [Tarodo de

la Fuente & Alais, 1975; Snoeren et al., 1984; Crearner. 1985; van Hooydonk et al.. 19860;

Famelart et al., 1996; Gastaldi et al., 1996, 19971 suggest that after an initial decrease below pH

6.7 (local minimum of voluminosity near pH &O?), the evolution of the voluminosity parallels

the increase in mobility (spin-spin relaxation time, T2)of water protons in skim milk, especially

klow pH 6.0 [Roefs. 1986; Roefs et al., 1989; Mariette et al., 19931.

The sharp transition (relative T2 maximum) near pH 5.2 is also manifest in some rheological

properties (loss tangent, or tan 6= G "/G', and elastic modulus G ', as defined in Section 3.4) of

rennet-induced skim milk gels at 20°C (this concems gels that have completely formed afker

acidification in the cold, addition of rennet, and subsequent quiescent wanning) [Roefs, 1986;

Roefs et al., 199061, and rems to fit with observations of acidified milk (no rennet) by electron

micmscopy [Heertjc et d,1985; Gastaldi et al., 19961 and indirect observations [Attia et al.,

19881. The peak in viscous-like behaviour ( t a 6) near pH 5.2-5.4 secms to correspond to the

optimum for 'meliability' andfor 'stretchability' of curd [Walstra, 19901. One may provisionally

conclude that the bonds keeping the casein particles together are wcakest, or fewest, at pH 5.2-

5.4 when a large amount, although not al1 of the CCP has k e n relessed: at pH around 5.3 (i-o.,

the pH of mon varietics of cheeses at the end of manufacture (Hill, 1995a]), nearly al1 of the
inoiganic phosphate in milk is solubilized, whemas eu. 14% of the Ca is still pnsent in the

casein particles [van Hooydonk et al., 198th (thennitcd skim milk, HCI-acidification at 4*C,

equilibration for 12 h in the cold, and subsequent wanning to 30°C); also Evenhuis & de Vries,

1959; Heertje et al., 19851. It rcrnains uncemin, however, whether CCP dissolves out to the

sarne extent and at the sarne rate in commercial practice.

At still lowet pH, a series of complex interplays of partial disintegretion-rearrangement-

teassociation of the deminetalized 'micdles' (relics theteof) seems to take place [Rose 1968;

Heertje et al., 1985; Roefs et al., 1985; van Hooydonk et al., 19860; Desobry-Banon, 19911 till

increasing electrostatic attraction (combined with hydrophobic interactions moefs & van Vlict,

19901) among casein molecules keeps the newly fonned (re-aggregated) casein complexes more

tightly together again at the isa-electric pH, around 4.7-4.6 at 20°C.

Recent biochemical, microstructural, and rheological investigations by Gastaldi and co-

workers [1996, 19971 (bacteriological and GDL acidification at 20°C) confirmed earlier findings

and interpretations of the sequence of events leading to aggregation and ultimately gel formation,

only the authors made a point of the existence of a 'micellar fusion or transition state' between

pH 5.5 and 5.0. [Note that some of the (rheological) results given by Gastaldi et al. appear

questionable and rnust therefore be interprcted with caution.] It its noteworthy that as the pH is

shifted fiom close to neutra) (2 6.0) to more acidic values & 5.01 the particles probably have

increasingly complicate and indeterminate (surface) arrangements before extensive clustering

and permanent (irreversible) gclling take place. How much of rnicellar (surface) churctenstics

are actualiy retained on the pathway to dcstabilization and agjpgation m a i n s unclear.

(b) Changes on Heatinn Bevond Pasteuci@m. Exposure to high hcating also changes the

average composition and sutc of association of the casein micelles apprcciably, which in turn

modifies their coagulation behaviours in ways that still de@ complete explanation. The

magnitude and revcrsibility of the changes genemlly depcnd on the scverity of the matment
applied and on the physico-chernical environment [sa Lucey, 1995; Mulvihill & Gtufferty,

1995; Singh, 1995 for teviews]. At temperatures in the region of 75-8S°C and above for a few

minutes amund physiological pH, most milk serum proteins (esp. PLg) denature progressively

and then bind (specifically) to micellar surface (u-casein) [Zittle et al., 1962; Hindk &

Wheelock, 1970a; McKenzie et al., 1971;Elfagm & Whcelock, 1977; Pearse et al., 1985; Singh

& Fox, 1987u,b; Jang & Swaisgood, 19901. The lower the pH at heating, the stronger the

association and the more PLg(-a-La)/u-casein compkxes adhere to micellat surfaces [Kudo,

1980; Heertje et al., 1985; Visser et al., 19861.

Micrompically, one can observe casein particles with 'ragged' and 'fuzzy' surfaces, and

'filamentous appendages' projecting imgularly Frwn the particles [Kallb et al., 1976; Davies et

al., 1978 (95OC-10 min)]. (In contrast, the hairy layer of native micelles is too thin and not dense

enough to be resolved by transmission electron microscopy.) Phenomenological descriptions for

heat-induced changes et the surface of casein particles in yoghun milk have been proposed by

Parnell-Clunies [1986] and Mottar et al. [1989] among others. Following the view o f Mottar et

al., PLg would initially bccome associated with the casein particles, nsulting in the formation

of an imgulat superficial stnicture of high (aliphatic) hydmphobicity; a-La would start to

deposit at higher intcnsities of heating (e.g., 90°C-IO min), covering the layer of PLg and

multing in smoother micellar suifaces of decreased hydrophobicity.

The degm of whey protein denaturution depcnds on the intensity of heating [Reoic &

Kurmann, 1978; Dannenberg & Kessler, 1 9 8 8 ~ 4and is influenced by a number of factors,

including pH, ionic (Ca) composition, md lactose content [Jelen & Ramay, 19951. Sevete heat

matment at ultra high temperatures (2 100°C)or for longer perids leads to sizable physico-

chernical modifications of the caseins as well, and to changes in the polydispersity and average

size (diameter) of casein particles relateci to limited aggtegation and dissociation phenornena

[Crcamer & Matheson, 1980; Snocren et al., 1984; Andenon et al., 1986; ûalgleish et al., 1987;
Moharnmad & Fox, 1987; Anema & Klostermeyer, 1996; Dalgleish et al., to be published].

Singh & Fox [1989] statcd that mild heating of milk to 90°C produces only minor changes in the

average dimensions o f the casein micelles, in agreement with the results o f Raynal & Remeuf

119981for casein particles in cow milk pre-heated in the range 75-90°C for 0.5-10 min. Based on

-
measutable increases in the viscosity of milk on heating (8S/90°C for 1 10 min), Jeumink [1992]

and Jeurnink & de Kniif [1993] surmised that only temporary clustering among heat-denatured

micelles occurs under such heat loads, possibly mediated by unfolding of WLg.

The interactions which stabilize the insoluble whey proteindw-casein complexes seem to

involve disulphide, hydrophobie (especially in the initial stages of complex formation? [Haque &

Kinsella, 1988; Jang & Swaisgood, 1990]), ionic, and hydrogen bonds, although many

researchers have considered covalent (sulphydryl) interactions to be prcvalent [Hill. 1989 for a

review; Gallagher 8c Mulvihill, 19971. The pmence of two half-cystines in para-K-

one at the boundary between the flexible C-terminal hair and the more rigid
casei~*ncluding

core (Figure Z.l)-implies that the whey pmteins must wom their ways through the entire dcpth

of the hairy layer before they may nact covalently with the sulphydryl groups o f K-casein

(oligomers).

About 20-30% o f total K-casein dissociates h m the micelles during or soon af?er thermal

treatment of milk at 85-95°C f0.r 5-10 min at pH 6.7 (as estimatcd at 20-30°C) [van Hooydonk et

al., 1987; Law, 1996; Anema 8 Klostermeyer, 19971. Hat-mediated depletion o f K-casein

seems not to k connected dircctly with the interactions between P L g and K-casein [Aoki et al.,

19741, though the presence o f whey proteins appun to k conducive to the dissociation (Kudo,

19801. Heatingmay also k accompanied by a small rcductiono f the content of micellar pcasein

m e r t j e et al., 19851.

The net negative charge of the w e i n particles as measured at ambient temperature seems to

increase slightly in the range o f pH 5.5f6.06.7 following (scverc) hcating (e.g., 90°C-30 min; 2
100°C-15 min) m l i n g & Dickson, 1979; Schmidt & Poll, 1986; Ancma & Klostermeyer, 1996,

19971, which suggests that the complexes of casein and denatured pLg(-a-La) may be slightly

mon negatively chuged than the original micelle surfaces (PLg cames a negative charge of -10

at pH 6.6 [Basch & Timasheff, 19671). (One ought to account for the small reduction in the pH

of mik cauxd by heating when interpreting the results.) Changes in micelle hydration

(hydrophilic pmpeiiies) with severe heating (à 110°C-20 min) appear to be marginal, at least if

the pH of milk samples is readjusted to its original value afier thermal treatment [Crearner &

Matheson, 19801. If anything, hydration may decrew slightly.

There are early suggestions that the micelles are the sites for deposition of Ca phosphate

h m milk senim Fox, 19811 and numerous reports confinning that the quantity of Ca phosphate

in the casein particles d a s incrcase on heating [Shalabi & Fox, 1982; Pouliot et al., 1989;

Dalgleish, 19896; van Dijk, 1990~;Wahlgm et al., 1990; Zhang & Aoki, 1996; review by de la

Fuente, 1998). The 'precipitation' of Ca phosphat-d concomitant reduction in soluble Ca

and inorganic phosphatb-depend on the intensity of the heat treatment and on the pH of the

milk [BnilC et al., 1978; Fox, 19821. Pouliot et al. [1989] have estimated that about 60% of the

soluble Ca is precipitateâ afler 10 s-1 min holding of k s h skim milk at 90°C. Heat-induced

complexes of Ca phosphate probsbly have (casein-binding) propcrties different h m those of

indigenous CCP [van Hooydonk et al., 1981; Lucey et d,1993aI.

IH-NMR spectra obtained for suspensions of casein micles at temperatures ktween 60-

98°C and around neutral pH show that abovc about 70°C parts of the casein molecules in the

micelles becorne morc flexible ~ o l l e m a& Brinkhuis, 19891, as though the (ovenll) structure

'melt' (revcrsibly) to somc atrnt. [Sec also Singh et al., 1996 for indirect evidence.] This

apparent relaxation or djustment may be important in rendering milk micelles morc or kss

liable to simultuicous denaturational changes in, e.g., whey proteins and salts induccd by

heating, d o r to the cffects of, cg., pst-acidification. The existence of a critical temperature
for appreciable modification of the casein puticles in the range 70-80°C around physiological

pH is also wggesteâ by the results of various investigations B16b et al., 1976; Bonomi &

Iametti, 1991; Bonomi et al., 1991;Home & Davidson, 19930; Iametti et al., 1993; Lucey et al.,

1W8e; Dalgleish et al., to be published).

No doubt the physico-chemical organization of the casein micelles in milk is highly sensitive

to acidification and heat-treatment beyond pasteurization. Still, despite a vast literature on the

acid and/or heat-modification of the casein particles in milk, including changes in the status of

semm proteins and Ca phosphates, we have linle insights into what molecular (re)arrangements

underlie the multiplicity of micelle chmcteristics.The cornplexity of the casein particies and the

lack of detailed information on their structure, and in particular on the disposition and mutual

relation of the components on their surface, is one of the factors that have hindered an

understanding of the mechanisms of milk gel formation. The effects of lowering the pH and

heating shall be discussed furthcr in relation to the formation and propenies of milk gels.

2.2. Formation and Propertics of Milk Gels

2.2.2. Studics on Gel Formotion in Acidifled Milk

In most (mechanistic) studies on the effects of lower than physiological pH on the renneting

reaction, milk is partly acidificd chemically and the pH remains constant during coagulation

[Rowland & Soulides, 1942; Kelley, 1951; Ashworth & Nebe, 1960; TuszyRski et al., 1968; Jen

& Ashwonh, 1970; Humme, 1972; Cheryan et al., 1975; Hossain, 1976; Olson & Bottaai, 1977;

Kowalchyk & Olson, 1977; Rarnet & Weber, 1980; Marshall et al., 1982; Shalabi & FOX, 1982;

Stony & Ford, 19826; Mehaia & Cheryan, 1983a; Pierre, 1983; van Hooydonk et al., 1986b,

1987; Carlson et al., 1987a,b; Korolcnik & Maubois, 1988; Kim & Kinsella, 1989a; Zoon et al.,
1989,1990; Shanna, 1992; Hyldig, 1993; Shanna et al., 1993; Schulz et al., 19976; Mpez et al.,

19981.

Haiwslkar dk Ka16b [198 11, Roefs [1986], Rocfs et al. [1990a,b], van Vliet et al. [1989,

1991~1,van Vliet & Keetels [1995], and Hammelehle et al. [1998] have looked at the structural

and mechanical properties of gels of skim milk formed by acidification with HCI or citnc acid to

pH 4.3-5.8 at 04OC and subsequent moderated heating to around 30°C, with and without

addition of mui* enzymes (Le., acid casein gels similsr to, but not quite identical to yoghurt-

like preparations).

de Kmif & Roefs [1996] examined the initial phase of acid-induced floçculation at

temperatures below 1SOC. Information is also available on the formation of acid gels by gradua1

and quiescent acidification at and above 20°C, whether by adding a gradually hydrolyzing

acidogen (most commonly GDL) to simulate fermentation by lactic acid bacteria (LAB)and

minimize the possible occurrence of localized pH gradients marwalkar & KalPb, 1980; Hcertje

et al., 1985; Roefs, 1986; Jablonka et al., 1988; Kim & Kinsella, l989b; van Vliet et al., 1989;

Bringe & Kinsella, 1990; Banon & Hardy, 1991, 1992; Home & Davidson, 19930; Desobry-

Banon, 1991;Desobry-Banon et al., 1994; Amice-Quemeneur et al., 1995; van Vliet & Keetels,

1995; Gastaldi et al., 1997; de k i f , 1997; Lucey et al.. 19970-6, Lucey et al., 1998w, 1999;

Home, 19991 or, more seldom, by bacterial action (Parnell-Clunies, 1986; Famelart & Maubois,

1988; Parnell-Clunies et al., 1988; Schulze et al., 1991; Biliaderis et al., 1992; Rohm, 1993;

RUnnegârâ & Dejmek, 1993; Benpigui et al., 1994; Vlahopoulou & Bell, 1990, 1992, 1993,

1W5;Vlahopoulou et of., 1994; Amicc-Quemeneur et al., 1995; van Made & Zoon, 1995aJ;

Gastaldi et al., 1996; de Bmbandere et al., 1998; Lucey et al., 1998& Chen et al., 19991.

In comparison, few otudies have k c n dediutcd to simultaneous minet and (Iactic) acid

coagulation Fhembte, 1986; Notl et al., 1989; Dalglcish & Hom, 1991ab; Noël et al., 1991;

Tranchant et al., 1999a,b]. Andotal (lugcly fatual) observations on the subject have km
reportcd by van Hooydonk et ai. [1986b], Zoon et al. [1988a, 19891, Aîtia et al. [ 19931, Caron et

al. [1997], and Schulz et al. [1999].

A variety of (complementary) methods have boen implemented in attempts to monitor

coagulation objectively with no or minimal mechanical perturbation of gel-setting, including

small deformation oscillation theometry [nviewed in Thomasow & Voss, 1977; van Hwydonk

& van den Berg, 1988; G m n & Grandison, 1993; O'Connor et al., 19951, thermal conductivity

[Hori, 1985; de Brabandere et al., 1998; Laporte et al., 1998; Tjomb, 19991, elecaical

conductivity or conductometry [Dejmek, 19891, turbidimetry [Payens, 1978; Surkov et al., 1982;

McMahon et QI., 1984a.c; Bringe & Kinsella, 1990; de Kruif, 19931, reflection photometry

[Hardy & Fanni, 1981; Hardy et al., 1981, 1985; Hardy & Scher, 1988; Banon & Hardy, 1991,

1992; Ould Eleya et ai., 19951, rehctometry ~ o r o l c n i ket al., 1986; Famelart & Maubois,

1988; Korolczuk, 1988; Korolczuk & Maubois, 19881, i n k d absorption [Laporte et al., 1998;

Tjomb, 19991, static light scattering (Bauei et al., 1995; Lehner et al., 19991, conventional

dynamic light scattering and diffusing wave spectroscopy [Dalgleish et al., 1981a.b; Walstra et

al., 1981; Dalgleish & Home, 1985; Dalgleish & Home, 1991a,b; Home & Davidson, 1990,

1993a,b], and ultrasonic wave propagation Everson & Winder, 1968; Marshall et al., 1982;

Benguigui et al., 1994; Gunasekaran & Ay, 1994; Bruneel, 1998; Tjomb, 19991. Not many

techniques are suitable to follow al1 the stages of gel (curd) formation. Also, as pointed out by

Jclcn [1997], despite the relative ease of pcrfoming such measumnents, the meaning of the

measutcd values-and their comlation/rclationsips to the underlying causes of the differences

and variations in diffcnntly coagulated milk systems-arc typically more dificult to understand.

The pmperties of (physical) biopolymer and food gels, including casein gels, have bscn

discussed by Clark & Ross-Murphy [1987], Clark [1991], and Doublicr et al. 119921; theoc

tcviews encompass rheological and sûuctunl characteristics and the applicability of various

gelation thmries. [Sec also Brinka & Schem, 1990, for fundamental accounts of the physical
and chemical principles of ml-gel phenornena.] Also, some aspects of (skim) milk gel formation

and pmpcrties such as pore size and distribution have been cxplained sani-quantitatively using

the concepts of fiactal geometry (Le., selfisimilarity of dynarnic patterns at different length-

sales) [Bremer et al., 1989, 1990; Home, 19890.6; Home et al., 1989; Walstra et of., 19911.

o w f k -Envmatic RoteoQsfsarid Aggrcgation of Caseln

2.2.2. Rennet C~(~guIrilion

During the cnzymatic phase of the renneting of milk, chymosin specifically splits off the C-

terminal part of ic-casein molecules around the casein micelles (primary proteolysis), thenby

gradually diminishing the 'electrosteric' repulsions and the ability of the particles to ricochet.

Rennet-altered micelles can subsequently approach one another and may flocculate, i.e., 'stick'

together. A balance of. chiefly, hydrophobic effects (plus some van der Waals attraction forces)

and electrostatic interactions (including Ca bridges) is thou&t to keep the @ma)casein micelles

sggregated [Schmidt & Payens, 1976; Payens, 1979; Bringc & Kinsella, 1987; also Zoon et al.,

l988a,b; van Vliet et of., 1989; Peri et al., 1990; Home, 1998; Lefebvre-Cases et al., 1998).

nie kinetics of the nnneting process are difiicult to interpret because two (partly

overlapping) reactions are involved [discussed in detail in van Hooydonk & Walstra, 1987;

Dalgleish, 1992, 19930; Holt & Home, 1996): the primary cnzymatic reaction appears to be

essentially firot-order (Le., its rate is directly proportional to the concentration of the substrate) in

milk over the pH range 6.2-6.7 nthcr than of the Michaelis-Menten type DJitschmann & Bohnn,

1955; Carlson, 1984; van Hooydonk et al., 1984, 19866; de Kruif et al., 1992; Hyldig, 1993;

Bauer et al., 1995; Leaver et al., 1995; Lomholt & Qvist, 1997; L@ez et al., 19981; the

secondaty flwculation stage can bc describcd by von Smoluchowski [1917] kinetics for

diffusion-controlled dimerirntion [Payens, 1989; Lomholt et ai., 19981.

Flocculation cm k thought of as a dynamic equilibrium ktween aggrcgated and un- (dis-)

agpgated particles. The reactivity of the micelles, Le., the probability of effective (listing)
encounters, at first rcrnains low (the rate of flocculation is virtually zero), and then incrr~ses

rapidly as a critical proportion of the K-caseinhis k n converted to parer-casein. Flocculation

becornes perceptible at the so-called (visual) clofting or coagulation tinr (CT), when this

fraîtion is about 70-85%. at least at physiological pH and mund 30°C (Payens et ai., 1977;

G m n et al., 1978; Dalgieish, 1979; Chaplin & Green, 1980, 1981; Carpenter, 1981; Green &

Morant, 1981; van Hooydonk et al., 1984, 1986b; Femn-Baumy et al., 1991; Shacma, 19921.

(Milk for most cheese varicties is renneted at ca. 31-34OC end pH S 6.6, although the action of

nnnet is optimal at around 40°C, pH 5.1-5.5 Fox Br Mulvihill, 19901. The temperature

coefficient, QIOOC, is ca. 1.5-3.0 at pH 6.7 ôetween 1-30°C DJitschmann & Bohren. 1955;

Tuszyfiski, 1971; Mehaia & Cheryan, 1983a; Carlson, 1984; van Hwydonk et al., 19841.) The

retatively low ef'ficiency of the flocculation reaction in practice (no excess rennet) is ascribed to

the fact that the flocculating particles still have a iimited nurnber of reactive regions denuded of

haia ('bare patches' or 'hot spots') at their surface. The initial (induction) period during which

then is little apparent change in fluidity is rcfemd to as the lugphcur.

Not only the relative rates and extents of proteolysis and aggregation (i.e., the conditions of

coagulation) detemine CT, they alro are key factors in relation to how gelation will progress

which in mm will influence the spatial distribution of the casein particles in the network, Le., the

basic stn~ctumof the rcsulting gel, hence its iheology and susceptibility to 'weeping' or

'wheying off (Le., synemis). Thus, faster rates of aggrrgation and gel formation tend to go

along with coarscr (more inhomogeneous) network structures [Gmn et al., 1981; Roefs, 1986;

Green, 19871, conceivably because of 'imgular' (coarse) aggregation m o o p & Peters, 19751.

However, the relation ktween rate of gel fiming and gel structure scems not to be maintaincd if

the composition/stnicture of the casein micelles is alcmd drastically, for instance by

acidification. Whcther the numkr of junctions in the nctwork or the numkr of bonds per
junction (or both) is incrcased with a high rate of aggregation, as has bem suggested by van

Hooydonk & van den Berg [1988], is still a matter for speculation.

(a) Fffccts of Concentration of Renne$. A higha concentration of minet enzymes at constant

(near neutral) pH leads to a shorkr time for the onset of milk coagulation as well as a higher rate

of geUcurd firming [Hossain, 1976; Ramet & Weber, 1980; Garnot & Olson, 1982; McMahon &

Brown, 1982; Marshall et al., 1982; Tokita et al., 1982; Bohlin et d., 1984; McMahon et al.,

19846; Lee, 1986; Zoon et al., 1988a; Lopez et al., 19981. Storry & Ford [1982b] and Bohlin et

al. (19841 did not observe substantial variations in firming rate, presumably because of the

limited range of rennet concentrations they investigated (about 0.3-0.7 mL.kg-1 and 0.3-0.4

mL.kg-1, respectively, Le., 0.03-0.07% v/v). Supposedly, the effect of rennet on the kinetics of

gel finning is psrtly related to the amount of casein macropeptide hairs still to bc rekased after

gelation, the percentage of GMP released at the onset of gelation increasing with increasing

rennet concentration [van Hooydonk & van den Berg, 19881.

Contradictory results exist about the effect of rennet concentration on maximum 'firmness'

or dynamic moduli (Le., a response signal which is supposed to be comlated with fimness) of

rennet gels, as estimated by dynamic rheometry. Hossain [1976], Ramet & Weôer [1980], Garnot

& Olson [1982], and McMahon et al. [1984b] did not find marked diffennces in gel strength

afier a few hours of ageing. Calculations of gel strength by McMahon et al. [1984b] using a

Scott-Blair and Bwnett-likc equation yielded incmsing ultimate (or long-terni, Le., at quasi-

quilibrium) gel strengtb with decmsing remet concentration, however. These worken, like

Riunet & Wekr 119801, monitorcd gel formation using a Fomiagraph and only put of the curve

of curd empirical finnncss us. time was uscd for curve-fitting. At longer times, the predicted

values of finnness werc higher thrn thox m w u d . van Hooydonk & van den Berg [1988]

showed that the increasc in fimincos as estimatcd with the Fonnagraph alrady legs behind that

measured with an Instron Universal Testing Instrument 5-10 min after the onset of gelation; so in
f a neiîher the measured nor the predicted values may ôe accurate. Rowland & Soulides 119421,

Olson & Bonsni [1977], Burems [1978], HoB et al. [1979], Stony & Ford [1982b], van Dijk

(19821, Bohlin et al. [1984], van Hooydonk & van den Berg [1988], Zoon et cil. [1988u], and

L6pu et cil. [1998], on the other hand, reported (limited) increases in (final) gel fimncss

(modulus) with increasing the concentration of Ennet. The teosons for the latter effect ternain

largely hypothetical. The apparent discnpancy betwecn the tesults may stem fiom differences in

the performance (accuracylsensitivity and maximum deformation applied, the latter pouibly

affecting the behaviour of gelling milk) of the measuring devices used and experimental

conditions (e.g., temperature). The efTects of rennet concentration may also be confounded by

inherent changes in CT.

(b) Effects of Low oy. If the pH of milk is loweted h m physiological value at a constant

concentration of rennet, the activity of minet enzymes increases vumme, 1972; van Hooydonk

et al., 198661 and micellar aggregation can start at a lower average degm of proteolysis of K-

casein [Pierre, 1983; van Hooydonk et al., 19866; Carlson et d.,1987a.61. In fresh skim milk at

30°C, the optimum pH for the action of rennet was found around pH 6.0; the onset of

aggregation (as infemd by viscometry) was at about 60% and 30% conversion at pH 6.2 and 5.6,

tespectively [van Hooydonk et al., 19866; also Lbpez et cil., 19981.

The reactivity of micelles that are completely converted into pu-casein micelles seems to

depend littlc on the pH, incrrcioes with Cal+ concentration, decrases with incteasing ionic

stmngth (NaCl), and rises madcedly with tempetaturc (QIOOC 16), especially frorn 15 to 30°C

[Dalgleish, 1983; Kato et al., 1983; van Hooydonk et al., 1986b,c]. Although it hm ôeen shown

that pH does exen some effkct on the rate of aggrcgation of (fully) pmteolyzed micelles

[Cheryan et al., 1975; Kowalchyk & Olson, 1977; Mehaia & Cheryan, 1983a; Kim & Kinsella,

1989~1,no unequivocal results have k c n publishcd up till now on the specific effcct of pH at

constant activity of Ca2+. Nevertheless, mults on finning rate of rcnnet-induced gels at a stage
when the enzymatic miction is essentially complete tend to confim the limited pH-dependencc

of the aggregation reaction [Gmn & Cnitchfeld, 1971; Zmn et al., 1989J.

A confounding factor in the aggregation of casein particles rnay bc the otac of MCP [Shalabi
& Fox, 19821. Roefs and CO-workers [1985] suggestcâ that pH-induced dissolution of MCP may

actually decreme the efficiency with which casein micelles coagulate but that the effect rnay be

offset by the concomitant incnase in the concentration of fm Ca2+. In fact, reducing the CCP

content of miik by lowering the pH, while maintaining the Ca2+ activity constant results in the

inhibition of rennet coagulation, which does not occur below pH 6.2, Le., when ca. 3(r/. of the

CCP has been solubilized [Shalabi & Fox, 1982; also McGann & Pyne, 1960; Pyne & McGann,

1962; Zittle, 1970; Zoon, 19881. van Hmydonk and co-workers [1986b] also pointed out the

importance of the concentration of CCP in the micelles (rather than ionic Ca) for the renneting

properties. Thcy surrnised that soliibilization of MCP may have a negative effect on the

accessibility of r w i n to m e t enzymes. Somc specific (structural) features of the casein

system mu* be involved which d e p d sîrongly on CCP.

Hossain [1976] and Stnry & Ford [1982b] reported that the ultimate finnness of rennet-

induccd gels i n c n a d with decteasing r e ~ c t i n gpH in the range 6.7-6.4 [also Mpez et al., 1998

(6.7-6.2) and N e l et al., 1991 (6.6-6.0)]. On renneting at about 30°C, Kelley [1951], Jen &

Ashworth [1970], and Zoon et al. [1989] found a maximum in gel finnness (dynamic elastic

modulus) n c u 6.0, 5.9, and 6.1, nspcctively; behwen pH amund 6.2 and 5.7, gel firmness

d e c d [Zoon et al., 1989], thus confinning findings of o t h r workers mowland & Soulides,

1942; Ashworth & Nek, l9d0, Olson & Bottazzi, 1977; Ramct & Weber, 1980; Storry & Ford,

198261. Zoon and collaborators also showed that tan 6(= G " G ') was not substantially affected

by pH above 6.0 [ a h L6pez et al., 19981 but a higher value was found at pH 5.7; the relaxation

time h m stress-relaxation measunments for gels at pH 6.7 and 6.3 was alsa largely unchangeci,
but it was shortcr a< pH 5.7. niew obsemtions may be contmted with those of Marshall et al.

[1982] in tems of (maximum) rate of curd fming of renneted milk at 30°C [also Kelley, 1951;

Tuswski et ai., 1968; Kowalchyk & Olson, 1977; Storry & Ford, 198261: a 6.5-fold incmase of

firming rate ktwecn pH 6.7 and 5.8 was reporte& a pH 5.6 the rate seemed to fall. (Note that it

is possible that the techniques used by Marshall et al. [1982] may not have been sensitive enough

for comparison with fundamental rheological studies.) Most of the above results have k e n

interpreted in tems of changes in the micellar-rnim partition of Ca phosphates at decreasing pH

values and charge neutralization.

Current reaction schemes for rennet coagulation hardly account explicitly for the variations

in average composition/morphology of the coagulating particles brought about by acidification

and, more generally, by processing milk. It is known that the stability of milk toward, e.g.,

renneting is a hinction of micelle size (which is related, arnong other factors, to the amount of K-

w e i n ) [Schmidt, 19801. pH-induccd changes in micelle polydispersity may influence the

mechanism(s) of gel formation as well [discussed by Holt, 1985b). The rate of aggregation of

renneted micelles seems not to be affected by the size of the particles, although small micelles

seem to become labile at an earlier stage of hydrolysis [Dalgleish et al., 1981a,b; Brinkhuis &

Payens, 19841, which can be explained in an intuitive way on purely geometrical grounds malt,

19856; Dalgleish & Holt, 19881: at constant surface thickncss, the smaller the diameter of the

particles, the p t e r the curvatun of the surface, and the smrller the minimum dimension of the

reactive regions on the s u r f i requircd for flocculation to occur. The effect of particle size on

CT is not entirely clear, but thcm secms to be no substantial difletences, at Ieast when the largen

and smalkst micelles arc rennctcd [ E b d et al., 1980; Dalgleish et ai., 19810; Ford &

Grandison, 19861. The dimension of casein micelles appears to affect the physical and

microstrvctunl characteristics of gels formed by minet action at amund neutml pH [Waagner

Nielsen et al., 1982; Niki & Arima, 1984; Chahed, 1985; Ford & Grandison, 1986 Niki et al.,
1994a.b; also Remeuf et al., 19891. Niki and collabontors noted that the rate of gel finning,

'finnness', and elasticity of rennet gels obtained h m solutions of resuspended small micelles

were higher than those for gels prepared h m large particles at the same concentration of casein.

They further obseived that gels of smaller particles had somewhat smaller pores and that rennet-

altcred micelles seemed to fuse and cluster more extensively in cornphson to larger ones. This

may indicate, as may k anticipated intuitively, that the same number of crosclinks can be

formed more readily by a large number of small particles than by fewer large ones.

The properties of the polymeric surface layer of mainly K-caseinare particularly relevant to

discussions of coagulation, affecting not only the destabilization and aggregation processes, but

also the specificity of chyrnosin towards its substrate [Payens & Visser, 1981; van Hooydonk &

Walstra, 19871. One would expect that charge effects modify the average conformation of the

polyelectrolyte hairs: as the pH is reduced, progressive titration of the acidic haia would cause

them to 'curl up' somewhat, which would also reduce the conformational &dom of the

(physically intact) macropeptide segments if they 'adherc' more closely to the micelle con (at

lest for a range of pH within which micelle 'surface' and 'core' are not too obscure notions:

quid of the hairy layer below 5.2-5.0?)

Further complication is added by the fact that, below neutral pH, chyrnosin may well adsorb

ont0 @ara) casein particles, meaning that the hydmlysis of K-caseinmay not be quitc random

any more and that some oufice inactivation of die enzyme molecules may occur. Adsorption

seems to inccwc with deccwing temperature and pH and appears to bc enhanced by [Ca2+]

[Stadhouders & Hup, 1975; Holmes et al., 1977; van Hooydonk & Walotra, 1987; de Roos et al.,

1995; Dunnewind et al., 1996; Larsson & Andr(n, 1997; Larsson et al., 19971. It is plausible that

under pnctical (mildly acidic) conditions of curd making the adwrkd chyrnosin molecules

create bue patches of para-u-casein by attacking adjacent molecules of (non-randomly

distributed) K-caseinone after the other by a 'catch-and-razor' mechanism [Brinhuis k Payem,

19851 kforc thcy desorb and diffipe away, nther than by pmducing randomly distributcd

individual molecules of pu-K-casein. A fwthr rcaching implication is that more coagulating

enzymes may be retained in the draincd curd so that (relatively slow, pHdependent) secondary

protcolysis may take place, which may influence cheesc ripening. It is well known indeed that

distribution of chymosin between curd and whey is detennined by pH at draining.

(c) Effccts of Pm-Heatitipp. It is notorious that heating milk of natural pH at temperature x time

combinations that cause extensive denaturation of the whey proteins markedly affects renneting,

iesulting in prolongeci clotting times, weaker gels, and diminished (rate of) syneresis. Texture

and flavour defects are a h typically encountered in cheeses pmpared fiom high heat-treated

milk, e.g., 90°C-15 s to l4O0C-4 s marshall, 1986; Banks et al., 1987; Banks, 19881. The

literature on rcnnet coagulability of pre-heated milk has been reviewed by Lucey [1995] and will

only be summarized hem.

CT incnases with the severity of pre-heating [Momssey, 1969; van Hwydonk et al.. 1987;

Dalgleish, 19906; Femn-Baumy et al., 1991 ;Lucey et d..1993~;Ghosh et al., 19961. (Standard

pasteurization treatments, e.g., 72OC-15 s or 63OC for 30 min, in cornparison, result in a slight

reduction in the pH and little change in renneting propcrtics with only ca. 5% of the whey

proteins kcoming amciatcd with the casein particles Fox, 1969; Lau et al., 1990; Femn-

Baumy et al., 1991; Lieske, 19971.) The rcnnetability of high heated milk deteriorates tùrther

during subsequent s t o n p in the cold (so-calld rennet hysteresis) [Mattick & Hallett, 1929;

Momssey, 19691.

One of the questions at issue is wh&r impairment of the renneting propcrties of pte-heated

milk stems esscntially h m ictudation of the enzymatic or aggngation rcuction, or both. Some

workers clairned that the chymosin-catalyzcd hydiolysis of K-caseinis h d l y affected by heating

(75 or 8S°C-30 min) (Marshall, 19861, others that hydrolysis is incomplete, which slows down

subsequent flocculation (mode1 sy~umscontaining casein micelles and PLg or a-Lasubjectcd

to high temperatures for long periods; e.g., 90°C-1 h) [Hindle & Wheelock, 1970~;Wilson Br

Whcelock, 1972; Wheelock & Kirk, 1974; Shalabi & Wheelock, 19761. Reddy & Kinsella

[1990] reported that heating (8S°C- 15 min) suspensions of casein micelles in the presence of P

Lg reduced both the initial rate and apparent extent of hydrolysis. The results of Femm-Baumy

and CO-workers[1991] (whok milk pn-heated at temperatures x times between 70°C-1 min and

160°C-O.1 s) and Leaver and CO-workers[1995] (whole milk, 72- I4O0C for 15 s-5 min) echoed

the a b v e conclusion,

The generally held view is that specific proteolysis is slowed down but that the poor

coagulability of heated milk (e.g., 8S°C-15 s; 90°C-1 min; 90°C-1 min; 70/120°C-5 min) is

related mainly to the impriired gel-fonning properties of the renneted 'micelles' 'sprinkled' with

(partly) denatured whey proteins m e , 1945; Morrissey, 1969; Damicz & Dziuba, 1975;

Marshall, 1986; van Hooydonk et al., 1987; Singh et al., 1988; Shanna, 19921. Decreased

sensitivity to enzymatic proteolysis appears to pertain largely to those molecules of r-casein

which are relatively poot in carbohydrate [Walstra & Jenness, 1984; Lieske, 19971.

It is dificult to disentangle the factors responsible for the inhibition and its reversal.

Association of whey protein aggregates with casein micelles either via complex foxmation with

K-casein andor via hydmphobic or ionic interactions at sites depleted of K-caseinappears to be

critical (Kannan & Jenness, 1956, 1961;van Hooydonk et al., 1987; Dalgkish, 19906; Reddy &

Kinsella, 19901. Addition of K-casein to milk diminishes the deleterious effats of thermal

processing (80°C-10 min), presumably bccaux whcy protcins now react primarily with K-casein

in the semm during heating, thus affécting the casein micelles less [Pearse et al., 19851. Complex

formation is expected to cause some stcric hindnnce to rennet enzymes and modify the balance

of electrostatic interactions between enzymes and substrate. Since PLg pioducts likely bind to

para-K-casein,they rcmain after hydrolysis and likcly interferc with interactions arnong rennct-

converted pseudo-miccllcs. Because PLg chains have an estimated contour lcngth mater than
twice the thickness of the surface laycr [Holt & Horne, 19961, they may conaibute another

category of haia on the surfaces of the particles (that is, if polyrncric pmducts do form and

interact with micellar surface upon heating under the conditions as in standard milk. The results

of Comdig [199S] and Leaver et al. [1995] oam to suggest othenuise as there seems to be a

limit to the binding of PLg to casein particles). Hence the partial inhibition of flocculation and

the resulting reduction in connotative properties of gel such as 'strength'. 'firmness', or 'tension'

at unadjusted pH-î.e., disruption of the continuitykohesiveness of the gel network [McMahon

et of., 19931.

Heat-induced reduction of soluble Ca2+ probably hampers further the flocculation pmcess

and ensuing development of a f

in gel. The higher stability of the casein particles imparted by
diminished cleavage of wasein andor delayed flocculation seems to bc compensated for to

some extent by the increase in micellar Ca phosphate (and also, maybe, by the sensitivity of

denatured PLg to Ca2+) [Harper, 1976; Walstra & Jenness, 19841. Evidence is still lacking,

however, on how excess Ca phosphate may modify the structure of the particles. One suggestion

would be that additional MCP reduces the essentiel flexibility (and net charge) of micellar

caseins and contributes to the decrease in stability by lessening the ability of the particles to

'adapt' to changing environments. Reduction of the amount of hydrolyzable K-casein does not

seem to cornlate directly with the rcnneting bchaviour of hcated milk (70/120°C-5 min) [van

Hwydonk et al., 19871.

It is not yet clear either what causes rennet hysteresis. It has been surmiscd that the

phenommon originates fiom the slow solubilization of hcat-pnçipitatcd Ca phosphates during

cold storage me, 1945; Momssey, 1969; van Hooydonk et of., 19871. In other quarters it is

thought that complexation of PLg with K-cwin is the overriding factor b n r n & Jennns,

196 1; Lucey et al., 1993a,b]: structural reamngemcnts of the whcy proteindu-casein complexes
[Sawyer, 19691 may occur during stonge of hcatcd mik, which may result in dditional stctic

hindrance.

The adverse efEcets of high tempetaturc on nnncting, both CT and gel strength, may k

counteracted to =me extent by decreasing the pH afkr heating, adjusting the pH to low values

and then reneutralizing to the original pH of the milk (either immediately or aftcr maintaining

the milk at low pH for some the), andor adding calcium chloride (CaC12; e.g., 0.02-0.04% wt.

milk) after heating [nviewed by Lucey et al., 19941. Addition of low concentrations of CaCh

teduces the pH of milk and increases the [Gaz+], both of which enhance the rate of flocculation

of renneted micelles. Limited addition of Ca2+ does not affect apprcciably the enzymatic

teaction, provided the pH is comctcd to its original value [Surkov et al., 1982; Mehaia &

Cheryan, 1983a; Walstn & Jenness, 1984; van Hooydonk et al., 1986~1.Reducing the pH very

likely rcduces charge repulsion, solubilizes heat-induced Ca phosphate, increascs soluble Ca2+,

and incteases the activity of chymosin. Acidified and mieutralized ('refomed') milk also has an

increased [Ca*+] [Singh et al., 1988; Lucey et al., 19961.

Predictably, the acidification-neu~lslizationprocedures mua also mediate fundamental re-

conformation of the casein molecules and particles ('remicellization'), but the underlying

mechanism(s) have not becn established precisely. Banks & Muir [1985] suggested that

disruption of the micellar architecture at low pH somehow renders the hidden or maskcd K-

casein more susceptible to m e t hydrolysis and enables the cosein particles rcformed on

(immediate) nneutralization to be more Fully integratcd in the curd. But the results of van

Hwydonk and his CO-workcrs[1987] (heat treatment at 70/120°C-5 min) secm to tell a different

story. According to thesc authors, the tnnsfer of Ca phosphate h m the heated p~rticlesto the

m m at low pH (5.5 or 6.0) and the 'reprccipitation' caused by adjusting the pH back to 6.7

(either dimctly or ofter holding at low pH for 24 h depcnding on the intensity of heating) may k

the main operative mechanism. Cycling the pH would I d to the reformation of Cdphosphate
complexes with composition and properties more like the original MCP, which would explain

the improved mnetability. The results h m acid-base buffering curves suggest that the

fonn-end concenûation-of Ca phosphate 'precipitated' on neutmlization of acidifki

(severely) heated (2 100°C-IO min) milk differ h m indigenous MCP and heat-induced Ca

phosphate, however [Lucey, 1992; Lucey et al., 1993aJ.

Clearly, rennet coagulation of the casein pacticles modified by heating-acidification-

neutralization is the result of a complicated multiple progression process driven by divergent,

panllel, and convergent streams of acting forces. Much remains to be explained on the

mechanisms by which the functionalities of the casein system are modulated by changes in CCP

composition and structure and by alterations of particle (surface) properties.

2.2.3. Gel Assembk) und Syneresis

Milk gel formation is a continuation of the initial flocculation of casein particles (that is, if

flocculation proceeds unhindered, Le., under quiescent conditions and with negligible

sedimentation of the particles). In fact, aggregation and concomitant reduction of overall protein

surface ana continue throughout chcese-making and in the early stages of ripening, and so

gelation and pst-coagulation events (Le., aging of the casein gel and curd formation) such as

strengthening, coarsening, and shrinkage (synercsis) of the gel, are facets of the sarne basic

process of casein aggregation [reviewed by Green & Grandison, 19931. The interactions among

the casein foms at various stages of milk clotting may not be identical, however, even if

substantial overlap is expected.

So-called rennet and acid milk gels, although basically composed of maciornolecules

(caseins), bchave differently f m pmpcr 'polymer gels' and secm to k M e r thought of as

rathcr disordered 'particle gels' [van Vliet 81 Walstra, 1985; Home, 19991. Extensive

microscopy, rheometry, and pcrtncametry studia [Oreen et al., 1978; Walstra et al., 1985;

Bremer et al., 1990; Rocfs & van Vliet, IWO; Rafs et cil., 1WOa.b; van Vliet et al., 1989,
1991~1have indicated that casein gels are heterogeneous (and dynarnic) at severai levels: (i) at

the scale o f the elementuy c w i n particles themselves, (io at the level of the casein 'chains' or

'strands' ( a d 'bundles' thereof) and 'nodes' formed by (partly fuseà) flocculated particles, and

(iii) at the level of the whole network.

(a) Earlv Gelation Events. Gels fonned by the action of rennet change considerably with time.

Detailed phenomenological investigations using (transmission) electron microscopy k l a b &

Hanvalkar, 1973; Green et al., 1978; Green & Motant, 19811 and dark field microscopy

[Ruettirnann & Ladisch, 19911 revealed that the flocculated 'micelles', which can still be

distinguished clearly at the time of visually detemined coagulation and by the time a continuous

(imgular) reticulum forms, gradually loose their individuality (they fuse) over a period of

several hours. Knoop & Peten [1975] o b m d that 2 h after the addition of rennet the fusion of

casein particles seemed to be festa at pH 5.8 than at pH 6.6. Measurements of model systems of

artifcial micelles made up of r-casein by small-agie neutron scattering at pH 6.7 have also

been interpreted in ternis of the coalescence of the particles within few hours of chymosin action

[de Kmif Br May, 19911. Additional evidence for the apparent re-stnicturing of casein aggregates

during renneting of diluted milk has k e n obtaincd using light scattering techniques pauer et al..

19953.

The chernical nature of the 'bridges' joining the particles and the fate of the bridging

material on micelle fiision rcmain largely mysterious, but an incteasing proportion of the surface

of the piriicipating particles appears to k involveci. One may argue that a small region around

the contact am can be considercd as a kind of polymer gel but then, nothing is known about the

(viscoelastic) properties of the hypothetical micro-gel and of the casein particles themselves. The

junction areas pmbably consist of several bonds of differcnt and variable natures: cg., first

interparticle van der Waals attraction, then al= simple sait bridges and hydrophobie interactions,

and Iatet CCP 'bridges'. Pmci-rasein mtains two cystcin icsidues ofter cleavage by minet; the
sulphydryl groups may facilitate intmnolecular bonding on oxidation or exchange reactions with

disulphidt bonds [Hsrwalkar & hllib, 1981; Hashizume & Soto, 19881. The phosphate cluster

on the N-terminal end of Fasein may play some part either by engaging directly in (specific)

ionic interactions (Ca2+ bridges) andor by holding the kcasein molecules in a defined

orientation at the 'micelle' surface wun et al., l982a,b; Pearse et al., 1986; Pearse & McKinlay,

19891.

Conceivably, the particles attain close contact (since most of the GMP has k e n removed)

and re-organization of Ca phosphates and casein molecules occurs to produce increasingly more

compact (stable) stnictures, hence the steady increase in gel strength (dynamic moduli) for more

than 6 h at and above 30°C [Zoon et al., 1988a,b; Benguipi et al., 19941. Particle fusion indeed

appears slower and less pronounced in acid gels (which contain intact ~ocaseinand virtually no

CCP)than in m e t gels [Glaser et al., 1980; Knoop & Buchhcim, 1980; Raefs. 1986; Roefs et
al., WOa,b]. Also, the casein particles in pie-heated milk (e.g., 8S/9S°C-1O min) appear to

'sinter' less extensively than those in unheated milk on acid development by lactic acid

fermentation [Knoop & Petcn, 1975; Kalhb et al., 1976; Davies et al.. 1978; Harwalkar & KaIhb,

1980; Parnell-Clunies et al., 1987; Mottar et al., 19891. By extrapolation, rnaybe the (principally)

acid coagulation which occurs in making acid-cuid cottage cheese also results in slower and less

complete aggrcgation of casein than is obtaind in the manufacture of other types of checxs.

Perhaps this is rclated to the relativcly high stability of the particles of cottage curd against

fusion and drainage of moisturc (syneresis).

Progressive coalescence of the aggregated pu-casein particles may be related to the

apparent second maximum in the rate of gel firming v5. time curvc [Steinsholt, 1973; Stony â

Ford, l982a,b; Schulz et al., 1997u] and to the rcporied distinct stages (flocculation and gel

formationfconsolidation) in the gel kuembly FuSW<i, 1971; Hady & Fanni, 1981; Hardy et
al., 1981; Suikov et al., 1982; Johnston, 1984; McMahon et al., 1984t1.c; Hardy & Scher, 1988;

Korolczuk, 19881, at least in non-heat-trcatcû, renncted milk.

(b) Gelation as a Multidusic Procesr. That gel assembly may be descrikd as a two-stage

process stems h m the observation that not al1 the casein micelles have been fully converted into

pma-casein micelles at the moment of gel formation. Under standard conditions, about 90% of

the paiticles are incorporated into the gel at the visual rennet Cf [Dalgleish, 1980, 19811. The

particles and small clusten thereof that are 'fm' (unaggregated) at or after CT may aggregate

differently ('less randomly') from those coagulated initially and the properties of the final gel

(curd) may be affected by the amount of casein fm at CT. This ought to be considered in

explanations of gelation mechanisms. The increase in the proportion of gelled material and

numôer of cross-links within the gel most probably contributes to the marked increase in gel

smngth, at least in the early stages after a gel has formed, because one may expect this

phenomenon to be complete afier roughly twice to thfice the time needed for detectable gelation.

(i) Storry & Ford [1982a] noted that the rate of finning (first derivative of firmness with

respect to time, as measuml as yield force at small deformation with an lnstron at pH 6.4 and

30°C, starting h m raw milk) of strictly rennet gels as a function of time rcached a clear

maximum first Ca. 10 min after visual clotting, then decteased over the next 10- 15 min to Ca.

80% of the maximum value, afier which it either rcmained constant or increased slightly for a

fuicher 15 min and dccreased steadily themafier. [Inaiguingly, apart from Schulz et al. [1997a]

who used a Paar Physica-Rheoswing hometer for mersuring viscosity at pH 6.4 and 32OC, no

such an effect appeared to have k c n noted by workcrs who studied the dynamic rheology of

renneting milk under seemingly similar conditions. We note that Kim & Kinrlla [1989b], also

using an Inotron, did rcgistcr apparcntly biphasic (i.e., distinctly non-monotonic) coagulation

protiles reminiscmt of those reportcd by Stony & Ford, but thcn, coagulation wlls by gradua1
acidification when relatively high amounts of GDL werc usai at and above 40°C (set also Lucey

et al. [199&11 and Section 7.32b).]

Storry & Ford [1982a,b] attributed the fint apparent maximum to the aggregation of casein

particles, and the second one to the incorporation of yet essentially unaggregated casein into the

pre-existing coagulum. (Note that in remet gels it is unlikely, however, that there are clearly

separate processes, at least after visual CT.)The two phases as defined in this way responded

differently to assay conditions [Storry & Ford, 1982bJ:the rate and amplitude of the first phase

increased with decreasing the pH (6.6-6.0) at 30°C, whilst pH appeared to have relatively little

effect on the second stage. In contrast, temperature (25-40°C) had marginal effect on the fint

phase (shallow maximum of maximum timing rate around 3S°C?) at pH 6.4, while the second

phase depended strongly on temperature, king pronounced at 2S°C and almost disappearing at

3S°C and above. At 30°C and pH 6.4, the concentration of rcnnet smned to have little influence

on either phase, but the rates of both phases increased with increasing concentrations of added

Ca2+ [O-0.04%] and casein (0.7-a%].

(U) The viscosimetric and rheometric data of TussyRski [1971] and Johnston [1984], and the

turbidimetnc and photometnc data of Surkov et al. (19821 (diluted skim milk), Hardy et al.

[1981], and McMahon et al. [1984a,c] (undiluted skim milk) also suggest that rennet gel

formation is a muitiphasic proccss. The studies hem deal mainly with initial events of c w i n

micelle aggregation, Le., bcfore or won atter visual coagulation. The findings point to the

existence of two distinct phases of aggregation, viz., formation of micelle clustcn vs. formation

and reinforcement of a gel network attet a fcw minutes, both affectcd by the concentration of

CaC12 (0.022- 1.1 1%) and the temperatun (25-3S°C) at pH a n d 6.45 (Hardy & Fanni, 1981;

Hardy et al., 1981;McMahon et al., 19844~1.

The phrasology used by Surkov et al. [1982] is not clear, but the ttported observations also

reflect changes oçcurring prior to or amund the gel point. The authors suggested that (unheated)
enzyme-altered micelles undergo (intmmicellar) coopcrative transition in 'quatemary structure'

(highcr-order organization) consecutive to extensive pmteolysis o f K-casein to yicld clot-foming

particles (activation energy, E, m 191 kJ.mol-1 and Qipc 1 12; renneting pH rii 5.6? and 25-

37°C). The nature o f the transformation (remiceHization?) is not specified but a clear dependence

on [CaC12] (0.066-0.20%) was noted, particularly below CU. 0.13%. Perhaps a little shrinkage of

the renneted particles is involved. Hardy & Fanni [1981], McMahon et al. [1984u,c], and

Korolczuk [1988] also have sunnised that some structural rearrangement of the renneted

micelles may occur just before or upon coagulation. I t is ternpting to envision a (highly

hypothetical) mechanism whenby 'inside (hydrophobie)-out' phenomena would occur so that

previously buried sites in the individual or (partly) aggregated particles get exposed on the

surfaces upon re-organization. The so-transformed particles would then undergo gelation

according to a Smoluchowskian mechanism (Easr 34 kl.mol-1 and Q~OOC

a 1.6) [Surkov et al.,

19821. These values of Eaand Qlpc are similar to those quoted by Tuszyfiski [1971].

(c) n i e Phenomena of Svneresis. Even after gel formation, additionaljunctions among the casein

'particles' (aggngates thercof) in the network cm k fomed kcause the constituting particles

are expected to contain numerous active sites smeared out over their surface. Thus, unless the gel

i s mechanically constnined (e.g., by clinging to the walls of a clean vessel), the network tends to

contract and becomes more compsct, cxpelling whey, a pmcess known as syneresis (or, as de

Kmif & May [1991] put it, 'apinodal dmixing' or decomposition o f the h e i n gel into a water-

rich and a protein-rich phase) [rcviewed by Pcarsc & McKinlay, 1989 and Walstra, 19931. Since

the particles kcome progmsively more immobiliad in the continuous Ca pu-caseinate

matrix, this implies that-existingcms-links have to k bmkcn or defonned loçally before new

ones may fonn. Since the stress proâuced by the formation o f new bonds must be relaxed, the

cornpliance of the network must affect the rate of contraction or shrinkap.

 Only limited shrinhge is expected if the gel is fonned at nst (no extemal pressure applied)

and constrained geometrically. But then, so-called microsyneresis may take place, which means

that at a local scale there tends to be a segregation into dense and less dense regions, leading to

wider pores on average, Le., coarser network structure, which may be reflected in an increase in

the pemeability of the gel with time and, in extreme cases, a decresse in its dynamic mechanical

modulus (apparent 'softening') and changes in its optical properties [Tuszyflski et al., 1968;

McMahon et al., 1984a; Walstra et al., 1985; van Dijk & Walstra, 1987; Korolcnik, 1988;

Parnell-Clunies et of., 1988; Schulze et al., 19911. Coanening of gel structure cm be expected to

facilitate (macroscopic) syneresis.

The mechanisms and kinetics of syneresis of the forming curd particles are pmicularly

dinicult to establish, especially on a small scale. Rational understanding of the (complicated)

effects of changing processing conditions is still limited, although there is considerable

information on the influence of various factors [reviewed in Walstra et al., 1985; Walstra & van

Vliet, 19861. To be sure, the distinction betwccn 'gel formation' and 'syneresis' is somewhat

arbitrary in practicc because casein aggregation and changes in the state or extent of aggregation

of the particles are likely concurrent evcnts and probably enhance each other. Actually, a number

of variables affect milk gel formation and syneresis in the same direction. Thus, increascd acidity

(pH 6.7-S.2), temperature (2 20°C), and pre-heat treatment intensity tend to have substantial

effects on both processes, while added CaC12 and fat contenthomogenization genemlly have

moderate effects [Gmn & Grandison, 19931. Under otherwise the same conditions, i n c d Ca

phosphate in the casein (pscudo) micelles appcars to d u c e syneresis, presumably because of the

rigidity it imparts to the gel; minet concentration stems to have a negligible effect, at lcast if

cutting of the gel is at the same 'ficmness' [Walstra, 19931. If syneresis w m a purely physical

pmcess, gel strength at cut would be expected to affkct synmsis with the pdiction that

nlatively strong gels with high water-holding capacity and protein hydration indices should k
more resistant to synercsis than weak gels; thm are conflicting observations on this point,

however [discusscd in Pearsc & McKinlay, 19891. The rôle of (paru) u-casein remains a largely

open question, but it is possible that either or both proteins may be involved in specitic

interactions that are an integral part of both the fonnation and synetesis of milk gels.

Syneresis behaviour appears to be intimately related to the dynamic character of the casein

network [van Dijk, 1982; van den Bijgaart, 1989; Roefs, 1986; Roefs et al., 19906; van Vliet et

al., 199 la; van Vliet & Walstra, 19941. The focus hem is on inherent or endogeneous, i.e.,

unaided syneresis. In ordinary (skim) milk gels, ongoing rearrangements of the network of

(para) casein particles involving the relaxation of interparticle bonds and ultimately the increase

in the numbcr of junction points are thought to k the Ieading cause of syneresis. Secondary

aspects such as shrinkage of the building blocks themsclves rnay promote syneresis, especially if

the pH falls (nduction of the net charge on the particks in the gel) or if the temperature riscs (or

both) while or a h r the gel is fomed. A drop in pH during synetesis may enhance the rate of

syneresis to a greater extent than is found whcn the pH is previously brought to the wme value

[Emmons et al., 19591. Changes in casein solubilityhydration seem to be negligible during gel

fonnation and synercsis [Ruegg et d.,1974; Lelièvre & Creamer. 19781, except perhaps in acid

milk gels, particularly those produccd by the action of lactic acid cultures, because slow

proteolysis by starter enzymes may rendet thc cascinate particles more rcactive.

Milk gels formed solcly by acidifîcation show comparatively littk syneresis, that is, if kept

still during gelation and lefi undisturbcd at a pH neu 4.6 and 30°C [vm Dijk, 19821. This hm

been related to the more permanent character of acid gels relative to rennet gels Bnguigui et

al., 1994; van Vliet & Walstra, 19941: in acid gels m-structuring would take place mainly at a

very local suk,e.g., within the strands of the network, w h c m in rennet gels the strands would

break and refom at mother place. (The influence of the mcâhod of acidification on gel strengdi

and syncrcsis hm becn discusd by Fox & Muivihill[1990].) In gels of (pasteurizcâ) skim milk
s o u d by yoghurt bacteria to a pH in the range 3.84.5 (as is typical of the manufacture of set

yoghurt), however, extemal pressure or cutting at 3 or 6OC induced extensive syneresis

[Harwalkar & Kalib. 1981. 1983; Modler et al., 19831. This may point to a different temperature

dependence of syneresis in acid milk gels (pH < 5.2) and in gels at highet pH.

Pre-heating of milk at high temperatures (a 90°C), with or without increasing protein

concentration (by membrane proeessing or fortification), increases gel firmness and effectively

reduces synensis of yoghurt-like products [reviewed in Mulvihill & Grufferty, 19951. This is

usually atttibuted to the fner microstnicture and the higher effective volume fraction of the

denatured whey proteins-casein matrix. It is ofien envisaged that the reduced propensity of the

casein particles in heated milk to fuse leads to a more even distribution of the particles

throughout the gel and a ktter immobilization (holding capacity) of the dispersed liquid phase.

There also is a trend to high heat-treat milk (e.g., 90°C for 2-3 min), possibly with increasing

protein concentration. as a prelude to cottage cheese-making [Kalab, 1979; Fox, 1993a.6;

Sinding Andersen, 19941, although synemis is desirable in the manufacturing of such products.

In pasteurized skim milk clotted bclow a pH of ca. 5.0 at 32°C. as in making cottage cheese

by the short-set method, the addition of rennet 1.5 h after inoculation with 5% starter bacteria

was found to enhance syneresis considmbly (as well as coagulum strength, both as measured at

the moment of cutting), the morc so whm morc rennet WPS added (0.5-4.0 mL per 1,000 Ib of

milk, Le., ca. 1.1-8.8~10-4 % v/v) [Emmons et al., 1959; also Attia et al., 19931. Milk that had

been cultural to a very low pH (i 4.6) exhibited only weak syneresis, even f i e r renneting

[Emmons et al., 19591. Emmons et al. and Thunton & Gould [1933] alro noted a retardation of

curd finning when excessive amounts of mnnet w m uscd relative to starter culture. Another

important huiction of muicting is to rcduce 'matting' of the acid-cutd flakes on cutting and

cooking (Thurston & Gould, 19331, a phenornenon which might k associated with the reportcd

readiness of acid-set casein curds to drain and givc denset, fimer particles after cutting
compared to acid-minet cuids mishop et al., 19831. Proôably, these observations underline a

gradual shifi from acid-set gels to enzyme-set milk gels; they are of imporiance for the

production of fnsh (acid) cheeses and for the still-limited fundamental understanding of

combined enzymatic and acid coagulation (see Section 2.2.7).

2.2.4. Physicd Chamcteristics ofMUk Geh

As mentioned in the foregoing discussion, an essential difference between rennet and acid

milk gels is that the sbucture of the overall network appears to remain approximately constant

(more permanently stable) with ageing time for acid gels, whereas it semis to bccorne gradually

more inhomogenous for rennet gels, at a faster rate for higher temperature or lower pH, or both.

(What we are concemed with here are acid gels obtained by slow acidification at temperatuns

above 20°C.) In many respects, the two types of gels look comparable (espccially just after gel

formation), however, despite the fact that the structurai elements and the dominant patterns of

interactions among them must be different [dealt with in Roefs, 1986; Bringe & Kinsella, 1987;

van Vliet et al., 1989; Roefs & van Vliet, IWO; Home, 1998; Lefebvre-Cases et al., 19981:

unaged gels look rather similar micnwcopically (ie., coarse-stranded particulate networks), and

have roughly the same hactal dimensionality, pcrmeability constant, and pore size distribution

[van Vliet & Walstra, 19941.

Another distinctive feature of rennet vs. acid casein gels is their mcchanical behaviour. The

relation betwcen rheological and endogcnous syncrcsis behaviour of casein gels hm been studied

by van Vliet and CO-workers[1991a] and Lucey et al. [1997a,c, 199ûu,b,e]. For relatively short

deformation t imes (Le., high deformation fiucncies) the vismelastic characteristics arc similar,

but for longer times standard rennet gels are more liquid-like (higher tan 8) than acid-induced

gels. A h , (pre-hcatd) acid milk gels tend to have a lower elastic modulus and break (yield) a a

h i j i r stress and smallr m i n (dcfmation)i.e., they am stronger and more brinle

( ' s h o r t e r ' ~ a nrennet gels when measured over similu practical conditions [van Vliet et al.,

1989; van Vliet et al., 19916; Walstra, 1993; Lucey et al., l9Wa,b; Lucey & Singh, 19971. mis

characteristic may k related to the relatively permanent character of acid gels.

2.23. The Use of Mil& Concentmted by UI~ruPltratioion

Important featwes of rennet and acid gel formation, synensis, and rheology are affectcd by

concentration of the colloidal phase of milk (Le., caseins, whey proteins, colloidal salts, and.

occasionaily, fat) by membrane processes such as ultrafiltration (UF) [Gamot, 1988; Fox &

Mulvihill, 1990; Guinee et al., 19921. Since concentration and high heat pre-treatments tend to

have opposite effects on cheese-making parameters, it bas been wggestcd that combining the

two processes may be desirable [Maubois et al., 1972; Casiraghi et al., 1989; Green 1990u.b;

Hyldig, 1993; McMahon et al., 19931.

At fixed concentration of rennet, increasing the casein concentration of unheated or

pasteurized ntentates up to Ca. 3-fold seerns to have liîtle effect on the CT (some slight

decreasedincreases have been reported) [Dalgieish, 1980; Gamot 8t Corn, 1980; Mehaia &

Cheryan, 19836; Shama, 1992; Sharma et al., 1993; Guina et al., 1996; Camn et al., 1997;

Samuelsson et al., 19971and on the extent of aggregation of nnneted micelles amund neutral pH

and 30°C [Green et al., 19831. The rate of enzymatic hydrolysis demases slightly [van

Hooydonk et al., 19841, however, presumably because of a ntardation of the effective diffusion

of the enzymes, but this is out-weighted by knhanced flocculation. The rate of gel firming and

final finnncss ôoth incrciise substantially, and littk, or even no, apprcciable syneresis occuro.

Also, atypicd ( c m ) structures dcvclop as the concentration factor incrcases, which cm k

perceived as textunl defats of the final pmduct [Gmn et al., 1981; Lakhani et al., 19911.

Although CT remains csscntially constant with incicesing the concentration of milk at fixed

levels of minet enzymes, a decmasing paccntage of the casein micelles are hydrolyzed, hence

incorporated, into the gel matrix at the point of visucil coagulation ncar neutml pH palgleish,
1980; Gamot & Corre, 1980; Shamia, 1992; Sharma et ai., 19931: while Ca.90% of the particles

in standard milk are incorporated into the gel at the CT, only 85%, 60%. and 50% are integrated

at the corresponding stage in 2, 3, and Cfold concentrates, respectively. This must affect the

early stages of gel formation and synemsis but the mechaniszns by which gel properties are

controlled are not yet clear. Changes in the ionic properties of milk retentates during UF,

including the repartition of Ca phosphate ktween micellar and saum phases [BNK & Fauquant,

1981; Walstra & Jenness, 19841, and their increased bufiering capacity [Brulé et al., 1974;

Covacevich & Kosikowski, 1979; Mistry & Kosikowski, 1985; Gastaldi et al., 19971 also ought

to be allowed for. Variations in the size of the casein particles ('coalescence'?) may also occur

[Walstra & Jenness, 19841.

A study on the effects of pH (6.0-6.8) in the temperature range 28-37OC on the renneting

properties of UF retentates (1- to Cfold) obtained fiom skim milk has k e n published by Sharma

et al. [1993] [also Waungana et al., 19981. CT by viscometry was found to decrease with

lowering the pH and increasing the temperature. In lx-3%concentrates fiom pasteurized milk,

the average K-caseinhydmlyzed at CT ranged from CU. 91.63% at pH 6.8,80-57% at pH 6.4, and

7046% at pH 6.0, irrespective of coagulation temperature. Gel strength incrrased with

decreasing the pH.

At about neutral pH and 30-32OC, the CT of high heated milk (85°C-15 min; 8U1W°C-2

min prior to UF)declincd markedly with increasing protein concentration [ S h m a et al., 1990;

Sharma, 1992; Guinec et al., 19961. Heat mamient (a 8S°C-15 min) incteased CT by 100% in

2x, 3 1% in 3%.and only 27% in 4x milkr; the lx heated control did not gel under the conditions

of the assay [Shanna et al., 199q. The rate of gel timing and gel firmness were d u c e d by pn-

heating [Sharma et al., 1990; Guinec et ai., 1996; Waungana et al., 1996, 1998; Pomprasirt et a . ,

19981. The initial rate of the enzymrtic rcaction w u rcduced in lx and 2x prc-heated

concentrates compued to unheatd concentrates, while the c f f a was minor in 3x and 4x

retentatts [Sharma et al., 1990; also Femn-Baumy et ai., 1991 (70°C-1 min to 160°C-0.1 s)].

The above studies make it clear that the rennetability of (ultra) high heated milk can be about
restored by subsequent concentration. n i e precise reasons for the irnprovement are not so well

understood. Shama et al. [1990] speculated that it originates from increased [Gaz+]. Similarly,

Fenon-Baumy et al. [19911 invokeâ electmchemical mechanisms (reduction of the net negative

charge of the casein puticles on increasing concentration, i.e., ionic strength). Brulé and

collaborators [1974], however, reporteci that et a given pH and with the type of equipment/üF-

membranes they used, the distribution of micellar us. soluble Ca was hardly affected by UF (W.

concentration factor 1-4).

The main effects of increasing protein concentration on the formation of acid milk gels shall
be highlighted in the following section.

2*2*6 Aggregation on Lo wering the pH -Acid CoaguiationO/ Milk

Considerable research effort has k e n devoted to understanding the conversion of milk to

acid gel products [reviewed by Fox & Mulvihill, 1990; Mulvihill & Grufferty, 1995; Lucey &

Singh, 1997; Home, 19991 but still a great deal has to be leamed, especially with respect to the

nature of the coagulating particles. The effects of acidification at incubation temperatures above

30°C after thermal pmcessing m particularly relevant to the manufacture of fermented products

and directly acidified hrsh cheeses. Besides, information gleaned from studies on the pmperties

of acidified pre-heated milks may translate into a better understanding of the phenornena

involved in the irnprovement of the renneting khaviour of such milks by lowering or cycling the

pH. Insightp into the reactivity of the casein particles in a dynamic pH environment shall also

illuminate important aspects of coagulation by concomitant acidification and rennet action.

As pointed out initially by Hcertjc and his CO-worktn[198S], (partial) disniption of the

intemal stnicture of (pre-huted) c w i n micelles on extensive dissolution of CCP appears to play

an important part in the loss of stability, rather than acid gelation king driven simply by

neutralization of charges at the isoelcctric pH. [Sec also Zabodalova & Patkul, 1982;Roefs et al.,

1985;Roefs, 1986;Visser et al., 1986;Benguigui et al.. 1994; de Kmif et al., 1995;Gastaldi et

al., 1996; Lucey et al., 1997a;Tarodo de la Fuente et al., 1999 for tentative phenomenological

interpretations of the cascaâe of events leading to the formation of acid milk gels.]

(a) Effects of Pre-Heating. Pre-heat treatrnent (8S°C-IOmin) at physiological pH seems to have

little effect on the overall release of colloidal Ca phosphates (as measund afier storage for 22 h)

compared to non-heat-treated milk [Dalgleish & Law, 19891 when the pH is lowered in a

controlled way (GDL) at 4, 20, or 30°C until precipitation [Law. 1996; Singh et al., 19961.On

acidification of pn-heated milk most of the micellar Ca phosphate is thus solubilized klow a pH

of about 5.2 [also Visser et al., 19861.

In heated milk, Law [ 19961 found that, unlike at 4 and 20°C, the dissociation khaviour of

the caseins at 30°C was not markedly affccted by acidification, which is at variance with the

shallow maximum near pH 5.5 observed for a11 levels of dissociated caseins in raw milk at the

same temperature [Dalgleish & Law, 19891.Similar results wcre reportcd by Singh et of. [1996]

on acidification at 5 vs. 22OC of skim milk pre-heated at 80 and 90°C for 5 min. Thus, in heated

milk, if anything, solubilization of inorganic material at 30°C secms to be accornpanied by a

gradua1 re-incorporation of serum proteins (some caseins plus denatureâ whcy proteins) into the

residual casein particles (or newly fomed aggrcgates?), as if the propensity of the caseins to

leach out h m the micelles on acidiQing wcre in fact diminished by pre-heating. The clear

temperatun dependence of pH-induccd pmtein dissociation points to hydrophobic interactions

(possibly mediatcd by whey piotcins) king a major deteminuit, in compkment to attractive

electrostatic forces. The question &ses why prc-hcat trcatment of milk should favour association

of the caseins-whey proteins on lowdng the pH at and above 30°C, and to what extent the

distribution/amngement of cascins in the aggregating particies is affectad by acidification. It

may be envisaged that incorporation of heat-denatured whey proteins within the micellar

structure (Le., the introduction of additional cross-links through hydmphobic interactions with

tlic cascins) somchow consolidates the casein particles [Singh et al., 1996; Lucey et al., 1997~1.

It is not certain either how pre-hcating may affect the kinetics of pH4nduced solubilization of

CCP subscgucntly.
Taking note of the results of Lin et al. [1972], Roefs et al. (19851, Griffin et al. [1988], and

Rollema & Brinkhuis [1989], which suggest that some size-detennining structural framework is

maintained when (most of) the CCP is removed fiom raw milk micelles (rather than the caseins

dispersing completely), the more so at temperatures above 20-2S°C. one may take the view that

yoghuit-like gels consist of cwin colloids whose intemal and surface arrangements may be

comparable to thosc of the original micelles in ternis of the location of the individuel casein

components, despite their heving notably different (minera1 and protein) compositions [also

Horne & Davidson, 1993~;Holt & Home, 1996; de k i f , 19971. The dominant interaction

forces that maintain particle structure are expected to difl'er considerably, however. How much

of micellar characteristics are retained by the aggregating casein particles m a i n s a

speculativband somewhat controvcniai-matter (notice the apparent divergence between the

a5oremcntioned view and the view pmposed by Heertje et al. [1985]).

One is led to wondcr, in particular, what the outer surface of such re-fonned casein entities

lwks like and how it contributes to particle finctionality. In fact, Iittle is known about the rôle of

K - c w i n at low pH. The observations of Roefs and CO-workers(1986, 199061 suggest that K-

casein still play an important part in stabilizing the casein particles in standard reconstituted

skim milk against coagulation by rcnnet at pH 4.6 and S O C , as if K-caseinwcre integrated once

more ont0 the particle surfaces following substantial dissociation uound pH 5.0 at low

temperature [Dalgleish & Law, 19891. The stabilizing cffcct is supposed to stem h m the small

residual negative chuge of the macropeptidcs and h m their ovcnll hydrophilic character,
which may give r i r to some steric repulsion at acidic pH. This is expected to offer a barris to

the fusion of acid-coagulated casein particles. The behaviour of Pcasein [Heertje et al., 1985;

Visser et al., 1986; HwaIkar 4% Kalhb, 19881 (isoelectric pH = 5.2) remains elusive; givai its

amphiphilic pmperties, pcasein rnay supplement K-caseinif it (n)deposits on the surface of the

particles upon acidification. Fstudies of Law [1996] and Singh et al. [1996] (also Dalgleish
& Law [1988] for unheated milk) showed, however, that at above 20°C no (or little) preferential

dissociation of micellar kcasein occun at pH values between 5.5 and 52.1

Onder otherwise identical (tempentuie) conditions, pre-heated milk shows evidence of acid

gelation at higher pH than unheated milk (Grigorov, 1966; Kokb et al., 1976; Heerije et al.,

1985; Kim & Kinsella, 19896; Home 8r Davidson, 1993~;RUnnegArd & Dejmek, 19931. In raw

milk acidified by slow hydrolysis of GDL at 30°C and measured by diffising wave

spectroscopy, Home & Davidson [1993u] mported values for the pH at the onset of gel

formation of CU. 5.0; these increased to about 5.5 in milk pre-heated at 90°C-10 min and a

transition in gelation behaviour was obsemd at 7S°C as the duration of heating app1ied was

extended h m 10 min to 2 h. It is possible that the condensation reactions of u-casein with the

whey proteins that occur upon thermal treatment above 7S°C lessen the ability of K-casein to

(sterically) stabilize the casein pseudo-micelles, allowing the particles to coagulate at a highcr

pH and nepive charge (and higher content of CCP?). Perhaps some increw of particle

(surface) hydrophobicity is involved. Heat-induced dissociation of r-casein may also sensitize

the casein particles to pH (Cd+)-induced aggregation. Dcnatured whey proteins may also play a

role through increasing the concentration of gelling pmtein andfor initiating early aggregation

owing to the relatively high i~oclectricpH of pLg (a 5.3 [Kinsella & Whitehead, 19891) 1e.g..

Lucey et al., 1997u, 1998c,el.

(b) Effects o f Pmtein Conccnb*tion. Acid coagulation of UF retentates appean to ôe a multi-

stage pmcess, much as in d a r d milk [Biliaderis et al.. 1992; Gastaldi et al., 19971. The
physico-chemical investigations of Gastaldi et al. [1997] dealt with fortifed milk systems [i.e.,

total solids werc varied dinetly by addition of skim milk powder to a concentration of 10

(control) to 20% wlv], but such observations are likely to be relevant ta gel development in

acidifying UF retentates as well. Intercstingly, incrcasing total solids of milk h m 10 to 20%

was reported to shifk the onset of gelation (GDL,20°C, as assessed by dynamic rheological

measurements with a Vixoprocess rheometer) taward values of pH lower by about 0.2 pH unit

(pH 4.86 vs. 4.65). A simiiar shift along the pH scale was obsewed for the experimental values

of micellar solvation, nlease of colIoidal Ca and phosphate, pH-induced dissociation of micellar

casein, and buffering capacity of milk, especially kfore gel formation kcame noticeable.

Measurable increases in percentage of micellar Ca phosphate with increasing dry rnatter at a

given pH during acidification were also reported by B ~ l &t Fauquant [1981] on increasing

pmtein concentration by UF. Regardless of the content of total solids, the effects of lowering

milk pH at 20°C on the properties studied seemed to reach their maximum between pH 5.5 and

5.0 [Gastaldi et al., 19971. The apparent stabilizing effect of increasing total solids against pH-

induced changes in the micelles was ascribcd primarily to the higher mineral content of casein

particles in milk enriched with total solids, the more highly mineralized particles supposedly

having to be bmught to lower pH to mach a given state of aggregation.

Given the many uncertaintics about the evolution in composition and structure of the

aggregating spccies, it is questionable whether acid coagulation of milk micelles can be

meaningfully interpreted within the conceptual fnmework of the 'hairy Iayer' model. Factors

othr than the envisagcd collapse of the K-crseidwheyprotein hain or filamentous appendages

in the poor (acid) solvent must corne into the picturc, such as rc-structuration of the surface to

create adhercnt patches of non-K-casein andor denatumd whey proteins. Another possibility

would be the desoption of w-ersein during the time taken for the particles to attain mutual

contac+-conccivabty, the acidifed puticles are mon loosc, hace dynamic, than native
micelles and may exchansc proteins with the m m more readily [Holt & Horne, 19%]. Fmm

the mults of Lucey et al. [199&] it is unlikely that soluble (denaturing and likely polymeric)

semm proteins may pmtake dircctly by acting as bridging agents bawccn flocculating casein

particles. Instead Lucey et al. [199&] found that heat-dcnatured serum proteins aswciated with

ciioein particlcs were the main source of bridging material.

2.2.1. Combined Rennet und Acld Coagulaîion of Mil&

Combined enzymatic and acid coagulation of milk gives rise to additional physicoîhemical

complexities. nie crucial influence on gel-forming reactions of (intemlated) factors such as the

relative rates and end-products of renneting and acidification, themal history, and protein

concentration make hem even more difficult subjects for systematic studies, particularly if

quantitative analyses of gel formation are to be profitiibly applied and related ta milk gel

technological properties. Direct information on rennet-acid gelling systems is relatively scarce,

but interesting (pseudo-linear) viscoelastic and spectroscopie investigations have b a n published,

essentially concumng in showing that milk gel development and dynamic properties are

appreciably modified by the conditions of concentration of remet vs. acidifying bacteria

[Lehembre, 1986; van Hooydonk et al., 19866; Zoon et ai., 19880, 1989; Noël et al., 1989;

Dalgleish & Home, 19910,b; N d l et al., 1991; Schulz et al., 1999; Tranchant et ai., 1999a.61.

When enzymatic protcolysis and continuous acidification both contribute to coagulation of

standard skim milk, a singular dependence of the dynmic elastic modulus on time (hence pH) is

observed, with an optimum ca. 2-3 h afier the onset time of increase in rnodulus (pH at onset of

gelation * 5.9-6.3 with 80-9OQhK-caseinhydrolyzed) m u n d pH 5.6-5.5, and a clear pessimum

(i.e., a local minimum) 1-2 h later near pH 5.0-5.3 kfore a secondary rise in modulus [van

Hwydonk et al., 19866 (0.001% v/v remet, inspecified starter concentration, pH at renneting

6.6 and 25OC); N d l et ai., 1989 (CU. 0.02% vfw ccnnet, 1% v/w Streptococcus lacfis ssp.

diacetylactis, pH at icnneting r 6.0 and 30°C); N&l et al., 1991; Schulz et al., 1999

61
(measurements of viscosity under unspecifed conditions)]. The region of pH at which the

pessimum is mched seems to coincide with the pH at which an apparent transition state in the

cawin puticles occun (Section 2.1.30) and with the pH marking the border-line between acid

and rennet gels.

(0 In a small defonation rhcologiul study on the influence of calcium on the clotting of

acidified and renneted skim milk at fixed levels of rennet and starier organisms, Noël et al.

[19891 made the following observations. First, the duration of the Iag phase was not substantially

affected by increasing the concentration of CaCl2 (090.04% pet wt. of reconstituted skim milk).

[In control milk renneted at pH 6.6 with about hvo times more rennet, the lag stage was

shortened by addition of CaC12, the effect king most pronounced below 0.016%.] In conûast,

the times associated with the first maximum and pessimum values of gel firmness (as measured

as shear stress) increased with the concentration of CaC12, with a plateau between 0.004 and

0.008% CaC12. Second, the maximum rate of fiming of rennet-acid gels increased moderately

with small additions of CaClz ($0.004%) but decreased considerably above 0.004% CaC12. [The

effcct was not as pronounced in rennet gels and the optimum concentration of CaC12 was m n d

0.016%.] Third, gel rigidity at the optimum and pessimum increased slightly with addition of

CaC12 in the range 0-0.004/0.008% but decreased markcdly above 0.016% CaC12. [A similar

trend was observed for the (m id-range) finnness of rennet gels at a n d 'quilibtium' .]

The pH-induced demineralization of the casein network was invoked to account for the

apparent M i n g down or softening in the development of the firmness of rennet-acid gels. The

effects of Ca wcrc explained on the basis of the concentration of soluble (rather than addcd) Ca.

The authors concludcd that addition of Ca. 0.004% CaC12 should provc most beneficial to

combined rcnnet-acid coagulation of cheese milk (pH at renneting = 6.0). compared with ca.

0.016% CaC12 for strictly enzymatic coagulation at pH 6.6. These concentrations correspondcd

to a compromise between most desirable fimness/elasticityof geVcud and rate of fiming.

 Noël and collabontors [199 11, also dopting a dynamic rheological approach, showed dia at

f i x d dosage of stcuier, the value of pH (6.6-6.0) a the moment of remet addition had the most

significant (mainly linear) effect on ovcrall coagulation kinetics. They also noted and quantificd

a significant interactive effect betwcen pH at renneting and rennet concentration (149~104%

w/w). The kneficial impact of adding m e t on increasing the amplitude of the local maximum

in gel firmness was found to k reseicted to the range 1-30~104%rennet. Coagulation

temperature (30-34OC) had a marginal influence on the panuneters studied.

(il) Dalgleish & Home [1991a,b] wmed to fibre optic dynamic light scattering to establish

the gelation profiles of cultured and renneted pasteurized (undiluted) milk without disturbing gel

assembly. They t w identified a distinct pattern of behaviour apparently typical of situations in

which renncting and acid 'prccipitation' were appmimately 'balanced' (le., 3.3~104%v/v

rennet, 0.06% wfv starter, pH at renneting = 6.6, and 30°C). This was contrasted with the timc-

dependent optical characteristics of gels formed under 'extreme' conditions of concentration of

rennet and acid-foming bacteria [6.6x10-r% v/v (high) rcnnet, 0.03% w/v (low) s u e r , 33OC

(approximately nproduced in Figure 7.3.1 under Section 7.3 of Chapter 7); and 1.6x10-4% v/v

(low) remet, 0.09% w/v (high) starter, 2S°C, respectively]. The mcasurements were obtained

semi-continuously over ca. 5 hours. At the lowcst concentration of rennet, the authors estimated

that the extent of breakdown of wasein was no more that ca. 10% of the total.

The changes in scattercd intensity and apparent particle size were discussed in tems of the

relative mobility of the pseudo-micellar scattering ccnten (and soluble material) within the

casein rnatrix, and tentatively relatcd to the viscoelastic propcrties of the gelling milks,

independently fmn the oboemtions of NMl and CO-workers(1989, 19911. Bath sets of

obscivations sccm to be wncerncd with analogous mctions, however, cven if it is not clear how

the findings compare, direct cornparison ktwcen the responws measunxi king complicateâ, in

part, by difierences in coagulation conditions and, pouibly, by confounding timc-rclated effects

(see Chapter 7, Section 7.3.2~for fuithet discussion). Interestingly. the light scattering technique

also appeamd to k sensitive to the rcsponr of the 'micelles' to rennet hydrolysis in the pre-gel

phase, Le., beforc any s i p of getation was noted. The appumtly 'fimst'-and presumably

most elastic-gels (i.e., those for which the diffisivities of the aggregate particles became the

most restrictcd and only rapid local motions remained observable) were formed when gelation

was predominantly by rennet action and occumd in the pH m g e 5.35-5.45. In the intermediate

rennet-acid situation (pH at gelation = 5.1). a distinctive reaction profile was observed but the gel

seemed to assume properties resembling those of mainly-rennet gels, except for the lower

apparent rigidity in the final stages of aggregation. As well, albeit on the basis of measurements

of tan 6 (= G'YG'), Noël et ai. [1989] concluded to an apparent (structural) similarity between

rennet-acid and rennet coagula. r o be sure, rennet-acid gels typical for h s h cheeses retain

distinctly better ability to synerese and drain than strictly acid gels, as discussed under Section

2.2.3c.l

The foregoing studies confimicd that fine gradations exist in the ways milk gel stmcturcs
develop as the specific contributions of mnneting and acidification are varied, although most of

such investigations lacked cleu control experiments in which coagulation is by rennet alone or

by acidification. Reorganization of the (aggregate) casein particles consequent to the

solubilitotion of CCP is expected. but it is aiIl vague what perticular events take place in the

pte-gel andlor port-gel phases and how cosplation equation(s) ought to be rcfined to describe

the setting of milk when coagulation is by muieting and concomitant, continuous d e c m of the

pH. Givcn the time-sale of the changes undcr investigation, the coagulation temperaturesi, and

the incrcasingly acidic conditions, the possibility of (spondic) synerctic piocesses modiQing the

evolution of the systems king tested ought to k kcpt in mind. Even if no exudcd liquid is
perceptibk macmscopically, 'rnicropockets' of whey may separate out within the gel as the

netwotk assumes more 'stable' States.

This brings us to the present work. For one thing, we asked whether we could estimate the

effects of lowering milk pH fiom physiological value to ca. 5.5 on the surface properties of

diluted, essentially unaggregated casein particles h m unheated and pre-heated milk, principally

thmugh the use of photon correlation spectroscopy (Chapter 4). We then sought to characterize

rheologically the coagulation behaviours of nnneted milk under different conditions of

biological acidification, with or without pre-treatment of milk, mort importantly heating a d o r

increasing concentration (Chapters 5 through 7). Key principles of the major techniques

implemented shall be highlighted next.

3.1. Dynamic Light Scattering (DLS) -Photon Cornlition Spechoscopy (PCS)
3.1.1. Particle Slu by &won& Light Scatterhg

(a) Princi~ksof Memurement. The'basis for determination of particle size and particle size

distribution using dynamic light scattering techniques is the scattering of light by particles

moving randomly under Brownian or diffisive motion. Light is scattered by particles in

suspension because of ncarly elastic collisions (almost no energy change) between photons and

particles. The intensity of light scattered at a given angle frorn the incident light is detemined by

the geometry of the collision and by the dynamic and morphological properties of the scatterers.

Although al1 psriicles scatter light, scattering is pndominantly by particks of larger than

molecular dimensions (e.g., biocolloidal particles) whose rehctivc index differs fiom that of the

sunounding medium. In simplificd milk systems (little or no contribution fiom fat globules and

somatic cells), the casein particles are primuily responsible. Scattering by serum protein

molecules is negligible and does not interfen with the measurements because, as compared with

the micelles (107-109 mol. wt.), whey proteins have a low mokcular weight (CU. 1. W . O x 101 Da

[Walstrn & Jenness, 19841) and are of small size.

The Malvem Photon Comlator Spectmmeterm (Malvem Instruments,Inc., Southboro, MA.

USA; Figure 3.1) we used to measurc the si= of casein particlcs is typical of modem DLS
spectrometers. The instrument consists of an hclium-neon laser light source which p a s ~ s

through a ample chambcr with temperature-controlled watcr bath and electric heater. The light

scattered by the sampk i t Mme angle h m the incident b e m (900 in Our expcrimcnts) is

detected by a photomultiplier tube mountcd on a variable scattering angle tumtable. F e angle

of scatter B detemines the distancescale (as defincd by @QI under Section 3.1.16) which can be
probed by the light scattered.] The amplified signals (photon counts) fiom the photomultiplier

are digitized and pmcessed by an autocorrelator intdaced to a microcornputer. The spectmmeter

is provided with computer program for computer control, data acquisition, and reduction of the

data to size parameters. Instrument whlp and run conditions are detailed in Chapter 4 under

Section 4.2.7.

holder - / -

HsNe laser source

Figure 3.1. Block diagram of the Malvem Photon Comlator SpectrometerTY(Malvem

Instruments, Inc., Southbro, MA,USA)

Difisive motion of sample particles in the incident laser light causes the wavelets of light

scattercd by differcnt particles to interfcre with u c h other. The rcsulting intederence patterns

cause the number of photons collected, i.e., the total ekctric field and hence the scattering

intensity at the photomultiplier to vary with time. (Light detectors acnully respond to the

intensity of scatterrd light, not to the electric field.) The main challenge in DLS experiments is

the derivation of quantitative information h m a fluctuating signal. The changing with time of

scattering intensity (Le.. the rate of intensity fluctuations) is rclated to the hydrodynamic
pmpcrties of the particles, that is, to how quickly the particks move in relation to each other:

small, rapidly diffising particles yield fast fluctuations, w h e m larger particles and aggregates

generate relatively slow fluctuations. In DLS it is theref~rethe appurent (merage) trunsIationaI

d i p i o n coeflcient D (m2.s-1) of scatterers in solution that is measured experimentally, and

fiom this an apparent (average) hydrodynumic diameter dh (usually quoted in nm) cm be

derived using the relation of Stokes-Einstein [Einstein, 19561, assuming that the particks are

spherical:

(Equation 3.1)

where k~ is Boltzmann constant (N.m.K-l), T is the absolute temperatun (K)(i.e., &BT is the

thermal energy), and qo is the viscosity (Pa.s) of the suspending medium. Equation 3.1 is

rigourously valid for dilute suspensions in which the interactions between particles can be

neglected. Also, preâictions via the Stokes-Einstein relation nfer to the properties of a large

number of particles; the detailed dynarnics of an individual particle undergoing Brownian motion

cannot be predicted. Suitable levels of dilution are defined through experimentation, precautions

king taken to presewe as much of the native structure of the particles as possible.

(b) Ex~erimentalDetermination of Autocomlation Functions and Difision Coefficient [Hallett,

1994; Dalgleish & Hallett, 1995). Modem DLS spectrometers analyse time-dependent

fluctuations in scattering intendty through the technique of outocorrelu~ionunui'ysis [Abbiss &

Smart, 19881. [ h l y spectrometers in cornparison analyocd the frequency specmim of the sipal

from the dctector, Le., the so-callcd 'bcating eff=td uising h m the small (Doppler) fiequency

shih. The two approaches are quivalent, howevcr, because the îùnctions calculated in each

case, vk., the b a t firquency function and the clcctric field autocorrelation function (to k
defined Iater) ais interrelated.] This means that the arriving photons are comlated instead of

king averagcd as is the croc in static light scattering experiments.

Since most ment instruments operate in the photon counting mode (hence the narnc 'photon

comlation spectroscopy'), the fluctuations over time of analogue intensity (numkr of photons)

are first encoded in a sequential strram of numbers called 'bins', each of which corresponds to

the digitized value of the scattered intensity measured during a small unifonn time interval t

refened ta as the sampling or sumple time (an instrumental setting; also known as comlator bin

time or time per comlator channel). Sampling times are typically very short (of the order of ps

to ms) because they must be considerably shorter than fluctuation times for the data to be

meaningful. This is related to the size of the particles or aggrcgates king measured.

The computational hardware of the autocorrelator then generates a function called the

intensiptime uutocorreIutionfiniction,Cl(@,usually composed of between 64- 100 channels and

dispfayed live during size measurements with the Malvem spectrometer. Construction of Cl(r)

from the string of numbers obtained &er the formation of bins is by multiplying the number of

photons in the bins together and summing them according to a specifk set of rules [outlined in

Dalgleish & Hallett, 19951. ris the comlation delay timc between the bins of intetest (ris given

by r = kxt, with k the numkr of delay channels separated by the sample time, Le., al1 channels

are arrangcd to look at succcssivcly largcr time spans). The intensity autocorrelation function

descriks the fluctuations in absolute scattering intensity. A plot of the full conelation hinction,

i.e.. a plot of photon counts per comlator channel against delay time p d u c e s an exponentially

decaying function with a theoretical asymptotic limit (badine or background value) as z

appmaches infinity equal to the time-average intensity q u a n d . (I(l))X The characteristic decay

or relaxation time is roughly indicative of the typical fluctuation tirnc of the signal and hence

containî information about the diffisionfsize of the particles: if the particles are small, the
correlation funetion decceascs quickly, whereas if they are large, the function decreases more

slowly.

The experimental intensity autocorrelation function cm ôe nonnalized by dividing by the

background and cm be related to the scattering electric field autocorrelation function, gW(r),

through the Siegert [1943] relation:

(Equation 3.2)

in which g(z)(z) denotes the nonnalized intensity autocomlation function.

The electric field autocorrelation fbnction inferred fiom C,(r) is important because it is the

huiction that can k h v e d at theoretically for a set of scatterers and it is ficquently chosen for

interpretation of s i a results. For a sampk containing monodisperse small particles [i.e., particles

of identical dimension, small compared to the wavekngth & (nm) of the incident light], g(O(r)

has the form of a single decreasing exponential:

(Equation 3.3)

in which the decay constant (LI@"-/

is related to the particle translational diffbsion coefficient D

and to the magnitude of the scattering vector Q (nm-1) as defined by the experimental scattering

arrangement, IQ 1 = (4nndh) sin(0/2), no (dimensionless) king the refhctive index of the

suspending fluid and B the scattering angle. D is obtained by fitting the experimental data to the

exponential coneiation function and, if the particles are non-interacting sphens, it con k

converted to partick size invoking the Stokes-Einstein Equation 3.1.

(c) b a l vsis of Autocorrelation Functions for P s ~ e r s eSvstew. Actually, most systems of

interest, and notoriously casein puticles, exhibit pol'isperse pmpertiw (i.e., their size is

disîributed). Each spccics in a polydisperse ssmple contributes its own diffusion coefficient to
the autocomlation function acconling to its mess fiaction in the system. In this situation g(l)(r)

becomes a sum or distribution of single exponentials over al1 the sizes present, with pmper

weighting factors <vr related to the relative abundance of particles of a given sin, which for small

particles gives:

(Equation 3.4)

where a is an experimental constant, i is an index of size, and m is the number of classes of

particle size. The initial decay, king dimctly related to the lmroge diJiaion coeflcient D o f the

particles, is of particulsr interest. If particle size distribution is continuous, the discrete

summation in Equation 3.4 can bc nplaced by:

(Equation 3.5)

Here G('describes the distribution of decay times FI,with T =Dpl.

In practice, the ill-conditioned natute of inversion of Equation 3.5 makes it dificult

mathematically to recover information about the pmpcrtics of size distributions hom the

intensity autocorrelation hinction. Several alternative mathematical procedures of varying

sophistication sueh as exponcntial sampling have km dcveloped to tackle the pmblcm and

derive partick size distributions pmpcr [discussed by Hallett, 1994). The method of cumuîmts or

moments ana&sis Il<oppel. 1972; Pusey et al., 19741 actually circumvents the pmblem of

inversion a d , for simple n m w distributions, pmvides a relatively simple and powemil method

for detemining the uverage hy&d)narnic size of particles in suspension. In cumulants analysis
of the data, the initial decay time is determined by a fit with an expansion of g(O(r) fiom

Equation 3.5 into moments (cumulants) of the fonn:

(Equation 3.6)

in which the p-, are the various momentq. An average value of D, and hence particle

hydrodynamic diameter, weighted by the intensity of the lighr scattered, is obtained fiom the first

moment, (0.Moments analysis of the experimentally determined intensity comlation function

Cl(@ is by fitting a second order polynomial in r to the logm-thm of the measured function after
subtraction of the background walvern Ltd., 19911.

Details of the principles and desips of DLS, and its applications to f d s (mostly milk-

based systemq including emulsions) are described more fully elsewhete [e.g., Chu, 1974; Holt et

al., 1975; Berne Br Pecora, 1976; Dickinson & Stainsby, 1982; Home, 19846; Burchard, 1994;

Hallett, 1994; Dalgleish & Hallett, 1995).

3.1.2. Application to the Stu@ of Patticle SurJace Structure

Conventional photon comlation spectroscopy applies to the study of (highly) difute, non-

interacting systems. The technique is non-invasive and relatively rapid, and is sensitive to srnall

changes in diametcr (of the order of 5-10 nm and up) in the size range (< 800 nm) of milk

micelles. In the initial phase of rcnnet action, undei conditions of physiological pH of milk, the

apparent average hydrodynamic diameter (dh) of raw milk micelles, as defined in Figure 2.3,

decrcascs masurably as the polymeric sîabilizing layer mund the pmicks is king removed

cnzymatically, as pictural schanaticaliy in Figure 3.2. nie obscrved reduction in mean particle

diameter afier enqmatic action (Mnallows for estimation of the apparent thickness of the
surface Iayer. This was demonstrated by Walstra et al. [1981] and substantiated by othcr workers

through the use of PCS [Home, 19840; Griffin, 1981 and several differcnt techniques, including
viscometry [Scott-Blair & Oosthuizen, 1961 ;Guthy & Novi&, 1977;de Kruif et al., 1992; Home

& Davidson, 19936;de Kruif & Zhulina, 1996; Alexander, 1997; Lomholt & Qvist, 19971.

Auange
hydrodynrmic 140
di8mt.r by
PCS (nm)
120 -
dd=2xthick~8of
th@ 8ufhc@kyar
100 '
I

A
nil

Tlma rfbr addition of nnnot at pH 6.7 (min)

Figure 33. Decrease in average apparent hydrodynarnic diameter dh of casein micelles as the
surface layer of K-casein macropeptide is broken down by the action of rennet enzymes
(chymosin). The pictorial shows the state of the micelles at different stages during the reaction:
with the 'hairy layer' intact at the star( of the reaction, with the layer (partly) removed as a
minimum or effective 'core' diameter is reached, and aggregating as the diameter increases once
most of the protniding polypeptide chains have k e n cleavcd. [The hydrodynarnic diameter of
non-renneted casein particles remains constant within experimental variation (horizontal
baseline).]

Such a behaviour is consistent with the glycomacropeptide (and possibly part of the para-u-

casein moiety) of r-casein existing in a suficiently extended state to provide a measure of the

steric component to stabilitition, and thus, renneting, together with PCS, is a useful tool for

indirect examination of the stnicture of the miccllar sunace, as detailed in Chapter 4. Likewise,

PCS rnay be used to probe the conformation or structure of adsorkd proteins in, e.g., emulsions

via determination of the apparent hydrodynamic thicknesses of the protein layers or aggregates

amund oil drop1et.s or latex particks [Fang & Dalgleish, 1993qb;Dalgleish, 19936, 1995;Tosh,

lm;Anema, 19971.
 With dense scattering media, such as concentrated particulate dispersions, scattering theov

is complicated by dependent scattering (for which scattering intensity is weakened by destructive

interferences of light scattcred by particles separatcd by less thon the wavelength of the incoming

light) and multiple scattering (for which the light is scatttred by a number of particles before it

maches the detcctor). Another form of laser light scattering, known as diffising wave

spectroscopy (DWS),has ôeen developed recently for use in undiluted rwbid suspensions.

Particle sizing by DWS actually depends on the occurrence of multiple scattering, the incident

photons experiencing a random walk (diffising wave) in the sample prior to king detected, to

provide information on particles in optically opaque (gelling) solutions. The technique relies on a

bifurcated bundle of optical fibres to p a s light into the m p l e and to collect light which is back-

scattered at angles close to 180' [Horne, 1989~;Home, 1991abl.Initial applications of DWS as

o partick siring technique contirmed that such phenornena as the decrease in micellar size upon

renneting at amund neutnl pH, which was originally measured by DLS in diluted milk [Walstrs

et al., 198 11, were not artefacts arising fiom excessive dilution [Horne & Davidson, 199361. This

gives additional confidence in the reliability and interpretation of other experimental approaches

using DLS as well. Although not yet applied fomally, fibre optic DLS may emerge as a

powerful technique for probing the heological properties of sensitive coagulating/gelling

systcms in a non-destructive way by acting as a zcntshear viscorneter [Home & Davidson,

1990; Dalgleish & Home, 1991a,6;Home, 199 1 a,6;Home & Davidson, 1993a,61.

3.2. Fluonmety
Pm& Metho&
3.2.l. hotein Hydmphobici@by Flu~r~scence

Fluoresccncc pmk mcthods may k the simplest type of mcthod for estimating protein

hydrophobicity (i.e., non-polar arcas; overview of the hydrophobie effect in Tanford [1980]).
Quantitative estimation of ('surf'ace') hydrophobicity by spectmfluorimetry relies on the affinity

of a fluorescent dye for the hydmphobic regions of proteins. The fluomphore docs not fluoresce

when it is not bwnd and fluorescence cm be detccted upon oclective binding of the dye to

pmtein. Short wave or W light is used to excite the fluorescent markers into self-luminous

molecules. During excitation at the wavelcngth of pnferential absorption, molecules are

transfemd fiom the ground state into an activated state; when they tetum to the ground state a

part of the absorôed energy is emitted as fluorescent light. Fluorescence is at longer wavelengths

than the corresponding excitation because some non-radiative loss of energy also occuis.

Two types of hydrophobic probes have ken used extensively, vu., l-anilinonaphthalene-8-

sulphonate (ANS, an anionic dye) and cis-parinaric acid (CPA) for momatic and aliphutic

hydrophobicities, respectively (&n-Na'lm, 1980; Voutsinas et al., l983a.b; Hayakawa & Nakai,

1985; Parncll-Clunies, 1986; Paulson & Tung, 1987; Mottar et al., 1989; perspective of

hydrophobic probe rnethods by Nakai & LiChan, 1988; also Lieske & Konrad, 1994, 1995 for

an approach relying on the specifk binding of the non-ionic detergent Twem 80 for estimating

hydrophobicity of (milk) proteins]. The unsaturated molecules of CPA are relatively unstable,

hence less convenient to use in practice than those of ANS. Emission of light by the bound fonn

of ANS (475 nm when excitation is at 380 nm) is outside the usual range for the intrinsic

fluorescence of proteins, which occucs mund 330 nm when excitation is set at 275 nm.

Consequently, sny effects seen at 475 nm should not arise (at least dircctly) h m changes in

aromatic residucs.

3.Z82- A n U i n d - N @ t h d e n d p h n e (ANS)-FInorimttty

Fluorimetric measurcmcnts of micellu cascin reported in Chapter 4 were perfonned

essnitially according to the method of Kato & Nakai [1980] in the absence of SDS. In this

method, each protein sample is serially dilutcd and meisunments of relative fluorescence

intensity (RH,as memurcd with a spectrophotometer) arc taken with and without ANS. A plot
of RF1 vs. protein concentration allows for the estimation of an index of pmtein hydrophobicity

(procedure and test conditions are detailed under Section 4.2.8; experimental plots are shown

undcr Section 4.4.1).

Fluorimetnc deteminations in Chapter 1 were conducted along the rcsearch line developed

Bonomi et al. Il9881 and Peri et al. [1990] for monitoring the coagulation of milk.

Modification of hydrophobicity of milk proteins is followcd through the binding of ANS and its

partition between a 'b'

(supernatant phase obtained aftcr centrifugation) and an 'aggregated'

(precipitate phase) protein fraction duting gel formation (sec Section 6.2.1Ob for experimental

details). Following this approach, Peri et al. [1990] and Iametti et al. 119931 were able to study

aggregation and curd-firming d u h g rmnet coagulation of milk. An analogous ligand-binding

strategy has k e n used to quanti@ thermal damage to proteins multiog h m heating milk

[Bonomi et al., 1988; Pagliarini et al., 1990; Saulnier et al., 19911, and to follow in real time

temperature-induced modifications in the hydrophobicity of milk protein fiactions [Bonomi &

lametti, 199 1;lametti & Bonomi, 19931.

It should be noted that the terni 'surface hydrophobf ity' for proteins tends to be used

ioosely because in many cases (especially not tightly sriuctured (globular) protein systems]

protein surface is ill-defined. Since the fluorogeaic reagent is certainly able to enter the

interstices in the micellar fnme (the porc width in the puticles king pmbably a fnu nanometers

[Ribadeau-Dumas & Garnier, 1970; Tarodo de la Fucnte & Lablée, 1987]), the hydrophobicity

characteristics measured in out wotk probably reprcsent an ovcrall effective or accessible

hydrophobicity, Hh nther han a surface pmpcrty pmperly speaking.

3.3. Sodium Dodecyl Sulphate-Polyacrybmidt Gel Electrophomia (SDS-PAGE)

3.3.L E ~ p h o r e ! Seporatiom
k of Ploteiw

Separation of colloids such as pmteins by electrophorcsis is based on the movement of

charged colloidal puticles in an electric field [detailed accounts by Harnes, 1990; HawcmR

19971. In sodium dodecyl sulphate (SDS) electrophoresis of denaturcd and 'reduced' proteins

bemmii, 19701, the proteins are separated essentially according to size (mol. W.), since

treatment with exccss of Le anionic detergent SDS (a potent protein denaturant and solubilizing

agent which binds the polypeptide chains at a constant weight ratio of about 1.4 g SDSIg pmtein

[Reynolds & Tanfonl, 1970a,b; Weber & Osbom, 19751) results in approximately the same

suflace charge density and shapc (a rad-like shape whose lengths vary with the mol. W. of the

polypeptides) for all SDS-protein complcxes. Since Le complexes are negatively charged over a

wide range o f pH, the bufiering system is not as critical as in native gel electrophoresis c h c d

out undet native (non-dissociating) conditions.

Separation is achieved through the sieving effect of a polyacrylamide gel, a synthetic

poIymer which enables support media to k cast with morc well-defined and reproducible pore

sizes than natural materials such as agarose and starch [Pharmacia LKB Biotechnology, 19901.

When an electric cumnt is applicd, the proteins move toward the anode through the stacking gel

and into the polyacrylamide scpention gel. In the polyacrylamide gel, the mololcces with lcugcr

molecular weights move morc slowly thnnigh the gel network, and thus, proteins separate into

bands.

Typical patterns of elcctmphorcsis gels (2û% homogencous ~ h f f i e l s @Phannacia

, LKB
Ltd., Baie d'UiîC, QuCbec, Canada) of bovine skim milk with and without m e t added are

show schcmaticdy in Figure 3.3. (Rcfer to Chapter 5 for details about sampls pmparation and

run conditions.)
 Origin
ar2-Camin (25,230 Da)
asl-Cawin (23,620 Da)
l-3-casein (23,980 Da)
K -Casein (ca.1B,55O Da)
P -Lactoglobutin (18.280 Da)
+ Para- K -casein (12,270 Da)
a-LacZalburnin (i4,tûû Da)
Diredon
of migration

Figure 3.3. Schematic picture of SDS-polyacrylarnide gel electmpherograms of bovine rnilk

proteins fiom unûeated (lefi lane) and partly renneted (right lane) milk on a 20% homogeneous
~ h a s t ~ e l (Phannacia
m LKB Ltd., Baie d'UrfC. QuCbec, Canada) [mol. wt. from Walstra &
Jenness, 1984).

Different migration profiles can k obtained depending on sampk preparation and run

conditions, including gel characteristics (sce Strange et al. [1992] for a review of clectrophoretic

rnethods used for analysis of milk proteins). Contrary to what would k expected from their

respective molecular weighio, for the set of experimental conditions described in Chapter 5, the

four main caeins scpuated as four rclatively distinct bands conrsponding 10, in the order of

increasing mobility, ad-,a,p, p, and K-casein. The whey proteins PLg and a-Lacould also k

distinpisheci, togethr with paru-K-cwin in minetcd milk.

3.3.2. DensitomctrIc Scanning und Quanti/Ication

Dcnsitometric scanning of the stained protein bands following clectrophoretic sepmtion can

be used to quantify the changes in optical density o f the bands. Essentially, the gel scanner is an

absorbanec spectmphotometcr d i a provides information both on the amount of material present

in a band and on the position of the band in the gel in the fonn of a rcad out of sbsorôance us.
position in gel. Cornputer-assisted detemination of the areas under the peaks by integration

allows quantification of the proteins.

3.4. Dynamic (08ciilatory) Rheometry

of Viscotlrcstic Materiah
3.4.1. Rheologicuil Cha~acterizatio~

In contrast to classical light scattering techniques, which operate mainly over

rnacromolecular distances, rheological measurements essentially ptobe the continuity of gelled

or gelling systems over macroscopic (supramoleculer) dimensions. In principle, two

complementsry approaches cm k adopted for studying the mechanical properties of viscoelastic

materials such as milk gels, viz., static (Le., stress relaxation and creep) and dynamk

measurements. Static (transient) experiments are carried out under steady shear or with a step

change or sudden application of strain (Le., defonnation) or stress. Dynamic (oscillatory)

methods involve the application of a sinusoidally varying strain or stress, as explained hereafter.

Becaux gelling systems show a time-dependent behaviour, dynamic testing is particularly well-

suited to monitor gelation transition and the oetting of a gel phase, as in the small defonnation

experiments described in Chapteo 6 and 7. Large defonnation (fracture) rheology, in

cornparison, is useful to establish the mechanical properties of the final gels.

Theoretical bases of oscillatory rhwilogy and the technology of its mcasurement are detailed

elsewhere [Ferry, 1980; Whorlow, 1980; Mitchell, 1980, 1984; Shoemaker, 1992; O'Connor et

al., 1995; Steffe, 19961; only essential concepts will k outlined hem. Rhcological tenninology

has becn accurately defined by Reina & Scott-Blair [1967] and Scott-Blair & Spanncr [1974].

(a) Princi~lesof Measurcment. Laboratory oscillatory rhcometers likc the Cmi-Med controlled

stresdstrain theometers (TA Instruments, New Castle, DE, USA; formerly Carri-Med Ltd.,

ûorking, LX)and the Nametre vibnting sphm viscorneters (Namem Co., Metuchen, NJ,USA)
work on the same principle: the rcsistance cxertd by a fluid/gel sunple to an oscillatory

defonnation is in eome way convertcd to a response signal. Frequency, geometry, and measuring

system cm bc entircly differcnt.

Most common rotationcil rhsomcters (e.g., Bohlin VOR, Carri-Med CSL, Contraves, and

Den mer) masure the parameters of viscoelasticity h m the drag force on a rotating body

(typically a disk or a cylinder-like geometry) that is placed ont0 or in the test sample. These are

'volume loaded' devices with container dimensions that are critical in the determination of

rheological pmpertics. With coaxial cylinders, either the inner one (Carri-Med CSL and Den

Otter rheometers) or the outer one (Bohlin and Contraves rheometers) is oscillated in a

sinusoidal mode. Vibrational instruments (e.g., Nametre and Bendix Ultra-Viscoson

viseometen) measure a viscosity parametet €rom the damping of the amplitude of vibration of an

immersed probe (usually a spherc, a bladc, or a cylinder) by the sumunding sample. Vibrational

viseometers arc 'surface loadcd' systems because they respond to a thin layer of fluid at the

surface of the probe. Some such instruments can also provide information on the viscous and

elastic pmperties proper Fitzgerald et al., 19901, but in many cases the (empirical)

measuremcnts obtained cannot readily be relatecî to the fundamental viscous and elastic

parameters detined undcr Section 3.4.1 c.

(b) Dvnamic Shear Stress. Shcar Strain. Shcar Rate. and the Conditions of Lincar Viscoclasticitv.

In both Carri-Mcd CSL and Nametrc apparatus, the type of defmation applied to the sample is

simple shcat; only the Carri-Med CSL (and in genenl thcorneters with a n m w gap me~suring

system) operates a well-defued adjustable shcar rates, howevcr. The time-dependent force pcr

unit suifacc am acting upon and within the sample, or slicm stress, a(r) (Pa), is associatcd with

a dynatnic deformation, callcd shea strain, >ir), cxprcssed as a relative defonnation (a ratio of

deformation to initial sample dimensions, i.e., r dimensionless number). In typical dynamic

experiments, the ranges of stress, sttain, and rate of strain [d~i)/dt]are adjusted to sufficiently

low values to ensure infinitesimal (leut-destructive) defomtion of fiagile (gelling) samples and

f i lfil1 the conditions of so-cplled finetu viscoelarlciily (LVE).

The linear viroelastic region is usually defincd as the region of stress-strain in which the
response of the material at my time t and at the selected fnquency of deformation (Le., the

reciprocal time-scak of a periodic dynamic measurement) is directly proportional to the value of

the applied force. Ideally, the conditions of linear viscoelasticity ought to be identified for each

iype of sample and run conditions, including rheometer dimensions. Only when working within

or near the boundaries of the linear viscoelastic domain can the data be meaningfully analyzed

within the mathematiial fhmework of linear viscoelasticity [Gross 19531. The mathematics of

non-linw viscoelasticity are complicated and limited progress hm been made in this area

[Gervais et al., 19824; Kobayashi et al., 1982; Bird et al., 19871.

(c) Sinusoidal Straining. Depending on the type of rheometer, either smin or stress is variai; in

either case, the parameters measured should be the same. For example, if a small oscillating

shear strain is applicd:

(Equation 3.7)

then the resulting stress response is measured, aloo varying harmonically:

+ [sin(~)cos6 + cos(^) sin 81

a ( t ) =u,sin(u 8)=c0 (Equation 3.8)

in which a,(radas-1)is the impwed anplar fkqucncy of dcforrnation (the samc fkquency as for

the memurcd stress; o = 2nxthe value of ficquency cxpmssed in Hz); A (dimensionless) and a0

(Pa) am the amplitudes of the m i n and stress waves, respectively; and 6is the observed phase
.angle (los angle or phase shift; in radian or degrce) ktwccn the defonnation uid stress (Figure

3.4). This phase differcnce originates h m the viscous propniics of the material.

Figure 3A. Cornparison of the idealizcd sheu stress msponses, a(r),of an elastic solid, a viscous
fluid, and a viscoelastic semi-solid under controlled oscillating shcar strain, No, when
defoimation (strain) is within the linear viscalastic range (theoretical curvcs takm nomi
Shoernakcr et al. [1992]). It is common to use amplitude of the input signal (minor stress) as
the ordinate, but cimplinide and strain are equivdent in an oscillatory (controllcd strain version)
test.

For an idcally elastic solid, 'dt)is in phase with fl),that is, Nt) is at maximum when Nt) is

at maximum and 6equals zero. For a purely viscous fiuid, 6 is d2 d i a n s (90') out-&phase

because Mi) is at a maximum whm the rate of strain is at maximum, which is the case whm )ir)

is at a minimum; then 6equals nR. For a lincar viscoelastic syrtcm havhg characteristics of both

a liquid (viscous flow)and a solid (elastic defonnation), Ghas an intermediate value k t w « n O

and d2 radians.
 In the lincar regimc, q is by definition proportional to and Equation 3.8 can k writtcn as:

o ( t )= [s(sin(air)cos S} + 5{cos@) sin 611 (Equation 3.9)

Y0 Y0

The fiequency-dependent elastic pert of the stress, that is, the part of the stress in-phase with the

strain, comsponds to the dynamic elarric modulus, also called storage M ~ U I ~GL' S
(Pa),
, which

is defined as:

(Equation 3.10)

G ' is a measure of the energy stored and subsequently released per cycle of defortnation. The

viscous componmt of the stress (also dependent on kquency), that is, the part of the stress out-

of-phase with the strain, corresponds to the dynarnic viscouc rn0rhrJu.sor lau nodulus, G (Pa),"

which is detined as:

(Equation 3.1 1)

G " is a measure of the energy dissipated as hcat pcr cycle of deformation. The ratio of G " to G '

is defincd as the loss tangent, tan 6;

G"(0)
tan &(a>) =-
G'W

tan 6 c 1 .O thus indicates a prcpondcrant clastic contribution to the visawlastic khaviour and

ton 6> 1.O indicates a prcpondtrant contribution of viscous effccts in the sample.

The relationship ktwecn the stress and m i n is defined as:

(Equution 3.1 3)
1
where G* is the complex moâulus (Pa), which inchdes the complete information of the

viscoelastic properties of the sunple material. In mathematical complex notation, this modulus is

represented as:

G (a)= G 1 ( o )+ iG"(a) (Equation 3.1 4 )

and its absolute value i s given by:

IG *(al=JG'(~>)*
+G"(cu)~ (Equation 3.1 5 )

i, defined as i2 = (-l)ln, is called the imaginary numkr ('imaginary' because it includes the

square root of minus one) and G ' and G" are referred to as, respectively, the real and the

imaginnry parts of G*.

An alternative to the complex modulus is the complex viscosity, q* (Pa.s), which is defined

BS:

=q'(cu) + itf '(a)

q (a) (Equution 3.16)

in which the real or dynamic (ordinary) viscosity (Le., the in-phase component of q*) is:

(Equation 3.17)
and the imaginary viscosity (i.e., the out-of-phasecomponent of q*) is:

(Equation 3.1 8)

(d) Intemretation of Rheolonical Data and Exnerimental Dificulties. Rationalization of

rhcological characteristics of biopolymcr gels in tcms of propertics at the more basic

macromolccular level is always somewhat conjectural. The nature of contributions to, e.g., G'
and G " is difficult to define in any grcat detail. One ought to nmemkr that only physical bonds

with rckxstion times within the experimental time-scak (recipmcal kquency) of observation

(deformation) contribute to both G ' and G ". The magnitudes of the moduli have km considered

to be proportional to the number of bonds effective in building-up gel structure [van Kleef et al.,

1978; Zoon et al., 1988~1.The values of moduli are expected to depend on the density and

homogeneity of the gel network and on the character of the cross-links. Assuming only one type

of bond or a constant proportion between the number of bonds of various types implies that the

ratio of G ' ' to G ' (i.e., fun 6)be independent of the number of bonds and mainly related to the

type of bonds [van Vliet & Walstra, 19851.

Various investigators have actually surmised that the value of tan 6 reflects the type of

interactions in milk gels pohlin et al., 1984; van Vliet & Walstra, 1985; Roefs, 1986; Walstra &

van Vliet, 19861. In that sense, ton 6 is a more sensitive parameter than G'and G" alone for

indicating changes in the nature of the bonds andfor in the relative contributions of the different

types of bonds. An increase in tun Gcan be interpretcd as an increase in the relaxation of bonds

dunng a deformation cycle (i.e., decrease in relative elasticity), the material behaving in a

relatively more viscous and Iess elastic way. One has to k cautious, however, when using tun 6

to discriminate baween different types of bonding and gel structures because it may be that a

shift in the type of interactions docs not rcsult in a change of fun 4 e.g., if the relaxation

behaviour of the interactions is similm. The frcguency-dependence of tun ôalso has to k borne

in mind.

With regard to the conditions of milk pl dcvelopment investigated in Chapter 7, changes in

the pH, Ca and phosphate content and ionic sücngth, and/or temperature may k thought to

modify mainly the type of interactions within the casein gels. Changes in rennet concentration

and casein concentration pmôably influence mainly the number of such interactions. Hcat-
induced interactions of whey pmteins with micellu casein probably modify both the type and

number of interactions.

Beforc penomiing useful viscoelastic measurements, potential pnctical pmblems specific to

the samples to be studied ought to be checked for, including the presence of bubbks in fluid

samples, drying-out of the material over long measurernents, and its propensity to synerese.

Exudation of syneretic Iiquid c m give rise to the formation of a film which prevents proper

adhesion of the (gel) ample to the surface of the measuring body, particularly in shear

experirnents witchell, 19841. Slippage along the measuring surface may thus be encountemd

and lead to unreliable (erroneous) measurements, unless ribbed or otherwise roughened sensing

elements are used to minimize the problem. The design of moâern rheometers usually

accommodates for possible sources of error related to their operating, e.g., inertia effects, non-

linear effects, end effccts, and fiiction betwcen instrument parts.

3.4.2. DynaciJc Testing with the Nametre RkeoIiner Rheomdmm

Several studies on setting (dairy) protein gels have been carried out with more or less ment

rnodels of the Nameûe Rheoliner RheometerN (Figure 3.5) [de Man et al., 1986; Parnell-Clunies

et al., 1988; Shanna, 1992; S h m a et al., 1989, 1992; Xu et al., 1992; Shanna et al., 1993; Tosh,

1994; Wang, 19991.

The rheometer is particularly simple to operate and requires no predetemination of the

measuring conditions as these are inherent to the design of the instrument. It masures a

viscoelastic pmpei<y called nomiml viscosity, Le., apparent viscosityxdensity or qw,,xp

[expressed in cPxg.cm-3, i.e., Pa.sxkg.rn4 (S.I. units) or cPxg.mL-1 (c.g.s. units)] through a

spherical siainleu steel sensor vibnting longitudinally at its natural frrquency around 650-670

Hz with a fixcd shear rate of between 40604175 s-1 [Nametre Co., 1972, 1987, 1993; aloo

Fitzgerald et al., 1975; Oppliger et al., 1975; Fittgcnld & Matusik, 1976; Fmy, 1977; Ferry et
al., 1979). (The Bendix Ultra-Viscoson vibrating reed viscometer described by Roth & Rich

[1953] and used by Marshall et ai. [1982] operates in an analogous way.)

Transducer Rheoliner console Cornputer

Sensor sphere (diameter 2.54 cm)

(+ thermocouple inside sensor wall)

400-mL thermostated glass beaket

(diameter 1O cm, height 13 cm)
Adjustabk ample holder

Figure 3.5. Block diagram of the Nametre Rheoliner 2010 Rheometerm (Nametre Co.,
Metuchen, NJ, USA).

The sensing sphere is set into sinusoidal oscillation of constant amplitude by way of a driver

coi1 cumnt. When the probe is immcrsed vertically in a liquid, the shear wave generated by its

surface dissipates in the medium; this damping effect increases the power (voltage) required to

keep the sphere vibrating. The output signal is obtained fiom the voltage developed across a

resistor in series with the magnetic coi1 and varies linearly with the product of qwpxp[Oppliger

et ai., 19751. The amplitude of the vibrations (i.e., the appiied defonnation) is fixed, small

mou@ (CU. 1 pm) so that gel stnicture is left essmtially undisturbed. Sample container is

sufficicntly large so that limited disturbance reachcs its walls; othcnwise the measurements

would k compliuted by rcflcction cffccts [Feny, 19771. A thennocouple locatcd inside the

sensor wall allows continuous parallel monitoring of the temperature.

 The instrument is calibnted to delivcr data as qqPxp products because mistance by the

sample ta the oscillating spherc is a function of both parametas. Since the density of clotting

milk is constant in first approximation (CU. 1.O35 kgxm-3 or g.mL-1 for regular separated milk at

20°C walstra & Jenness, 19841). the variable that is measured is equivalent to an apparent

viscosity. Howcver, because the system changes h m a liquid to a viscalastic solid during

testing, the readout fmm the instrument will k rcfemd to as Nametre consistency or simply

consistency. These tenns seem preferable to any viscosity term. To minimize the effects of

variations of fluid density, both the sample and the sensor ought to k kept at stable temperature.

In principle, viscoelastic parameters proper can be computed fiom consistency

measurements and the entered value of sample density in reccnt versions of the rheometer [see

the nlationships provided by the Nunetre Company, 1987, 19931, including dynamic moduli,

G*,G ', and G" (Pa); (apparent) viscositics, q*, q', and q" (cP or mP8.s); and cornpliances

(recipcocal moduli), P. J', and J" (Pa-1). Complex viscosity, which is a htnction of both in-

phase q ' and outsf-phase 7 " components, is considered a better index for characterizing the

rheological propertics of viscoelastic materials than consistency because consistency does not

tmly represent the data once the milk has k e n coagulated to a mi-solid gel.

3.4.3. Dynamic T d n g s with the Carri-Med Conmlled Stms RICeometr

Oscillatory testing usually appem as an option on advanced controllcd stresdstrain

rheometen such as the Carri-Med CSLn' theometers (Figure 3.6). which have been most

commonly used for churcteiizing the viscoslastic khaviour of dairy products.

The CSL LOO modcl uscd in the present study has a microprocessor controlled induction

motor drive coupled with a minimum fnclion, low incrtia air karing, and a high resolution

opticat encoder for a n p l u displacement (a parameter analogous to stmin) [Cam-Mcd Ltd,

1989u.bJ.The rate of shur is v u i d by djusting the motor speed and the shear stress is
measurcd using a toque spping or torsion bu. The stress is calculatecl h m the geometry of the

mwuring fixture and the deflection of die optical detector, which hm k n calibrated by

applying standard toques [sec the rclationships provided by Cam-Med Ltd.. 1989u,b]. ('Toque'

is defined as the moment of lords producing torsion; this is analogous to stress.) Like the

Nametre rheometer, the Ch-Med rheometer is available with software for computer control and

data analysis; reduction of the data to findamental viscoelastic parameters is accomplished

simultaneously with the measumnents.

Housing for high resoluüon

optical encoder for angular
displawment
Housing for fiküonless air
tmaring
Coaxial cylindem
(Mooney-Ewart geornetry)
Water jacket
Peltier dament
Hefght adjusting micrometer scak Cornputer

Figure 3.6. Block diagram of the Cam-Med CLS 100 Controllcd Stress Rheometerm (Carri-
Med Ltd., ûorking, UK).

In contrast to the Nametm, diffmnt measuring systems can k fitted to the Carri-Med

rheometer. Selection of a specifc fixtum and adjustment of conditions of operation are mainly

according to the characteristics of the sample to be testd, as sumrnarizcd by the manufacturer

[Carri-Med Ltd., 1989~;also Schimanski, 19901. The coaxial 'cylinder' attachmcnt used in this
study is shown schematically in Figure 3.7. (Note also the important difierence in sampk

volume, hence weight, uscd for experiments with the Nametre and Carri-Med rheometers.)

Toque (N.m)

Tnincation (gap)

Figure 3.7. Coaxial 'cylinders' with cone and plate end (Mooney-Ewart geometry; not drawn to
scale). RI = 2.40 cm; R2 = 2.50 cm; cone angle, a = 2O20'24"; immersed (shear effective)
height, H = 3.0 cm; gap ktween the üuncated conical end of the cylinder and the bottom of the
cup = 77 Pm; approx. ample volume = 7 mL [Carri-Med Ltd., 1989~1.

This 'cup and bob' arrangement proved M e r suited for in situ monitoring of gelation

processes starting hom liquid milk than a parallel plate arrangement with which the numerical

values of rheological p m e t e n could not be obtained reproducibly (probably because the low

viscosity smples tended to flow out of the gap initially). In the courid 'cylinder' system, flow

(shear) takes place in the annular gap betwccn inncr (rotating) and outer (stationary) stainless

steel cylinders of ndii Ri and RI.mpectively. Mooney calculated tmncation and angk reduce

so-called end eflects which arise fiom the differcntial flow patterns at the bottom of the rotating

cylindcr. The clearance gap betwccn the end of the cylinder and the bottom of the cup, as an

inüinsic part of the geometry, is fuccd.

 4. HYDRODYNAMIC S U E AND HYDROPHOBICITY OF CASEM (PSEUW)
MICELLES AND THEIR POSSIBLE RELATION TO CHANGES IN THE
STRUCTURE OF PARTICLE SURFACE BETWEEN pH 6.7 AND 5.5

4.1. Outlook

The focus in this chapter is on the variations in the average structure of dispetsed casein

micelles brought about by acidieing milk in the range of pH between 6.7 and 5.5 at 2S°C, with

or without pre-heating. Variations in the average conformation of surface (K-casein)molecules

are of particular interest since destabilization and aggregation phenornena in milk critically

depend upon the properties of the surface of the casein particles (outlined under Sections 2.1.2,

2.1.3, and 2.2.2 of Chapter 2).

Collapse of the so-called 'hairy' outer Iayer of K-cwin macropeptide upon acidification is

frequently invoked to explain destabilization of the casein particles and variations in

experimental parameters such as voluminosity (solvation), hydrodynamic size, and Gpotential of

the particles, as well as viscosity of milk [e.g., Home & Davidson, 1986; Banon & Hardy, 1992;

Ould Ekya et al., 19951. Then have becn little attempts (if rny), however, to specifically

measun the putative conformational transition under conditions that minimize the possibly

confounding effects of more global changes throughout the particles at pH below physiological

values. The influence of thermal pm-trrrtment of milk on the pH-dependent khaviour of

panicle surface has not been clearly defined cither.

Practically, quantitative estimation of the (combined) effects of mildly acidic pH and pre-

heat trtatrnent on miccllir surface structure was attemptd through determinations of apparent

hydrodynarnic dimeter of diluted casein puticles by photon comlation spcctroscopy (PCS),

with and without remeting, as outlined under Chapter 3, Section 3.1. (The main msults have

bmi summarized for publication [Tranchant & lklgleish, 1994; Dalgleish et al., wbmitted for
publication].) A penpherd study was conducted to estimate aromatic hydrophobicity of the

casein particles betwecn pH 6.7 and 5 S . An overview of simple prepamtion for particle size and

hydrophobicity meesumnents (detailed below) is given in Figure 4.1.

Al1 electrolytes of low moleculsr weight (these and ensuing studies) were of reagent or M e r

grade.

1. Fresh Milk and Re-Treatments

4Jm

Pooled, nfrigerated bovine milk fiom Holstein cows was obtained weekly fiom the Elora

Dairy Research Station of the University of Guelph. Unless otherwise indicated, the milk was

presewed with 0.02% (wlv) sodium azide (NaN3; Fisher Laboratory Supplies, Unionville,

Ontario, Canada) with no proteinase (plasmin) inhibitor added. The antibacterial agent NaN3

does not appear to affect the coagulation of milk by minet (van Hooydonk et al., 198601.

Fresh milk was separated (CU. 0.1% or less nsidual fat) by centrifbging at 1,380xg (3,000

rpm) for 30 min at 4OC (J2-MC preparative centrifuge and JA-14 rotor, Beckrnan Instruments,

Inc., Palo Alto, CA, USA). Residual fat w u rcmoved by filtration on a Buchner funnel (hrough

Whatman 934-AH glas fibre filters (Fisher Laboratory Supplies). All milk samples and

derivatives were kept at 4'C until testing time to guarantee unifonn temperature histotory and used

within seven days. A limitcd number of samples h m skim milk reconstituted fiom powder to

9% total solids (RSM;see Chapter 6, Section 63.1) were measurcd.

4.2.2. HcPring k e d u r e

Pre-heating of whok milk at its original pH at W°C was c h c d out in 125- or 250-ml

Erlenmeyer flasks in a water bath with continuous stimng. Heating time was 1 min, not

including the time needed to reach the desind temperature (CU. 5 min). n i e milk was then cooled
 Frah whok cow i U k pH 6.7 + 0.02% w/v NaN,

eat (90°C x 1 min) then

cool to m m temperature

Centrifuge at 2S°C
(23,500g x 30 min
36,500g x 30 min)
Centrifige et 2S°C
i
(23,5008 x 30 min
36,5006 x 30 min)
i i
Ultra- Ultra- Ultra- Ultra-
filtrate filtrate filtrate filtrate

Collect last pellet MUF Acidic Collect lest pellet MF Acidic

of sedimented pHi.6.7 MUF of sedimented pH 6.6 MUF
small 'micelles' small 'micelles'

Re-suspend 'micelles' Re-suspend 'micelles'

(CU. 125 g.Lolof M U F (CU. 125 g.L-1 of MUF
at pH 4 6.7) then filter at pH a 6.6) then filter

4
Dilute in filtercd MUF Dilute in filtercd MUF
at diffcrent values of pH at differmt values o f pH

100pLin ForachpH' 100 CL in For each pH

2.5 mL MUF dilute serially 2.5 mL MUF dilute sdally
with or without then add ANS with or without then add ANS
+
rennet enzymes

Partick ahe Hydropbobkity

+ +
rennet enzymes

Pirtiek a b c
+
Hydrophobkity
m ~ u m ~mcuuremenb
~ b m-urrmentr me88oremenb
@CS) (d!uorimt~) @CS) (nuorimcty)

Figure 4.1. Schematic of sampk prcpamtion for particle size and hydmphobicity mcuurcmentsby
photon correlation spcctroscopy (PCS)and ANS-fluonmeûy, respcctively (sec text for deuils).
rapidly to 2S°C in crushed ice and used dircctly in subsquent preparative steps. AAcr thermal

treatment, the original pH of the mik (CU. 6.7-6.8) was reduced by ca. 0.1 unit of pH, as

expected, and left uncorrectecl. The dmp in pH can be attributcd mainly to shifts in the

equilibrium of milk salts induced by hcating (decrease in the solubility of Ca phosphate with

concomitant release of hydrogen ions).

4.2.3. Casein Micelles of Reduced S&e Po&d&pecsity

Casein micelles of reduced s i n polydispenity were prepared from whole non-pre-heated and

pre-heated milk at unadjusted pH by two successive centrifugation steps at 2S°C using a

Beckman L8-70M ultracentrifuge and a Ti-70rotor (Beckman Instruments, Inc.), an approach

pnviously descrikd by Dalgleish & Home [1985]. Aftcr the first step (a23,500xg. or 20,MH)

rpm for 30 min), the supernatant was carefully withdrawn with a syringe and then subjected to

further centrihigation (a 36,50Oxg, or 25,000 rpm for 30 min). The last gel-like pellet of

sedimented material (small fiaction of the micelle size distribution) was drained and

immediately redispened in milk ultrafiltrate (MW,pH 6.7; Section 4.2.5) to a concentration

of about 125 g of wet micellar casein per liter of MUF,that is, ca. five times the level of casein

in ftesh milk. (MUF was prcparrd fmm the same milk h m which the casein particles were

separated hm.) The suspension was filtered twice through cellulose nitrate membrane filters

(MiIlipore Canada Ltd., Mississauga, Ontario, Canada) of pore size 0.80- or 0.22-pn before use.

The use of caxin puticles of relativcly small o h (average hydrodynamic diuneter 90-

1 loi5 nm at pH amund 6.7 and 25%) was cxpccted to facilitate monitoring o f the renneting

r e ~ a i o nby PCS. Thcre is genenl agreement that the diffise sterk layer of K-casein

macropeptide around native casein micelles hm an approximately constant thickness (essentially

independent on the size of the particles, at least whcn probed by renneting at amund neutral pH

[reviewed by Dalgleish & Halktt, 19951). Relative decreiss in particle hydrodynmic diameter

in the emly stages of the mnneting pmcess should thcrcfom i n c m (i.e., k estimatcd more
easily) with decreasing the original size and nmwing the distribution of sizes of the particles.

The use of MUF (an alternative to dialysed whey or dialysed ultracentrifùgate Parker & Home,

19801) not only allows study of the micelles in an environment which closely tesembles the non-

micellar phase of fksh milk; it also is essential to try to maintain the structural integrity of the

casein particles for subsequent cxperiments.

4*2.4. Pre-~cidflcatIonof MUk

The pH of raw and pre-heated skim milk was adjusted just kfore use at room temperature in

the range 6.7-5.5 by drop-wise addition of IM-HCI and vigorous stining to minimize localized

coagulation. The pH was measured after ca. 15 min 'equilibtation' (Accumet pH-Meter 915,

Fisher Scientific). Near complcte equilibration of milk pH may require up to several days

because of the complicated (relaxation) phenornena at work and, therefore, it would have becn

impractical for the prernt study. Different pH series were prepiued independently from different

bulk milks.

4.2. S. Milk Ultruflltrate (MUm

Milk ultrafiltrate (UF-permeate) was prepared h m unheated and pre-heated skim milk at

room temperature using a benchtop Amicon TCF high-shear ultrafiltration module (Amicon

PM10 membrane (mol.

Canada Ltd., Oakville, Ontario, Canada) fitted with a nusable ~iaflo@

wt. cut-off 10,000 Da; Amicon Canada Ltd.). This liquid contains lactose, salts, and proteins or

polypeptides of low molecular weight, such as the proteose-peptones, and u n be regarded as the

continuous phase in which the casein micelles are stable in fmh milk, except for its low content

in whey proteins (little or no a-lactalbumin and ~lactoglobulin).To remove traces of UV-

absorbing material (for fluorescence analyses), new membranes wcrc soaked in 5% (wlv) NaCl

for 30 min and rinsed with distiltcd watcr prior to use. Their cleaning and stoting was according

to the instructions of the manufacturer. Residence time of the milk in the 500-ml re-circulating

unit did not excecd 30 min in average. The tint 5 mL of pmneate was systematically discarded.
 MUF of different pH was prepared from partly acidified milk, so that its ionic properties

closely reflect the changes in mineral salt equilibria (especially Ca phosphate) which occur upon

acidification of milk. This is essential to minimizo premature dissociation of the casein micelles

upon dilution at pH values lower than physiological values. The pH of UF-penneates was

checked upon pepmtion and their dynamic viscosity qo determined at the appropriate,

controlled temperature using a Cannon-Fenske size nolOO capillary viscometer (kinematic

viscosity range 2- 1O mm2.s-1, or CS; International Research Glassware, Kenilworth, NJ, USA).

The classical equation of Hagen-Poiseuille provides the basis for the determination of viscosity.

The time for MUF to flow through the clean and dry capillary was cstimated in the ordinary way

[e.g., R i n i & Mittal, 19921 using 10 mL of test fluid. The viscosity of MüF was daived by
comparing with the efflux time for distilled water (viscosity standard) at the measuring

temperature.

4.2.6 Renneting of Resûpcnded MiceIIes

The rennet used was a commercial, single strength solution of calf rennet (Le., a standard

concentration such that 200 mL is suflicient to set 1,000 kg of milk in 20-30 min a 30-32OC

[Hill, 19941) obtained from Christian Hansen's Lsboratory Ltd., Mississauga, Ontario, Canada.

This was diluted Ca. 1: 1000-1:10 in MUF at physiological pH (= 6.7) or in distilled water at tirne

of use.

All enzyme solutions werc filtercd (0.22-pm membrane filter; MiIliporc Canada Ltd.).

Appropriate amounts were added to diluted solutions of tesuspended casein micelles and the

renneting reaction monitomd by PCS (sec below). Suitable amounts of rennet were defined at

each pH so that the aggregaiion phase of renneting at 25OC occurred within approximately 30

min of addition of rennet enzymes.

4.2.7. Photom Cornfation Spcctmscopy

(a) JnstMnent Setun and Run Conditions. Average hydrodynamic diameters dh of diluted casein

particles were measured by PCS using a Malvem 4700 optical system attached to a 7032 digital

correlator (Malvem Inrniments, Inc., Southboro, MA, USA; Section 3.1). The software provided

by the manufacturer allowed calculation of particle average difision coenicients D from the

accumulated intensity autocomlation functions using conventional cumulanis analysis. Average

values of dh were derived fiom the values of D, assuming that the particles measured were

spherical and oôeyed the relation of Stokes-Einstein (Section 3.1 ., Equation 3.1). The same

experimental autocorrelation functions were used on occasion to detemiine the distributions of

particle sizes using a software developed by Hallett et al. [1989] as outlined by West [1996].

Measumncnts were made at a scattering angle of 90° at temperatures ranging fiom 10 to

25iO.S0C. MUF (filtcred twice through a 0.22-prn membrane prior to use to minirnize

contamination by dust particles) was used as a diluting medium. Initial measurements showed

that the viscosity qo of M U F remained constant within experimental e m r between pH 6.7 and

5.5 (no effect of pH and/or pre-heating of milk at the 0.05 level of statistical sipificance).

Experimental value for qo at 2S°C was 1.013I0.020 mPa.s (or cP; average of n = 5 independent

replicates); 1.488I0.018 mPa.s at 10°C (n = 5). The refractive index no used in al1 calculations

was 1.333, that is, the value for water at 2S°C.

Experimental settings were adjusted by running rries of dilutions of the casein particles and

conelator sample times. The latter wcre adjusted so that the exponentislly decaying intensity

comlation functions rcached a stable background level within the number of channels observed.

Typically, 100 pL of muspendcd micellcs (Section 42.3) was added to 2.5 mL of filtercd MUF

and d i s p e d by inverting the disposable cuvette.

(b) Data Acquisition and Treatmen~At 2S°C, comlator sample and experimental times were set

to 35 p and 60 s, respectively (60 ps and 120 s at 10°C). [The experimental time defines the

duration of an individual size experiment.] In the study on the eflect of acidifcation on micellar

diameter in the absence of rennet, repeated sets of 20 individual PCS runs, each lasting 60 s (or

120 s), were made for each sample of diluted casein particles, and the lest 15 measurements were

averaged. [Under the conditions of dilution, pH, and temperature investigated, particle size was

found to be stable over the time scale of the assays but systematically decreased by about 3 nm

initially. Presumably changes in equilibrium between the particles and their environment

occurred upon dilution and some relaxation time was required for their adjusting to the effects of

dilution.] Standard deviations (coefficients of variation) for each set of mns were CU. 3 nm (3%).

For a given sample, the estimated spread of results among the mean values of diameter fiom

different sets of satisfactory runs was consistently Iess chan k5 nm.

1.2.8. ANS-Fluorimetry

Hydrophobicity of micellar casein was estimated fluorometrically in triplicate using the

aromatic fluorescent p m k ANS (hemimagnesium salt; Sigma Chemical Co., St. Louis, MO,

USA) according to the original method of Kato & Nakai [1980] (Chapter 3, Section 3.2) after

modifications. Resuspended casein micelles (Section 4.2.3) were diluted serially with M W of

various pH to final concenüations in the range 0.005-0.1% (wlv) at pH 6.7, 6.3, and 6.1; and

0.001-0.04% at pH S.S. ANS (8.0 mM dissolvcd in 10 mM sodium phosphate buffer, pH 7.0)

was addcd (10 PL) to 2 mL of diluted particles after temperature equilibrium was reached.

Fluorescence intensity (FI) was mcasurcd at ambient temperature with a Shimadzu RF440

Recording Spectrofluorophotometer(Shimadzu Corp., Kyoto, Japan) at excitation and emission

wavelengths of 380 and 475 nm, respectively. Insrniment sensitivity was set to unity. Readings

of FI were consistently taken a few seconds a f k addition of ANS to minimizc potential

denaturing effects of the fluomphore. Quinine sulphate (10 phd in distilled water) was useâ as an
extemal standard. Absorbanct of the cascin particles-ANS complexes was checked at 380 nm

using a Shimadm W-Visible Spectrophotometer (Shimadm Corp.). Ovenll absorbance did not

exceed 0.05 absorôance unit over the ranges of concentrations of micellar cascin used in the

calculations o f hydrophobicity.

Net FI of each sample was obtained by subtracting the FI of the sarnpk without ANS

(intrinsic FI) from the FI of the sample with ANS (extrinsincFI).The initial slopes of the plots of

net FI vs. micelle concentration were calculated by linear regrcssion and used as indicators of the

global accessible hydrophobicity Ho of the casein particles.

Both fluorescence and light scattering measurements in this study require dilution of the

casein micelles by a factor 10 to 103, and so the question arises as to how stable (and stnicturally

sensitive) the particles are to dilutions of this magnitude. Turbidity was checked by visuel

inspection of the cuvette contents before and afier the experiments. No unusual variations in

absorbance (turbidity) or fluorescence, nor decays in light scattering intensity or changes in the

average particle diameter wcre detected over the time frame of typical experiments.

Reproducible and satisfactory linear relationships (comlation coefficients r 2 0.99) were

obtained between FI and concentration of micellar casein. Apart h m an expected development

of cloudiness upon rennct coagulation of the casein particles, the suspensions appared to bc

'stable' (not hazy) under the cumnt expcrimental conditions. Parker & Home [1980] showed

that dialyscd whey and dialyscd ultracentrifbgate h m skim milk, whope properties arc similar to

those of MUF, could k used satisfactorily to dilute casein micelles by factors of hundreds at 20-

2S°C.

I.2.9. Statbticd Ancilyses

Simple linear rcgmsion analyses of primary fluomscence measurcments were carricd out

with a Casio FX-850P calculator. Statistical analyses of the data for particle hydrodynamic

diametet and hydmphobicity (analyses of variance with appropriate F- and t-tests) were d e d
out using procedure 'general linear models' (GLM) of the SAS/Statm software package [SAS@

Institute, Inc., 19961 with pH of MUF and pre-hcat treatment of milk as quantitative and

qualitative explanatory variables respectively. (Similm results were obtained when pre-heat

m e n t was quantifid as 20 and 90°C.) The pH was treated as a continuous variable.

Procedure 'mixed models' of SAS@was used on occasions because it cm handle data generated

from several sources of variation (i.e., the so-called fued and rundom eflects) instead of just one

(the fixed effects) for procedure GLM [Matthes-Sem, pers. communication; SAS@ Institute.

Inc., 19881. nie variations contributed by random effects were of minor importance in this study

but they had to be accounted for.

Statistical cornparisons were made using weeks as blockp to remove a substantial part of the

important week-to-week variability nlated to the use of fiesh milk, and hence reduce

experimental e m r and incnue the precision for estimates of matment means and tests of

hypotheses Fines & Allen, 1992; Kuehl. 19941. In renneting experiments, the value of average

micellar hydrodynarnic diameter kfore the addition of rennet (i.e., dhb) was used as a covmiate

in addition to blocking. Measumnents for particle diameter in the absence of rennet were

transformed as logarithm of (dk80) in an effod to make the distribution for experimental data

approximately normal.

Unless otherwise specified in the dissertation. replication implies the independent repctition

of a basic expriment with the use of different puent milks. n i e results for measurements

repeatd on sub-samples (typiully dve times for determinations of puticle size) within a given

week were averaged and subsquently subjcctcd to statistical analysis.

4.3. Raults and Direussion for Photon Cornlition Spcetroecopy

The resuits fiom statistical analysis of the variations in average hydrodynamic diameter and

hydrophobicity of casein particles diluted in MUF obtained from unheated milk at different

values of pH between 6.7 and 5.5 at 25-20Tare summarized in Table 4.1.

Table 4.1. Resuits fiom the significance testinga of the effects of MUF pH (6.7-5.5), milk pre-
heat treatment (90°C- 1 min), and week on the average hydrodynamic diameter of casein particles
(dh)and overall decrease in particle hydrodynamic diameter upon nnneting (Mm at 25T?and
particle hydrophobicity (Ho) at 20°C. (Statistical analyses for dh were perfonned using procedure
mixed models of SAS@.)

Factors dh Adhr HO

PH **
Heat *e
Week **
dh be/oe renneting (dM ntb
PH* **
pHxheat e*
dh before r e n n e t i n g ~ i i nt
dh before rennetingxheat nt - nt
a, No significant effect: *, effect sipificant at 5%; ** effect significant at 1%.
b~ffectnot tested.

4.3.1. Apparent Hydro@namic dia me te^ of C d n Putticla Diluted In MUF ut Dl#erent

Vulues of pH

Average apparent hydrodynamic diameters dh of casein particles as a fiction of the pH of

MUF in which they were measured at 2S°C are show in Figure 4.2, panel (a). The nsuhs

displayed are h m experiments with thm different starting milks. with or without pre-heat

matment. The statistical rndcls derived to describe observations gathered over a 15-week

perid are illustrated in Figure 4.2, pancl (b). [The curves for the statistical models do not fit the

data points s h o w because these points correspond to 3 different milks while the models were

derived using 15 different milks.] Afier making allowance for wcck-to-week variability, it could

be confidently concluded that Iictween pH 6.7 and 5.5, the pH of MUF had a highly significant
 115 -
: a. Exprirntntal data
115

110 - 1 Pre-heated
A 1- 110
0 0 "
- (H) particles
10s - R A -- 105
A a .
t?
100-:O O A 1.
100

95 - m I Unheated - 95
A a A particie ci es :
90 -?a - 90

85 ,' Y 85
5.5 5.7 5.9 6.3
6.1 6.5 6.7
pH of MUF

- b. Experimentil data
: rad statiatical modeis

'

Figure 4.2. Apparent average hyddynarnic diameter dh of

c w i n puticles dilutcd in MUF at 2S°C as a fhction of the
pH of MUF. nie rcsuits arc shown for experiments carricd
out ushg 3 different fmh milkr (differcnt syrnôols). Casein
particles were isolatcd h m the original unheatcd milks (filled
symbols) and fiom thc same milks aftct heat trcatmcnt at
90°C for 1 min (open symbols). The curves in panel @)
correspond to the statistical models derivcd to fit experimental
data obtained using 15 differcnt h s h milks.
(p < 0.01) main e f k t on dK hydrodynamic size of casein particks isolated h m b t h unheated

and pn-heated milks. The cuwature in the plots of dh vs. MUF pH nflects the sipificant (p <

O.Oi) quadntic effect of pH (i.e., second-degm tenn in the predictive quations).

On acidification to pH 5.5, there was an overall apparent decrease in the average diameter of

casein particles isolated fiom unheated milk of the order of 10 nm (Mh* a 9.2îî.2 nm, Le., Ca.

9% reduction; n = 15 replicates). (A similar evolution of hydrodynamic diameter was found for

casein particles from skim milk reconstituted to 9% total solids but the changes in size of such

particles were not considered in any detail in the present work.] These observations corroborate

previous reports of the effect of partial acidification on particle size as estirnated by DLS

analyses of-in most cases-diluted skirn milk [e.g., Roefs cf al., 1985 (RSM,8OC); Home &

Davidson, 1986 (fiesh and reconstituted milk, 20°C); Roefs, 1986 (RSM, 15-2S°C); Vreeman et

al., 1989 ( k s h rnilk, 30°C); Banon & Hardy, 1992 (RSM,30 and 42OC); de h i f & Zhulina,

1996 (RSM,30°C)]. For particles isolated from pre-heated milk, the duction in diametcr Adha

was 7.2f1.4 nm (Le., Ca. 6.5% reduction; n = 8). The average hydrodynamic diarneter first

declined steadily over the pH interval 6.7 to Ca. 6.0 and then leveled OR, or slightly increased,

between pH 6.0 and 5.5. Total intensity of the scattered laser light hardly changed with time at

al1 the values of pH investigated (experimental data not shown), confinning that there was no

substantial disintegration of the diluted particles upon exposure to increasingly acidic conditions

at 2S°C.

(i) In the interpretation of what might happcn to the casein micelles on partial acidification,

one ought to bear in mind that the ionic strcngth is not constant but increws gndually with the

contributions of Ca and phosphate ions (nfer to Chapter 2, Section 2.1.3~). Gradual

solubilization of micellar Ca phosphate probably weakens the miccllar assembly, but at

tempcratum above about 2S°C, therc seems to k little pH-induced dissociation of caseins, even

at values of pH a which most of the colloidal Ca phosphate has k e n dissolved [e.g., Rose, 1968;
Dalgleish & Law, 19881. Interprctation of the nsults obtained fmn PCS is complicated by the

fact bat size memurcments are based on the hyddynamics (diffusion properties) of the

particles. Thus if swelling (increase in porosity) of the particles occurs, this may result in a

decrease of apparent Stokes diameter (i.e., underestimation of actual particle diameter), unless

the effects of swelling are compensated for by increased hydration and drainage, and

concomitant virous drag. Variations in porosity may also modiîy the light scattering properties

of the particies.

A further complication is that changes in the salt composition of the serum may alter micelle

size distribution and this may inteifere with estimation of average particle dimensions by PCS.

Intensity weighted size distributions occasionally obtained for unheated casein particles diluted

in MUF at neutral and more acidic pH at 2S°C are show in Figure 4.3. No conspicuous

modifications of the distributions wen apparent at the lowest values of pH investigated but this

does not necessarily imply that the relative proportions of small and large particles remained

unaffected by acidification. lntensity weighted distributions tend to be biased towards the large

particles and so it is possible that subtle modifications in the details of the distributions were

missed. The results obtained by Vmman et al. [1989] using a combination of light scattering

and electmn microscopie techniques suggest that there may be an incrase in the number of

largest casein particles at the expense of the smaller ones ('dispmportionation'?) on nducing the

pH of f m h skim milk h m 6.7 to 5.6 at 30°C.

(Li) To explain the apparent pH-dependence of micellar hydmdynamic diameter, the relative

importance of colloidal Ca phosphate and electrostatic interactions betwecn pos itively and

negatively charged residues of casein molecules at the different pH certainly has to be

consided. The dccrcasc in paflicle hyddynamic diameter obsmcd ktween pH 6.7 and 6.0

sccms to pamlkl the dccrrise in particle voluminosity rs cstimatcd by ultracentrifugation

(occasionally by viscomeüy) betmcn 20 and 40°C p8tOdo de la Fucnte & Alais, 1975; Drrling,
 Particle diameter (nm)

Figure 4.3. Intcnsity distribution of particle sizes for casein

particles isolatcd from unhcatcd frssh milk and dilutcd in MUF at
(panel a) and (panel b) at 25%. The rcsults are shown
for particles of relatively small si= isolated from a single
reprcscntative sample of milk. Vertical lines indicate the average
values of hyddynamic dimeter.
1982;Snoercn et al., 1984;Creamer, 1985;vrn Hoaydonk et al., 198éu; Visser et al.. 1986;

Hallstmm & Dejmek, 1988a,b;Vmman et al., 19891 and the decreasc in dynunic viscosity of

reconstituted skim milk as measured between 15 and 40°C [Banon & Hardy, 1991, 1992;Ould

Eleya et al., 1995;de Kruif, 19971 in this range of pH.

It may be reasoned that between pH 6.7 and about 6.0 at 2S°C, the swelling tendency caused

by increasing dissolution of micellar Ca phosphate is dominated by the increased attraction

arnong charged groups. The ionization state of the carboxyl groups (pK B 3.6-4.5) is not

expected to change substantially in this region of pH, unless microenvironments andlor milk

history result in shifting of the values of pK. It is uncertain what happens with the casein ester-

phosphate grwps (pK s 6.5). Some decrease in negative cliarge is expected, but if these groups

are buried within micellar Ca phosphate, as was suggestcd by Holt et al. [1982],then may

actually be an increase in ionization when they are f m d on solubilization of Ca phosphate. nie

number of positive charges likely increases due to the protonation of the histidine residues (pK a

6.5). so that the resulting electrostatic attraction with negative groups may cause some tightening

of particle structure.

Between about pH 6.0 and 5.5, no hirther change in particle hydrodynamic size seemed to

occur. T d o de la Fuente & Alais [1975],Snoercn et al. [1984],van Hooydonk et al. (198601,

Famelart et al*[1996],and Gastaldi et al. [1996,19971 reponed a slight increase in voluminosity

at around 20°C in this range of pH, with a small but clear maximum near pH 5.3-5.4,although

this point rcmains controversial (e.g., apparently conflicting evidence at 30°C in Darling [1982]

and Vreeman et al. [1989]).Vreeman et al. [1989]concluded that swclling of the micclles may

occur on reducing the pH of milk to 5.5, when most of the colloidal Ca phosphate is solubilized

1e.g.. Davies & White, 1960;Bru16 & Fauquant, 1981;Piem & BrulC, 198 1; van Hooydonk et

al., 19860; Dalgleish & Law, 19891. In this region of pH, amino groups are expected to be

almost fùlly charged and the net negative charge probebly diminiohes mainly because of the
protonation o f carboxyl groups, although there rnay be some replenishment because o f the

mlease of negatively chargai ester-phosphate gmups. Some expansion of the micelles rnay result

if the effects of casein demineralization prevail. Partial loosening of the puticks (possibly

contributed by some concomitant loss of protein material) would make them more fie-draining,

and this rnay explain why the hydmdynarnic diameter did not increase substantially below pH

6.0.

It i s noteworihy that the hydrodynamic size of casein particles seems to remain essentiaily

constant when important-dthough not alC-colloidal Ca phosphate is mnoved by chelating

agents at around neutral pH [Lin et al., 1972; Grifin et al., 19881. This rnay k taken as an

indication that putative loosening of the micellar structure on acid-induced dissolution of Ca

phosphate contributed relatively little to the decrease in hydrodynamic size Mp evidenced in

the prcsent study. Structural changes concomitant with increasing charge neutralization(titration

of the acid groups on the csseins and panllel increase in ionic strength) may be preponderant.

(iio The above Iine o f explanation does not distinguish baween the effects of solvent

conditions on the casein particles as a whole and on their surface. Interestingly, between pH 6.7

and 5.5, psrticle hydrodynamic diamcter decreased to about the same extent as it does when

casein micelles are treated with rcnnet at amund neutral pH, Le., about 10 nm [Walstra et al.,

198 1; Home, 198461. It is tempting to speculate that the thickness of the 'electrosterically'

stabilizing surface layer round the casein particles is progressively rcduced as the pH i s lowered

frnn around neutral to 5.5, with a concomitant decrease in particle stability. kcrease in the net

negative charge of surface c w i n molecules (putative polyelectrolyte hairs of u-çcwin

macmpeptide) with pH would docrew local clectmstatic repulsion betwecn the hairs, which

would make them more susceptible to folding (or rctmction) into a more compact (les draining)

structure. Changes o f puticle surface pmperties rnay aloo result fmm changes in the interior

(involving caseins other than u-eucin). It rnay k notcworthy h t early (indirect) investigations
of the confornation of non-micellar K-casein by ANS-fluorimetry (Clarke & Nakai. 19721

pointed to a most pronounced effect of pH between 7.0 and 6.5-6.0 (phosphate buffer. 20°C).

Fiuorescence intensity (an estimation of apparent protein hydrophobicity) increased continuously

over this range of pH and decreased slightly between pH 6.0 and 5.5 [Clarke & Nakai. 19721.

n i e curvilinear component in the evolution of expcrimental hydrodynamic diameter in the

region of pH above 5.8-6.0 may thus bc related to important reduction of viscous drag as the

difise (drained) surface Iayer becomes thinner (smoother), that is, less drained. The limiting

value of hydrodynamic diameter k l o w about pH 5.8 may be taken as an approximation of the

apparent diameter of particle 'corn'. Notice that a nonlinear component (apparently symmetrical

to that for hydrodynamic size) also seems to be present in the evolution of particle &-potential

(electmphoretic mobility) in the earlier stage of acidification above co. 5.7 between 20 and 40°C

[.hg & Dickson. 1979; Banon & Hardy, 1992). That the overall decrew in particle

hyddynamic size Ml,may be less on partial acidification than on renneting at standard pH may

have to do with particle surface Iayer remaining more drained afier conformational collapse than

after its (partial) removal. Acidic and enzymatic treatments may also probe somewhat difkent

characteristics of partick surface as mentioned in Chapter 2, Section 2.1.2b.

(iv) To try to establish further the influence of pH on the average conformation of the surfhce

layer, peripheral experiments (results not subjectcd to statistical analysis) were conducted in

which particle hydrodynamic diameters werc determined in MUF in the presence of ethanol and

cornparcd to the values of diasneters obtained in the absence of ethanol at a given pH and 2S°C

(Figure 4.4). (Paiticle diameten measured in ethanolic solutions were corrected for the incrcasc

in the viscosity of MUF with added ethanol. Thc concentrations of non-aqueous solvent were
adjusted at each pH so as to correspond to the conccntntions beyond which diluted casein

particles started to aggregate mwurably [sec Home & Davidsan. 19861.) At pH 6.7. unhcated

casein particles diluted in MUF with 25% vlv ethimol showed a reduction in dh of up to 20 nm.
 5.5 5.7 5.9 6.1 6.3 6.5 6.7
pH of (ethanolic) MUF

Figure 4.4. Apparent average hydrodynamic diameter

d,, of casein particles diluted in MUF at 2S°Ca (open
symbols) and (filled symbols) (EtOH)
added as a fiinction of the pH of M W . The results with
EtOH-fke MUF are shom for casein particles isolated
h m 3 different unheated fresh milks (different
syrnbls) as in Figure 4.2; results with ethanolic MUF
are shown for particles isolated h m a single sarnple of
unheated milk.
At pH 6.1 and 5.5 in MUF-ethanol (10 and l%, mspectivcly), puticle hyddynamic diarneter

decreased by about 5 and 2 nm, respectively. (Kecp in mind that average values of particle

dimeter canicd e m of about 3 nm. Notice also that the pH values just quotcd nfer to those of

MUF alone. Upon addition of ethanol, a weakly protic solvent, it was found that MUF pH

increased by ca. 0.1 unit of pH, presumably because ethanol reactivity resulted in a lowenng of

the activity of hydmnium ions. Given the relatively acidic conditions investigated, it is possible

that oxonium ions C2H5-0@H2formed.)

In principle, the conformation of the outer layer of ~ î a s e i ncan also be probed by studying

the effect of ethanol on the hydrodynamic size of the casein particles because ethanol is thought

to induce collapse (dehydration) of the hairy layer through lowering solvent quality Forne,

1984a, 19861. This is likely connected with the decreased electrostatic repulsive interactions

between the charges on surface protein molecules in the lower dielectric constant solvent, mainly

because of counter-ion binding (i.e., limited dissociation of pmtein salts). Thus if the surface

layer exists in a kss extended conformation klow about pH 6 . ( k n d there are no confounding

interactions k t w n n the effects of pH and cthanol combine&, then it would be expected that

the difference in particle diarneter ktween ethanol-expoxd particles and their unexposed

counterpuî decreases with decteasing the pH fiom 6.7 to 5.5. At first sight, the results obtained

in this work seem to concur with this expectation [also malogous rcsults of Home & Davidson,

1986 (fnsh and reconstituted skim milk dilutcd in a solution containing Ca, imidazole, and NaCl

at values of pH between 7.5 and 6.0 at ZODC)]. It should k realized, however, that this line of

investigation is not without limitations. Differcnt concentrations of ehanol must k used as it is

established that the stability of miccllar c w i n ta ethanol strongly dcpcnds on pH [Home&

Parker, 1980, 1981a], and so, direct cornpuirons cannot k ma&. Given the magnitude of the

decrease in hyddynamic diamder a pH 6.7 attributabk to the cffeets of ethanol. it appears

n e c e s q to conclude thut cthanol probably induccd unifom conttactiom throughout the casein
particles. It is difficult, therefore, to ascertain what proportion of the overall decreasc in particle

hydrodynamic size originates fiom reduction of the hydrodynamic thickness o f the surface

region.

(v) The reversibility of pH-induced variations in micellu diameter was also investigated in

marginal experiments. Casein particles fiom unheated milk were diluted in MUF at pH around

6.7 at the level of dilution required for PCS.the pH of the suspension was then gradually brought

to 5.5 at room temperature. and subsequentiy raised back to 6.7 (NaOH) within about 15 min.

PCS analyses were carried out at 2S°C at different stages of acidification-neutralization on

aliquots of the suspension. I n a similar expcrimenf diluted particles were subjected to a pH cycle

5.5 + 6.7 $ 5.5 starting fiom MWF prepared at pH 5.5. In some cases the particles became
unstablc and did not 'survive' cycling of the pH as evidenced by the development of conspicuous

cloudiness. Important reduction of particle stability may readily be explained considering the

conjugated destabilizing effects of dilution and acidic environment, and time of exposure to

thesc unfavourable conditions. (It is possible that after direct acidification of MUF only marginal

stability of the diluted casein particles could be reached.)

When the particles did not aggrcgate, the nduction in apparent hydrodynamic diameter on

partial acidification could be essentially reversed by raising the pH to its original value. For a

typical expriment in which particle average diameter was 107 nm originally, values of diameter

at pH 5.5 and at pH 6.7 afler partial acidification were 98 and 110 nm, respectively. Over the

interval of pH of interest at 2S°C, reversibility o f the variations in particle hydmdynamic size

would be expected if such variations are essentially nlated to conformational transitions o f the

surface laycr as mediated by changes in the patterns of clçtrostatic interactions between the

chargcd polyekctrolytc 'haia'. ûvenll (ionic) composition of the particles i s unlikely to be

recovcrcd on neutralization 1e.g.. Luccy et al., -1996) but colloidal fonns of casein rnay persist
whose basic (surface) organization is comparable to that of native casein particles. (For these

particles in our work, there were no estimations of sudace layer thickness using tennet.)

4.3.2. Apponnt Hydrodynmic Diamcter 01Caseh Putticla Isolatcd fron Prt-Heatcd MUk

and the met of Low pH

Casein particles isolatcd fiom fksh milk pre-heated at 90°C-1 min had signiticantly (p <

0.01) larger rnean hydrodynamic diameters than the particles from unheated milk over the

window of pH investigated at 2S°C (Figure 4.2). The difference between dh for unheated and

pre-heated particles was about 10 nm on average. There was evidence (p < 0.01) of an interaction

between the effects of pH and pre-heating, suggesting that the trend in the variation of apparent

particle diameter with MUF pH may be different (slightly divergent at values of pH below about

6.0) for unheated and hested particles. [Similar nsuits (p > 0.05) were obtained when the

diameten of untreated and hcat-trcated particles were rneasured in MUF prepared frorn pre-

heated milk (not shown), suggesting that the composition of the penneate did not change

appteciably upon pre-heating milk at 90°C for 1 min an& that the changes did not have

measurable effects on the characteristics under investigation.]

Pre-heat tmatment of milk at 90°C for 1 min extcnsively denatures milk serum pmteins and

initiates their (covalent) binding to (essentially) micellar K-casein [e.g., Jang & Swaisgood.

19901, hence, modiming the surfice of the casein particles since that is where most of the K-

casein is located. It secms simplistic, however, to ascribe the systematic diffmnce betwecn the

hydrodynamic diameters of unheated and prc-heatcd casein particles to the cornplexrition

ktwecn heat-denatured un proteins (espccially PLg) and micelle structure solely. Although

it seems rcasonabk to expect that incorporation of additional proteins within or ont0 the micellar

structure would rcsult in an increase of particle hyddynamic sim. this increase may be kyond

the sensitivity limits of DLS measurcmcnts. Givcn the cxperimcntal protocol followcd, it cannot

k nilui out that fiactions of miccllar crsein of different sizc wcrc separated by successive
ultracentrifugation dcpending on wheâhcr the milk had undergone themal pre-treatment.

[Notice, however, that paiticles measured using diluted pre-heated skim milk (Le., no

ultracentrifiigation step) also seemed to have higher average hydrodynamic size than the

particles in diluted unheated skim milk. (Not enough such measurements were conducted to

further the analyses.)] The observeci differcnce in particle hydrodynamic size may also have been

contributed by alterations of size distribution resulting fiom pre-heating. Lim ited (tem ponvy)

clustering of the heat-modified particles, as suggested by Jeumink [1992] and Jeumink & de

Kniif [1993] for systems subjected to a heat load comparable to the one investigated herein, rnay

have played some part.

Analyses of the changes in hydrodynarnic size in the present work have been complemented

by measurements of particle size in differently diluted pre-heated RSM at mund neutral pH

using fiber optic quasi-elastic light scattering (FOQELS), in conjunction with conventional

dynamic light scattering (PCS)[Dalgleish et al., to be published]. It is noteworthy that the results

of these investigations point to a slight reduction of the appmnt diameters of the casein particles

following pre-heating of milk at temperatures behueen 60 and 90°C for about 5 min. The

decrease seemed most pronounccd (about 10-15 nm) afler heat treatment at amund 7S°C.

(i) Experimental evidence in this work suggests that the overall decrew in hydrodynarnic

dimctet between pH 646.7 and 5.5 at 2S°C may be kss for casein particles isolatcd fiom rnilk

prc-heated at 90°C- 1 min than for particles h m unhcated rnilk (Mh* m 7.2kl.4 nm and 9 . W .2

nm, mpectively; sec concumng evidence under Section 4.3.3). A possible interprctation of these

rcsults is that the surface Iayer of pre-heated casein particles is inherently thinner and/or leu

readily collapsibk on charge neutralization than the stabilizing layer of unheated particles.

Reduction of particle size afier heating may contribute.

In line with the interprrtation of micelle surface structure proposcd by Home & Davidson

[198q (Chapter 2, Section 2.1.26), it may k envisagcd that the interactions between denatwed
whey proteins and u-caseiwssibly compiemented by interactions involving indigenous

and/or heat-precipitated Ca phosphate~ontributeto cross-linking, hence rigidification of the

inner regions of the suiface Iayer. These regions may thus become more integrated into the core

of the particles, leading to thinning of the surface layer frorn within. The putative strengthcning

and the bulk contributed by the whey pmteins may d u c e the susceptibility of the surface Iaycr

to collapse andor enhance its ability to drain solvent after (partial) collapse (andfor removal

upon enzymatic hydrolysis). (Some differential charge effects may also corne into play.)

However, if the thickness of the sulface Iayer (iather than its actual resistance) is the prime

determinant of particle intrinsic stability vis-à-vis aggregation [e.g., Home & Davidson, 19861,

then its putative thinning on pre-heating milk at 90°C-1 min may render the particles in so-

processed milk l e s ~stable overall (although not necessarily able of efficient-pennanent-

aggregation on, e.g., mnneting, in particular under conditions of pH around neutrality). This

would concur, for example, with the increased susceptibility of pre-heated milk to acid-induced

aggregation and gelation (Chapter 2, Section 2.2.6~).

(Ii) Cettainly, it remains unclear how thermal treatments of milk of high but not excessive

intensity (e.g., 80-90°C for 1-5 min) affect micellar (surface) structure. Then is consistent

evidence in the litenthire that casein particles can acquire micmscopically thick (= 20 nm) and

dense, inegular surfaces upon pre-heating milk beyond pasteurization [e.g., Davies et al.. 19781.

However, it seems that rather severe conditions (long times) of heating (e.g., 90-9S°C for 10-30

min or 121.foc- 15 min) are required to induce these extensive modifications of particle surface,

likely thmugh interactions with denaturcd polymeric whey proteins. In the study of Davies and

co-workers, no appmciablc change of the appcarance of micellar surface was evidenced for

particles h m milk pasteurizcâ at high temperatutc for short time (98°C for 0.5-1.87 min). The

results of measuremcnts of particle si= in (undilutmi) pre-heated RSM using FOQELS, in

conjunction with PCS [Dalgleish et al., to k published], suggest that the 'eiectrosteric' surface
layer of casein particles in milk pre-heated ktwcen 80 and 9S°C for 1 to 5 min is not thick (5 5

nm). This suggestion would be in keeping with the fact that PLg molecules adsorbed at oiVwater

interfaces seem to fom layers which are about 2 nm thick [e.g., Dalgleish & Leaver, 19911 and

that thete may k a limit to the binding of heat-denatured PLg (and a-La)to micellar casein

[Concdig. 1995; Comdig & Dalgleish, 1996; Oldfield et al., 19981.

4.3.3. Effect of Rennef Actium on Pattick Diameter ai Different Values of pH

in renneting experiments, the evolution of the hydroâynamic diameter dh of diluted casein

particles was monitored as a hinction of time afler adding rennet under different conditions of

MUF pH at (typically) 2S°C. Characteristic such plots are shown in Figures 4.5 and 4.6. It should

be noted that different concentrations of rennet enzymes were used to extend the let@ of the

renneting miction, particularly at acidic values of pH. Decreasing the arnount of rennet added at

low pH compnisated for the increased rate of enzyme activity. The rate of change of apparent

micellar diameter with renneting time was slow enough therefore, so that the values of minimum

diameter measured upon renneting wen assumed to be essentially unaffected by the time

constants chosen for data acquisition (Section 4.2.76). The downward trend in dh was fairly cleat

but accurate estimation of the overall decrease in diameter Mg was sometimes problematic. It

proved usehil in genenl to smooth experimmtal data by calculating a three-point moving

average thmugh the actual measurements so that the values of apparent minimum diameter could

be discemed more easily (Figure 4.7).

(0 AAer accounting for week-to-week variability, it could k concluded that betwcen pH 6.7
and 5.5 at 2S°C, MUF pH and pre-hcat trcatment of milk had highly significant (p c 0.01) main

effêcts on the extcnt of hydrodynamic diameter decrcasc upon renncting. Therc also was a highly

signifiant (p < 0.01) positive cncct of particle initial diameter dhb, with no sipifiant (p >

0.05) quadntic or interaction effects betwecn the variables investigatcd (Table 4.1). As

illustmted in Figure 4.8, plots of Mn vs. MUF pH for casein particles h m unhcated and pm-
 O
130 - .Addition of O
minet O
120 -
110 - - + Oo
a
o o ~ p o o ~
100:-
90 - w
L
[Rennet] =
1:10 dilution
1O pl of a

e
'*O-. .- 5
110 -.
+ ooeo'J
O
O

a --O
"

4C
100 -. [Remet] =
O ti
O
2.0plofa . . - 5
90
- 1
a
Y
1:100 dilution
80 0 -10

O 10 20 30 40 50 60 70
Time (min)
Figure 4.5. Apparent average hydrodynamic diametet dh of
@ma) casein particles isolated from a single sample of
fksh milk as a fùnction of timc &r adding iennct enzymes
under conditions pf pf at 2S°C (filled
symbols). Experimental data w m smoothed by crlculating a
three-point moving average thmgh the values of db. The
instantaneous rates of change in diameter with time, i.e., the
slope of the curvts of dhvs. time are plotted as open symbols.
Hydrodynamic diameter d,, (nm) Hydrodynamicdiameter d, (nm) Hydrdynamic diameter 4 (nm)

'
L
& , O u i - - t 3 8
O
0 - 0 C & , o uO t Ur
r Od I ' , O u i - e h )
O O u i 0
O
Rate of change in d,, (ndmin) Rate of change in d, (nrnlmin) Rate of change in 4 (mlmin)
 O 5 10 15 20 25 30 35 40 45
Time (min)

Figure 4.7. Apparent average hydrodynamic diamet

@ma) casein particles isolated h m a single sample of
unheated k s h milk as a hinction of time after adding m e t
enzymes at pH 6.7 and 2S°C (filled symbols). Unavcraged
primuy values of dh and the comsponding instantancous rates
of change with time (Le., the s l o p of the curves of dh vs.
time, AddAt; o p syrnbols) am plotted in panel (a). In panel
(b), experimental data wem smoothed by calculating a thme-
point moving average through the values of dk.
 12 - b. - Simple regression r

Linear
lines for unheated

-
~ d < ( a ) 5.49pH+û.1SdU-46.41

5.5 5.7 5.9 6.1 6.3 6.5 6.7

Figure 4.8. Overaii deerrrsc in hydrodynamic diameter Ml of

@ma) casein particles isolated h m (filled symbols) and
(open symbols) milks upon the action of nnnet as a

-
function of the pH of MUF in which the particles were dilutcd at
2S°C. In panel (a) the rcsults are shown for experiments carricd out
using 18 diflercnt h s h milks togethcr with the lines for simplr
~ ~ o f M ~ a g a i n s t p H o f M The-- UF.
denved to fit experimental data accounting for the effect of
particle diameter kforc renneting (dM) arc illustrattd in panel (b).
heated milks were approximately lin= over the pH interval of interest. Averages of al1

replicates are showed in Figure 4.8, together with the linear models derived to fit experimental

observations. [In the predictive equations for Mf, initial values of dh (ix., dhb) of 103.6 and

100.0 nm, and 1 13.4 and 1 10.0 nm were used for casein particles fiom unheated and pre-heated

milk respectively. For each type of milk, the higher values correspond to the values of dhb

predicted by the statistical models (Figure 4.2); the lower values approximate average

experimental values.]

The values of MH ranged from about 10I2 nm (n = 18) at pH 6.7, as anticipated, to less

than 2 e nm (n = 15) at pH 5.5 for casein particles from unheated milk at 2S°C. [Experiments

with particles h m RSM gave comparable results.] For particles isolated h m pre-heated milk,

the initial reduction in hydmdynamic diameter showed a similor dependence on the pH, but was

systematically smaller by about 3 nm than for unheated particles (Mf = 7.0î1 .O nm, n = 8 at

pH 6.7; and < 2.ûi1.0 nm, n = 7 at pH 5.5). With partictes from pre-heated milk, the

characteristic shape of the primary curve of dh vs. renneting time remained (Figure 4.6), albeit

over extended periods of time at pH values close to neuûal. This concurs with the generally

accepted view that interactions between heat-denatured serum proteins and micellar casein do

not render the sessile Phe-Met bond of K-cwin compktely inaccessible to rennet enzymes.

Available evidence (Chapter 2, Section 2.2.2c, and Chapter 5) suggests that the extent of

enzymatic hydrolysis of K-casein is moderately affected by pre-heating miik beyond

pasteurization. It smns masonable therefore to relate the telatively low valucs of Mfl for

particles from prc-heated milk to, mainly, modifications of the nature/ttiickness of the surface

layer rather than simply.io a lack of enzyme action.

A possible nason for the obsewed reduction in Mg with pH is that at acidic pH the extent

of the decrcase in particle diameter could not k fully appreciated because premature aggregation

of the particles compensated for the effect of macropeptide mnoval on the hydrodynamic
diameter in the early stages of the reaction. To examine this possibility, renneting experiments

wem carried out at temperatures k h m n 20 and 10°C. Lowering the temperature decreases the

rate of aggregation whilst still allowing enzymatic proteolysis to pmeed-albeit at a slower

rat-algleish, 19791, thus minimizing overlapping ôetwcen the two stages of the renneting

process. The variations of Ad# with MUF pH estimatcd at temperatures below 25OC for

particles fiom unheated and pre-heated milks (data not shown) were similar within experimental

error to the variations estimated at 2S°CI

(II) Essentially, the results h m renneting experiments substantiate the hypothesis of a

progressive reduction of the thickness of the surface layer of casein particles as the pH is

lowered in the range 6.7 to 5.5. This is likely accompanid by a (proportional) reduction of

particle intrinsic stability. Modification of the stabilizing eficiency of the surface layer on

(partial) acidification may be modulated by pre-heating milk at 90°C-1 min if, as experimental

findings suggest, the effective thickness of puticle suiface layer is rcduced on pre-heating.

It is noteworthy that tentative explmation of experimental data in light of the geometric

model proposed by Dalgleish & Holt [1988] for the renneting reaction was not hlly satisfying.

In this model, the casein particles are regardcd as king (prirnarily) sterically stabilized by the

hairy layer and interactions among the particles cm only occur when areas of their core surfaces

corne into direct contact via (hydrophobic) ban patches (gaps) denuded of hairs. This allows the

area of the gap in the sudacc layer to be defined in ternis of the diameter of micellar core and the

thickness of the stabilizing layer. Probability that gaps of the critical size arc produced can k

calculated and thus the aggrcgation khaviour consquent on rennet action can k defined. This

simple model is relevant to the aggrcgation of (partly) rcnneted micelles at the physiological pH

of milk. It is possible that the signifiant positive cffect of particle initial diameter on the extcnt

of diameter dccreasc upon renneting A d ' cvidenccd in this work hm to do with luge casein

particles aggregating at a later stage of hydrolysis comparcd to smallcr puticles. (This is

suggested by expcrimcntiil evidence palgkish et al.. 1981a.b; Brinkhuis & Payens, 19841 and

predicted by the geometric model. Depcndcnce of the thickness of the hairy layer on the size of

the particles seems a less likely explanation for the observecl influence of initial particle size.)

Apparently, refinements of the geometric model are required to account for the effects of

decreasing milk pH, which promotes aggngation at substantially lower extents of breakdown of

r-casein than those predicted'by the model at pH below physiological values.

4.4. Results and Discussion for ANSFluorimetry

4.4.1. Pre- Tests

The reference solution of quinine sulphate was intended to be used for correcting the

readings of fluorescence intensity (FI) for instrumental variability. In al1 pH series, the

calculated values of Ho for casein particlcs diluted in MUF at pH 5.5 wcre about twice the values

of Hoat pH 6.7, but the corrected values (obtained fiom adjusted FI readings) were less precise

than the uncorrected ones. Under the instrumental conditions used, the solution of quinine

sulphate gave FI readings that gradually decreased ovcr the period of study (CU. 45% decrease

over five weeks). Changes other than those directly related to inherent sample and instrumental

variability presumably came into play (e.g., changes in the fluorescence propertics of the

reference solution). In any case, the correction factors used did not seem fully appropriate, and

thus, only uncorrected readings were consideted.

(a) Background Fluoresccncg Ideally, the fluorescence of the diluting medium ought to be

negligible at the wavelength pair chosen. In the present study, a fairly high background

fluorescencc-probably arising h m 'non-observed' species present in MUF (e.g., riboflavin

and small peptidcs>-ium measud, thus mstricting the usabk range of the spectrofluorometer.

Figure 4.9 shows typical FIdata for casein particles dilutcd in MUF at around ncutnl and acidic

PH*
 150 - -
.Y-
A
: pHof-
FI = S63.7f[cascin]+49.34
= 6.7
cxtrinsic (with ANS) R' = 0.994 1
s 100 -

E:

I
net R~= 0.9928 - & =4749

-50 .' . . i5

" ao qo *Z sdg e; d
s b8 Qe S,
d
Concentration of rnicellar casein (%w/v) in MUF

pHof- FI==~~~s.~[cuc~~]+s~.cH
' cxeinsic (withANS) R
' = 0.9177

FI = 117.78[cascin]+75.38
intrinsic (without ANS) R2= 0.9202

net R' = 0.9927 9& = l a

=9 -50 ,'
O - w V L
$ "
ô 8
O
O O O
Concentrationof micelIar casein (%w/v) in MUF
Figure 4.9. (x), - f
(O) and pCt (e)
&&& pfa au<icies setially dilutcd in MUF at
a and at c a 2 0 ' ~ . The results arc show for casein
particles isolatcd b r n a single rcpresentative sample of
unhtated fiesh milk prt-treatcd with 0.02%w/v sodium azidc
(NiNi). The lines comspond to the lineu rcgrcssions of FI
against concentration of micellar casein; Ho (dope)
comsponds to the ovcnll apparent hydrophobicity of casein
particles.
 Background fluonscence (with and without ANS) at pH 5.5 was about 20% higher than at

pH 6.7, which pmbably reflects changes in the ionization state of some chromophores.

Regardless of the pH, background fluorescence accounted for about 90% and 50% of maximum

intrinsic and extrinsic fluorescence, mpectively. To some extent, however, these drawbscks

were balanced by the fact that the diluting medium used throughout the study was well suited for

preserving the structural integrity of the casein particles.

(b) Effect of Sodium Aside. Fluorescence is particularly sensitive to interferhg substances. To

check for potential effects of the azide anion in the systems under study, separate series of

measurements were carried out with and without preservative. Over the range of concentrations

of rnicellar casein investigated. intrinsic and extrinsic FI for NaNi-treated sarnples were

invuiably lower thm FI for their untreatcd counterparts, irrespective of the pH (Figure 4.10). FI

for a blank containing MUF plus ANS was also reduced in the presence of NaN3, suggesting that

NaN3 had a slight fluorescence quenching effect. The effect of NaN3 on the calculated values of

H ,was small, howcver. Since the main interest hem was the (combined) effects of partial

acidification and pre-heating of milk on rnicellar Ho, the absolute values of Ho were of minor

importance and so,NaNj was u d in aII subsequent fluorescence expiments for practical

purPo=*

(c) Sensitivitv of ANS Fluorescence to the Chemiul Environme@ For many fluorescent species,

changes in the chemical environment lead to apprcciable changes in fluorescence emission. In

the present study of casein particles dilutod in MUF, interpretation of the results may k

complicatcd by several factors, including the sensitivity of ANS fluorescence to pH and milk

salts, and the possibility of diffemntial binding mechanisms of ANS. The quantum yield of

fluorescence of ANS has bem documentai to be esscntially insensitive to variations in pH in the

range 2.0 to 8.0, and to the pmnicc of Ca2+ ions [Gibrat & Grignon, 19821.
 Net FI =

449.50[casein)-7.82with NaN,
a
45 1.98[caoein]- 1 1.17 without NaN3

Concentration of micellar casein (%w/v) in MUF

*
: pHof MUF
Extrinsic FI
.

Concentration of micellar casein (%w/v) in MUF

Figure 4.10. E f k t of sodium mede (NaN3) on the

fluorescence intensity FI o f casein particles scrially diluted in
MUF a flend at c a 20°c a (filled syrnbols) and
YYjthPLP (open syrnbols) NaN,. The results are show for
casein particles isolated h m a single representative sarnple
of unhcatd hsh milk. The lines correspond to the linear
regressions of FI against concentration of micellar usein.
The possible contribution of the negatively charged sulphonate moiety of ANS to its interacting

with protein molecules ought to be kept in mind. Electrovalent interactions may play a rok in the

fluorescence properties of ANS, which may interfere with the detemination of overall

hydrophobicity. Caution is thus required in linking ANS fluorescence data directly to changes in

pmtein accessible hydmphobicity consequent to conformational changes.

(d) Effect of Dilution Renne on the Estimation of A ~ ~ a r eHvdro~hobicity.

nt As hydrophobicity

was estimated over different ranges of concentrations of rnicellar casein depending on the pH, it

was necessary to check that the changes in Ho were not obscured by troublesome dilution effects.

Comparative experiments (results not shown) indicated that the level of dilution was not an

important confounding factor hem.

4.4*2*Appatent Hydropkobici@of C d n PariicIts DUwcd in MUF at DiHemnt Vdues o/pH

The values of Ho for casein particles diluted in MUF at CU. 20°C were significantly affected

(p < 0.01) by changes in the pH of MUF in the range 6.7-5.5; no other statistically significant

effect on Ho was evidenced (Tables 4.1 and 4.2). For both unheated and pre-heated casein

particles, Ho i n c r e d approximately linearly with decreasing the pH, as illustrated in Figure

4.1 1. At pH 5.5, the values of Ho were about twice the values at pH 6.7. Averages of thm
independent determinationsare plotted in Figure 4.1 1, along with the linear relation derived to fit

experirnental data.

The measurements of 'surface' hydrophobicity of micellu casein in iaw and pre-heated

(reconstitutcd) milks mpottcd by Lieske (19971 also point to an increase, alkit cleuly

sigrnoidal, between pH 7.0 and 5.8. (in the work of Lieske, hydrophobicity was determincd

through the specific binding of the non-ionic detergent Twccn 80 to hydrophobie areas of the

protein molecules at 20% according to the mcthod of Licske & Konnid [1994, 19951. Perhaps

the sigrnoid-like charactcr of the changes in hydrophobicity estimatd by Lieske [19971 indicates

that coaperative underlying pmcesscs uc involved.)

Figure 4.11. Overali apparent hydrophobicity of casein
particles isolated from (filled symbols) and plk
(90°C- 1 min; open symbols) k s h milks as a function
of the pH of MUF at ca. 20°C. The means of deteminations
carried out using 3 different fmsh milks are plotted together
with standard deviations (vertical bars) and regression lines
of against pH of MUF (see also Table 4.2).
Increase in particle hydmphobic chancter on lowenng the pH may be related to the decrease in

particle voluminosity (hydrophilicity) reported by various researchers (see Section 4.3.1).

(0 n i e observed variations in apparent hydrophobicity are likely related to the physico-

chemical changes in the casein micelles that arc biought about by exposurc to increasingly acidic

conditions. Approximately linear evolution of Ho with pH in the present study may have to do

with the fact that, unlike for hydrodynamic size and c-potential (Section 4.3.1). changes in the

hydrodynamic properties of the casein particles are not reflected in the values of Ho. Detaiied

interpretations of the variations in Ho are more conjectural.

Table 4.2. Overall apparent hydrophobicity (arbitrary intensity unitdpercent wlv of micellar
casein) of cusein particles isolated fiom unheated and pre-heated (90°C-1 min) h s h milks and
serially diluted in MUF at different values of pH at eu. 20°C.

Casein partictes pHof M F

From unheated mil& 975 î 126 660 k 77 620 k 84 486 î 48

From pre-heated mil& 988 i 204 776 f 187 623 î 80 508 î 100
*
aPlus-minus values are means of triplkate deteminations standard deviations.
b~aluesof Ho for casein particles from pre-heated milk do not differ significantly (p > 0.05)
fiom the values of Ho for particles h m unheated milk.

At least two factors arc expected to affect the binding of the fluorescent probe ANS to

micellar casein, viz., the size (and number) of the hydmphobic sites on the casein mokcules, and

the ionic and polar characteristics near the hydmphobic regions. Intemlated changes in the

geomeüic conformation and arrangement of the caseins, the patterns of dominant interactions

among thern, and their net charge and charge density arc thus likely to influence overall apparent

hydrophobicity as estirnateci in the present study. It may be envisaged that perturbations of

particle structural equilibria on putial acidification lead to i n c d exposure (henee

accessibility to ANS molecules) of prcviously buried hydrophobic (aromatic) side chains of the

casein molecules. Greater flcxibility of the casein matrix, such as would occur if the casein

particles relax or loosen on progressive solubilization of Ca phosphate, may contribute to

exposure of such less polar midues. Apparent hydrophobicity may also be enhanced owing to

nduction of particle net charge on acidification. The surface layer of w-casein, although very

much exposed, may have fcw sites for the effective binding of ANS kcause this ngion of the

particles is relatively hydrophilic and contains linle ammatic residues (Chapter 2, Figure 2.1).

Upon treatment with rennet enzymes, aggrcgation of pma casein particles ensues, nt least in

part, h m nonspecific interactions between newly exposed hydrophobic regions on the particles.

Whether it involves (partial) inside-out phenomena andor neuûalization of charges, increaxd

hydrophobic character of the casein particles at pH below physiological (in conjunction with the

effects of partly collapsed surface layer) would promote aggregation and re-arrangement of the

(coagulated) particles. Specific enzyme-substrate interactions during the pre-coagulation phase

are likely to be modified alsci.

4.4.3. Apparent Ilydmphobici@ of C w l n Parllrla fronr Re-Heated Milk and the E a c t of

Low pH

As mentioned in the prcccding section, the values of Ho at 2O0C for casein particles isolated

fiom milk pre-heated at 90°C for 1 min did not differ sipificantly (p > 0.05) fiom the values of

Ho for particles h m unhcated milk. An expianation for this observation is not easily at hand,

espccially if the measunments w m biased by changes in the particles introduced by

ultracentrihigal separation. Undcr the effeft of mlatively high centrihigation accelemtion, it is

possible that the particles undcrgo deformations (e.g., partial structural collapsc), so that the

effccts of hcat on Ho may no longer k seen a b r centrifugation.

Exposurc to high tempartwcs would be expcctcd to inducc an immediate increasc in

mwurable protein hydrophobicity because hydrophobic patches which are initially buried inside
the native fibuchire are likely to becorne more exposed (accessible) in the course of heating as a

result of protein unfolding morrissctt et ut., 1975; Nakai, 1983; Bonomi & Iametti. 19911.

Although this argument is pmbably more applicable to globular (Le., relatively rigid) protein

structures, it would concur with evidence that the micellar structure appears to loosen duting the

initial stages of hcating at above about 70°C [e.g., Rollema & Brinkhuis, 19891.

T h e n are several suggestions to explain reduction in accessible hydrophobicity following

thermal treatment (apart fiom possible wefactual effects ancilor lack of sensitivity of the

rnethod). Part of the heat-induced (confocmational) modifications of the casein particles may be

ternporary, i.e., reversible to some extent. Since the present measutements of FI were made on

the final pmducts after heat treatment, it is possible that the hypothesized initial increase in

hydrophobicity escliped investigation. Exposun to heat may ultimately Iead to (mainly

imvenible) reamngements of overall micellar structure (e.g., compaction [Dalgleish et al.. to

be published]) which occlude measurable hydmphobic domains. In light of the results of

Vouuinas et al. [1983a,b], Bonomi & Iametti [1991], Law [1996], Singh et al. [1996], and

Dalgleish et al. [to be published], it may k envisaged that global tightening of the casein

particles following (moderately high) pre-heating of milk arises from strcngthcning of intra-

particle hydmphobic interactions in which (partly) denatured whey proteins may participate.

Another possibility is that intrinsic properties of the PLg and a-La asscçiated with micellar

casein modulatcs the hydrophobicity characteristic measured, e.g., thmugh enhancing

hydrophilic pmpcrtics.

It is frrgucntly cnvisagcd that heat-induced association of whey proteins with the casein

particles incrcases the hydmphobicity of rnicellu surfre and mduccs the hydration barrier

against agpgation, thus favouring aggngation and gelation [e.g.. Heertje et al., 19851. This

point remains largely unclear, however. Part of the ambiguity hem arises h m the dificulty in

linking experimental estimations of 'hydrophobicity' to a-1 changes in the hydmphobic

130
propertics of spcîific regions of the casein particles. Mottar et al. [1989]reported values of

(aliphatic) "surface' hydrophobicity of micellar casein decra~ingwith increasing the intensity of

pn-heat treatment of milk, vis., direct UHT, indirect UHT, and 90°C-IOmin. Similuly, Lieske

[1997] measured markedly lower values of hydrophobicity for micellar casein in skim milk

reconstituted h m high-, medium- (and to a lesser cxtent, low-) heat powder than for micellar

casein in raw or pasteurized milk. This was particularly obvious in the range of pH between 6.7

and Ca. 6.0; klow pH 6.0, comparable values of micellar hydrophobicity were measured for

low- and high-heat systems, which were substantially higher than the values measured at around

neutral pH [Lieske, 19971. Decreased effective (accessible) hydrophobicity of the casein-whey

protein particles in mildly and highly pre-heated milk may contribute to rendering such particles

less able of efficient (rennet-induced) aggregation under conditions of around neutml pH. It may

be interesting to check whether the hydrophobicity of casein particles changes upon heating in

the absence of whey proteins.

4.5. Summary Discussion

Essentially, the results presented in this chapter substantiate the generally held view of

'electrosteric' destabilization of milk casein particles, with progressive reduction of the effective

thickness of particle surface layer upon exposure to increasingly acidic mlvent conditions.

Confonnational collapsing of the 'hairy' Iayer in the range of pH betwem 6.7 and ca. 6.0 (and to

a lesser extent, 6.0 and 5.5) would result mainly h m the cffects of gradually decrcrscd net

charge, Le., increased elcctrostatic attraction ktween suiniee (K-casein)molcculcs. (There may

al= be rearrangement of particle surface as a rcsult of changes in the interior.) In renneted milk

systems, such modifications of surface elcctrosteric pmprrties likely modulate the stability of the

casein particles to aggregation also thmugh modifling the eficiency of specific enzymatic
proteolysis. Apparent incrrw of the hydmphobic charactet of the particles when the pH

decrcascs, as cxperimental observations occmed to suggest, may potentiate the destabilizing

effects of progressive titration and physical collapse of the s u r f a Iayer.

The surface layer of wein particles isolated from milk pre-heated at 90T-1 min appeared to

ôe similarly affccted by partial acidification, but seemed inherently thinner and/or differently

susceptible to (pH-induced) structural changes than the surface layer of particles fiom unheated

miik. Hat-induced reduction of the effective thickness of the surface layer-possibly thmugh

interactions between dcnatuted whey pmteins and K-casein-would be expccted to have

important repcrcussions in ternis of overall stability of the casein particks. What specific

modifications to the physico-chernical characteristics of particle surface are brought about by

thermal processing of milk and in what way they affect aggregation bchaviour remain to be

clarified.

We note in passing that ambiguities do m a i n also about the extent to which mon global

changes of the cascin particles contribute to the loss of stability and cnwing aggregation,

especiaily below pH 5.5 at temperatures above 20-25T, and the extent to which such changes

may be modulatd by pis-hcat trcatment of milk. Some researchers have suggested that the

structural integrity of the particks is lost at or amund pH 5.0 [e.g., Heertje et al., 1985; Visser &

Smits, 19851. (At temperatures above 200C this is the pH valw ai/klow which casein particks

form a gel.) Othea have taken the view that basic structural features are largely maintained

below pH 5.5, the more so at temperatures above 20-2S°C [e.g., Lin et al., 1972; KalPb et al.,

1983; Griffin et al., 1988; Home & Davidson, 1993a; Mulvihill & Gnifferty, 1995; Holt &

Home, 1996; de Kruif, 19971. To what extent pH-induced rmdjustmmts of particle surface

d o r interior occur along the pathway to destabiliziaion and aggregation. with or without

nnncting, is still a matter for spcculations.

 S. QUANTIFICATION OF RENNET HYDROLYSIS OF K-CASEMIN
CHEMICALLY ACIDIFIED SKIM MILK BY SDSIPOLYACRYLAMIDE
GEL ELECTROPHORESIS

S.1. Outlook

In Chapters 6 and 7, we resorted to sodium dodecyl sulphate polyacrylamide gel

electrophoresis (SDS-PAGE), as an alternative to liquid chromatography, for quantifying the

extent of K-caseinhydrolysis in differently nnneted and bacteriologically acidified reconstituted

skim milk (RSM). (Sec Chapter 3, Section 3.3 for theoretical aspects about SDS-PAGE.) Prior to

geîting into that part of the nsearch, we had sought to adapt a suitable methodology and to

assess its usehilncss by experimenting with fnsh skim milk nnneted at constant pH in the range

6.7-5.5 at 2S°C. Pnvious investigations of rennet hydrolysis of K-casein using quantitative

polyacrylamide gel electrophoresis include those by Chaplin & Green [1980] and Carpentet

[1981] ( f ~ s hskim milk, pH 6.6, and 30°C) [also Basch et al., 19851. Conditions of

electrophoresis differcnt h m the oncs used by thesc authors were adoptcd in the work outlined

henin. Also, both unheated and pie-heated milks w e n testcd in an attempt to evidence

documented (combined) effects of acidic pH and pic-heat ûeaûncnt on the kinetics of K-casein

hydrolysis in milk (reviewed under Section 2.2.2 of Chapter 2).

5.2. Experimental Detiiis

S.2.l. Fresh Mük and me-Tnaîmen&

Procedures for the pttparation, prc-huting (watcr bath, W0C-1 min). and partial

acidification (HCI,m m temperature) of frcsh separateci milk were as descnkd under Chepter 4,

Section 4.2. The same commercial liquid preparation of rennet enzymes was used also.
5.2.2. Renndhg, SqlJng, and Pnpamtion of MUk

Pre-wanned (partly acidificâ) separatcd milk was divided into 10 aliquote of 5 mL each that

were placed in test tubes. The aliquots were al1 treated with the arnount of remet necessary to

give a clotting tirne (CT) of about 15 min at pH 5.5, ie., 30 pL of a 1:lO dilution. (In this

chapter CT was defined as the time between addition of rennet and the appearance of visible

dots in the milk, and estimated visually by slowly rotating the tubes by hand, checking for the

formation of aggregates in the film of milk flowing down the walls of the tubes.) Test tubes were

topped with araf film@ and subsequently immersed in a water bath at ZSf l°C,with intermittent

stirring.

At predetennined reaction times, the enzyrnatic reaction was ended by adding 150 pL 1 M-

NaOH. (The use of urea was avoided because the cyanate pment in urea solutions can convert

lysine residues to products of altered charge density and molecular weight [Stark et al., 19601.

Problems may arisc on electrophoresis, cspccially if the extent of modification varies h m

sarnple to sample.) A sampk containing no renne @action time = O) was treated similady and

used as a blank. To ensure termination of rennet hydrolysis, the samples were immediately

prepared (denatured) for electrophoresis as follow.

Aliquots (100 pi,) of the NaOH-treatcd reaction mixtures wen pipetted into small vials. To

these, 150 pi, of a protein-solubilizing buffa made of 10 mM Tris, 1 mM EDTA, and 20 m M

imidazole, pH 8.0; 250 pi, of a 2û% solution of SDS; 100 pL of 2-mercapt~cthanol;and 100 pL

of a 0.05% solution of bmmophcnol blue wcre added. Vials w m tmnsfmed to a M i n g water

bath with continuous stimng for 5 min. Aîtcr cooling to m m temperature, the samples wen

immediately uxd or storcâ a -lO°C until elcctrophoretic analysis. All samples were analyzcd

within t h m days following eiuymatic mction.

 Gel EIectmphomb, Stdning, Densirometrir Scanning, a ~ Quanti/icutio~
S2*3* d

Gel electrophoresis was perfonned using an automated PhastSystemm (Phumacia LU3

Ltd.. Baie dlUrfë, QuCbec, Canada). Aliquots (1 pi,) of the mdied samples were loaded in

duplicate (intemal duplication) ont0 pic-cast 20% homogeneous ~ h a s t ~ e l s(Phannacia

@ LKB
Ltd.), which were run according to the recommendations of the manufacturer [Phannacia LKB

Biotechnology, 19901 with one alteration. Where indicated, the electmphoretic nin was stoppcd

afker a separation tirne equivalent to 130 volthours (Vh) instead of 99 Vh (standard separation

time) in an attempt to optimize separation of the different protein fkactions. Eight samples were

nin on each gel, their ordering on the lanes of the gels king randomized to minimize possible

systematic error such as end-effccts.

The gels w m stained in a 30% (vlv) methanol-10% (vlv) acetic acid-water solution

containing O. 1% (wlv) PhastGel Bleu R (a Coomassie R.350 dye), and then washed in 30% (vfv)

methanol and 10% (vlv) acetic acid. In the last step of development, the gels were given a sodc

in a solution of glycerol to impmve thcit prcservation. They were then Ieft ovemight to dry.

Scanning of the gels was carricd out the next day at a wavelength of 633 nm using an Ultroscan

XL laser densitometer (Phannacia LKB Ltd.). The m a under intensity peaks was detemined by

integration using the pmprietary software Gel Scan pmvidcd with the densitometer.

Pmtein fractions wen identified by comparing the sepration profiles with those obtained by

Dalgleish & Shanna [1993] and Sharma & Dalgleish [1993]. The whey proteins a-lactaibumin

( a b ) and ~lactoglobulin(PLg) sewd as an intemal refercnce for guantifying the K- and

para-K-casein bands. Relative pmtein concentrations were obtained by dividing the (inof the

integnted intmsity peak to tbat comsponding to the sum of the perks for a-Laand PLg. The

milk proteins a-,P., @ma)Y-casein, and a-La and PLg have bcni rcported to have similet

Coornassie blue-binding characteristics [Capenter, 1981; Collin et al., 19911 and ro the

densitometric readings weis not comted. A mugh check of cxperimentally derived

concentrations-that is, a cornparison of the proportions of the major casein and serum pmteins

to average values given by Alais & Linden [1991+confinned that the quantification was not

importantly biased by a differentirl dye binding effcct.

S.2.4. Stafisticaf Anai)ses

Trials were replicated on three independent occasions using h s h milk collected on three

diffennt weeks. Results are reported as averages of al1 (i.e., independent and intemal) replicates.

Progress curves for the enzymatic conversion of K-casein were fitted into first-order equations

using a cornputer pmgram developed by Leatherbarrow (19921. Least-squares fitting with robust

weighting was used to find the best curve and derive first-order reaction rate constant k in each

case. Testing for statistical significance of the effects of milk pH, pre-heat matment at 900C-1

min, and week on k, visually estimated clotting time, and percentage of K-casein hydrolyzed at

CT was carried out using procedure GLM of SASTY[SAS@ Institute, Inc., 19961 with blocking

on weeks (Le., similar approach to that described under Chapter 4, Section 4.2.9).

5.3. Results and Discussion

5.3.1. Plc- Tests

Preliminary experiments confimicd that raising the pH was an effective method of ending

the enzymatic reaction [al= Chaplin & Green, 1980). However, it was necessary to ascertain that

alkaline samples could k h z e n and s t o d satisfactorily. To this end, identical samples were

subjected to electmphorcsis immediately after stopping the enzymatic reaction with NaOH and

after stonge for five days. In thcsc paired experiments, the relative amounts of K- and para-K-

casein varied by about 15% with no obvious increasc or decreasc trend. Since the variations werc

of the same magnitude as diffcrenccs k t m c n subsampks and betwm, indepndent data sets,

the procedure was decmod adquate.

 asp.P, and K-caseinshowcd as relatively distinct peako in the
The four main caseins aa-,

scanning patterns (not shown). The peak for pa-K-casein tended to overlap irnprtantly with

that for a-La, which certainly affected accuratc quantification of the pcak areas. Increasing the

separation time did not substantially improve the distance betwecn the bands.

Pre-heated separated milk gave similar sepsrstion patterns as unheated milk. One notable

consistent feature, however, was that the densitometric profiles at 'zero-tirne' (unrenneted milk)

showed a small peak at the migration distance of (supposedly) paru-K-casein, suggesting that

there may have ken some thenno-degradation of K-caseinafter heating milk at 90°C for 1 min.

(The identity of the materiai in the band was not confirmed in OUT work; this could have been

done using, e.g., capillary electrophoresis.) The results of Hindle & Wheelock [1970ab],

Marshall 119861, and van Hooydonk et al. [1987] suggest some hydrolysis of the Phe-Met bond

of K-casein by heat on seven heat treatment of milk (several minutes at temperatures above

lûû°C). Possible heat-induced degradation of r-casein efter milder heat treatment is less

documented, however.

5.3.2. Kinlrcs o/K-Casein Hydto(us& in Skim MN& Renneted at Difletent Vdues of pH

Results fmm the signifcance testing of the effects of milk pH, pre-heat treatment, and week

on charactcristic parameters of the nnneting pmcess at 2S°C an summarked in Table 5.1.

Unlikc in the study of particle size reportcd in Chapter 4, week-to-week variability did not have a

statistically sipificant effsct (p > 0.05) on the kinetic parameters estimated hercin. The time

course of the conversion of K-ewinat differcnt values of pH in the range 6.7-5.5 is illustrated in

Figures 5.1 to 5.3; Figure 5.4 and Table 5.2 summuize the main featutes for unheated (and pm-

heated) skim milk.

 [ p H of milk 1

O 420 40 60 80 100 120 140 160 180

Timc a f k the addition of minet enzymes (min)
Figure S.1. Disappearance of K-cwin (filled syrnbols) and
appcarance ofpma-K-casein(open symbols) as hnictions of time
afler the addition of n m e t enzymes diffaantconditionspf
a of unheatd skim m i k at 2S°C. The results arc show for
experiments carricd out using a single sunpk o f k h milk
remetcd with 0.006%v/v rcnnet. The curvcs ate first-ordcr fits
of cxperimental &ta, k concsponding to the first-oder rate
constant of remet hydrolysis; m w s indicate visual clotting time
(average values sbown in Table 5.2).
138
 para -wasein
~ ~ 4 . 4 75 3

K-cascin
~~4.9676

0.0 -
t p H of milk

O para -K-cascin

*
pH of milk

T i c afier the addition of m t enzymes (min)

Figure 5.2. Semi-loguithmic plots of the progrcss curvcs of

.
rennet hydmlysis of K-caseinshown in Figure 5.1 The Iines
correspond to the linear regressions of the natuml logarithm o f
the dative amount o f K-casein (or pu-K-casein) against
tirne of renneting; m w s indicate visual clotting time
(average values shown in Table 53).
 Timc aftcr the addition of icnnet enzymes (min)

Figire 3.3. Contrasted time-coursesof the

&(panel a) and of the pfm-r-ygCjp(panel
b) under diffetent conditions of pH of unhcatcd h s h skim
milk at 2S°C. The rcsults shown am the same as in Figure 5.1;
the curves an firstsrdcr fts of expenmental data.
8 iso

pH of tennetcd milk

Figure 5.4. First-oider D ~ L Pfm M k

visualmhaand%KIcaseinhvdrolntdataas
functions of the pH of rcnncted skim m i k a 25°C. nK means
of determinations carricd out using 3 differcnt h s h milks are
plottcd togetber with standard dcviations (vertical bars) and
regmsion lincs o f CT against pH of milk (sec also Tables 5.2
and 5.3). Filled and open symbols =fer to a and
(90°C- 1 min) milks, tcspectively.
141
Table 5.1. Rcsults h m the significancetestingr of the cffects of pH, pre-heat treatment (90°C-1
min), and weck on characteristic parameters of the mnneting process in fnsh skim milk [0.006%
(v/v) rennet, 2S°C]. k = first-order reaction rate constant; CT = visual clotting time.

Factors

pHxpHxhear - 1+ -
8,No significant effect; *, effect sipificant at 5%; **, effect significant at 1%.

Table 5.2. Effect of pH on some characteristic parametersa of the renneting process in unheated
fiesh skim milk [0.006% (vlv) rennet, 2S°C]. k = first-order reaction nite constant; CT = visual
clotting time.

PH kx 103 (sol) CT (min) % ~Xaseinhydmlyzed

at CT

5.5 1.10 i 0.13 9.67 î 1.70 *

44.0 4.32
aArithmetic means of thm independcnt nplicatcs f standard deviations.

(i) All primary curves of (pu) r-casein vs. timc were fitted into an intcgrated first-ordcr

quation. For both unheated and pre-heated milks, the firstsrder fit was satisfactory at al1 pH

values when enzymatic hydrolysis w u monitorcd thnnigh measuring the disappcarance of the

substrate K-casein. In this case, the calculatcd values of the reaction rate constant (&) obtained

h m the thm sets of experimcnts agieed well. M e n the progms of the mction was
detemincd by cstimating the relative amount of the product pu-K-casein f o d , the quality of

the first-order fit was less (e.g., Figum 5.2) d the experimental values of k were more variable
(data not shown). These variations may rsflcct, at least in part, the diniculties encountered in

quantifjringpara-K-caseinjust discussed.

The systematic decrame in pu-K-casein in the late stages of the enzymatic reaction

presumably arose h m sampling problems caused by the building of a finn gel: thorough

(manual) mixing after the addition of sodium hydroxide was pmbably not suficient to break the

clots forming the coagula, and this may have led to a decrease in the amount ofpmu-w-casein

pipetted and thus an artifactual, apparent decrease in pwa-~mcasein.For these reasons, reaction

constants reportcd herein are from measurerntnts of the remaining K-caseinonly.

The results of ptevious worken [e.g., van Hooydonk et al., 1984, 19866; de KNif et d,

1992; Lope2 et al., 19981 also suggcst that the kinetics of the proteolysis of K-casein in milk cm

be adequately descrikd by a first-order reaction between pH 6.7 and 5.5 and 20-30°C. In fact, to

describe the teaction which occufo in mik, it secms to be possible to use either a firstsrder

formulation or a Michaelis-Menten mechanism with a relatively high value of Km [Dalgleish,

19921. There remains the question as to whether the Michaelis-Menten mechanism is a correct

formulation to use in any case. By using this fonnulation, an implicit assumption is made that

both enzymes and substrate arc able to equilibrate at al1 time, and this carries the implication thrt

enzymes and substrate are mobile throughout the solution. The proteases, king relatively small

in cornparison with the casein micelles, am frrc to move through the solution (at kast at around

neutral pH),but this cannot be eqmlly truc of K-caseinmolecules which are immobilized within

large particks and therefore move slowly in compuison to the enzymes. A factor which

complicates the interpmtation of kinetic puuneters is the prescnce of two enzymes in m e t ,

viz., chymosin and pcpsin.

(8) The influence of pH on the fitst-order reaction rate constant, the clotting time, and the

percentage of ic-casein hydmlyzed at CT at 2S°C is illustrateci in Figure 5.4. Lowcring milk pH

resultcd in in i n c m in the rcaction rate constant, as expccted. The main (lineu) effect of pH
on kwas significant at 1%. In line with the observations of van Hooydonk et al. [19866] at 30°C,

a maximum rate of hydrolysis was found in the region of pH mund pH 6.0; diffemnces in k

betwecn pH 6.1 and 5.5 were relatively small (less than about IV?? change). The curvature in the

plots of k as a function of pH concurs with the significant (p < 0.05) quadratic effect of pH.

Significant (p < 0.01) Iinear and quadratic effects of pH also existed for CT, in line with the

observations of Noël et al. [1991] pertaining to the coagulation of bacteriologically acidified and

renneted reconstituted skim rnilk between 3 1 and 34°C. Perhaps these quadratic effects may be

seen as an indication of the leveling off of the effects of relatively acidic pH. It may be

noteworthy that a curvilinear component was also evidenced in the evolution of experimental

hydrodynamic diameter of casein particles over the same range of pH at 2S°C (Chapter 4,

Section 4.3), and that the curvilinear evolution of reaction rate constant with pH seemed to

mimr that of panicle hydrodynamic size (Figure 5.5).

There are a number of possible reasons for the discrepancy between the above observations

and values of pH optima for rennet action in rnodel substrate solutions (between pH 5.4 and 5.1)

reported in the literature [e.g., Garnier et al., 1968; Humrne, 1972; Visser et ai., 19801. It is

likely that both the accessibility of the active site in the substrate and the interactions between

chymosin and substrate are affected by pH in a way that dcpends on the state of the substrate. If

the promision of the macropcptide segments frnn micellar surface u-casein is decrcasd around

pH 6.0, as the resuits of particle size mcasurements in Chapter 4 suggest, accessibility of the

labile bond of u-casein may be increascd-hmce enzyme efficiency favoureâ-ôecause of

diminished steric hindrance caused by the glycomacropeptide. (It is also possible that

accessibility of the substnte is decrcased but that the effect is compaisated for by increascâ

intrinsic activity of rennet enzymes.) A direct effect of the increase in ionic strcngth resulting

h m acidification may ôe a shielding of charges, which may diminish the electrostatic repulsion
be-n enzymes and -in particles. There may even bs adsorption of chymosin ont0 the
 First-otder rate constant of
Hydrodynmic diameter d,, (nm)
nnnet hydrolysis k x ld (s")
micelle surfbce et the lowest values of pH investigated, as pointed out under Section 2.2.2b of

the litcranire rcview. Perhaps this contributes to reducing rcnneting effïciency through reducing

thc pool of fkc active enzymes.

In both unhcated and pre-heated milks, the extent of K-casein hydmlysis at the visually

estimated clotting time at 25°C varied h m about W h at the original pH of the milk to about

45% at pH 5.5. These values pmvidc an estimation of the extent of conversion required before

para casein particles can aggrcgatc. As anticipated, a considerable fraction of the usasein had to

be hydrolyzed by rennet kfore the casein particles fonned visible clots at pH 6.7; decreasing the

pH promoted the aggregation at significantly (p < 0.01) lower extents of break d o m of K-casein

(hcnce shorter timcs). Such effects are well established and likely contributed by the decrease of

particle intrinsic stability that in expected to msult h m (partial) conformational collape of the

(K-casein)molecules at their surface at values of pH klow physiological (Section 4.3). Details

of the rennet-induced destabilization of casein (pseudo) micelles at acidic values of pH still have

to be clarified (quantified).

5.3.3. Rinetics of K-Casein Hydmiysb In Pte-Heated SMni MU&and the ENect o f b w pH

Some characteristic properties of the rcnneting pmcess in pre-heated skim milk are shown in

Table 5.3 and Figure 5.4 ( a h Table 5.1). Pre-heating milk at 90°C for 1 min not only

sipificantly affect4 the rate of enzyrnatic hydmlysis (p < 0.01), but also substantially delayed

the process of aggregation at undjusted pH, as nflected by the substantially higher values of

visual CT (p < 0.01). The extent of K-casein hydmlysis at the onsct of coagulation was not

sipificantly affectcd by pm-hcating (p > 0.05). As expacd, lowering the pH to 6.1, 5.8, or 5.5

impmved the mnnetability of pn-heated milk in tenns of k and Cf.The fact that klow pH 6.0

CT was similar for pm-hcated and unhcatcd milks concws with the statistically signifiant (p <
0.01) interaction between the effects of pH and heat on CT. No such an interaction efVect was

apparent for the reaction rate constant.

Table 5.3. Effect of pH on some characteristic parametersa of the renneting process in skim milk
pre-heated at 90°C- 1 min [0.006% (vlv) remet, 2S°C]. k = first-order reaction rate constant; CT
= visual clotting time; U = value of parruneter measured for unheated k s h skim milk under
comparable conditions of renneting.

PH kxl03 (sol) [% of^] CT (min) [% of U] % K-casein [% of U]

hydrolyzed at CT

6.3 0.5 1 f 0.06 [ndc] 48.0 I4.5 1 [nd] 80.0 f 3.23 [nd]

5.5 0.75 I0.04 [63] 9.67f 1 .25 [100] 45.0 f 7.63 [IO21
aArithmetic means of three independent replicates î standard deviations.
bo/, of U at pH 6.7.
CNot detennined.

Results from the present investigation support the commonly held view (Section 2.2.2~)that

rennet hydrolysis of casei in is slowed down in milk pte-heated at temperatures-times

equivalent to 90°C-1 min, but that impaired coagulability of so-treated milk at unadjusted pH is

mainly due to the inability of the (adequately) renneted casein particles to aggrcgate effïciently.

Apparently, the interactions between dtnatured whey pmteins and micellar K-caseinfollowing

thermal treatment of relatively high intensity hinder only partly the susceptibilitylaccessibilityof

*-casein to enzymatic hydrolysis so that hydrolysis proceeds to comparable extents in unheated

and pre-heated milks. Below pH 6.0 in this wodc, the effccts of pre-heating on delaying

appreciable coagulation appeucd to k largcly rcversed (Le., values of Cf comparable to those

for unheated miü resulted), dcspitc the rate of enzymatic pmteolysis icmaining lower than for
unheated milk. This suggests that under acidic conditions the casein jwticles in pre-heated milk

rnay oggregatc more neadily than the puticles in unheatcd milk. This would concur .with the

increased suneptibility of highly pre-heated milk to acid coagulation (mview under Section

2.2.644 and experimentsl observations under Sections 4.3.2 and 7.2.3).

Certainly, therc are misons other than the dcncwd efficiency of K-cwin hydrolysis that

may contribute to the impaired coagulability of highly pre-heated milk renneted at unadjusted

pH. Rennet enzymes rnay spiit the K-casein but the ennet-convcrted pseudo-micelles rnay k

unable to aggregate becaux of the whey proteins associated with their surface. Alternatively,

pre-heating rnay result in important changes of overall miccllar structure (rg.,through altering

the distribution of colloidal Ca phosphate) which rnay mder even extensively renneted particles

unable to aggregate eflïciently. That some stnictural factors rnay be involved is suggested by the

fact that the adverse effects of pre-heating milk cm be reversed to a large extent by lowering

(either pennmently or tempotanly) the pH as reviewed under Section 2.2.2~.nie details and

repercussions of such putative re-structuring of the casein-whey protein particles remain to be

made explicit.

5.4. Conclusions on tbe Usefulntss of the Metbod

Overall, SDS-PAGE proved to k a usehl alternative technique ?O chromatographie and

fluorescence techniques for estimating the cxtent of K-casein hydrolysis in separated milk

renneted at 2S°C. The automated PhastSystemm procedure and the availability of ready-to-use

gels allowed relatively simple and npid analysis of both K- and pma-uskwin. Accurate relative

quantification of the prduct para-K--in by photomctric scanning of the staincd protein bands

following electmphoresis was hamperd by the limitcd resolution betwecn this protein and a-La.

Quantitative analysis of K-cwin was found to give more diable msults piovided the
delimitating and adjusting of the boundaries of intensity p e h kfom integration was also

carried out in a consistent way.

SDS-PAGE was practical, if not for detaikd kinctic analyses of the primary phase of
rennetin-e rnsitivity was not sufficient to study the reoaion during its early stage*, at

le- for visualizing the pmgress of the enzymatic reaction and estimating characteristic kinetic

parameters. Attempted quantification of the effects of acidic pH andlor pre-heating on the

kinetics of rennet action at 25°C agreed well with established facts. This gave confidence that

SDS-PAGE could also be used sitisfactorily for complementing the rheological analyses of
combined remet and acid coagulation rcported in Chapters 6 and 7, despite differences in the

type of milk, acidification (and temperature) conditions. [Note that additional (unidentified)

peaks tended to be present in the scmning patterns for bacteriologically acidified and renneted

RSM. These p e h (prcsumably comsponding to microbial metabolites) werc not taken into
account because they did not appcar to hamper relative estimation of @ma) K-casein.]

Certainly, relatively important nplication was required to attain workable precision. [Con

factors, and in particulu the cost of consumablcs (PhastGels@), would have to be taken into

account if the procedure werc to be considered for routine analyses.] A convenient way to

increase the number of replication and keep experimental variability within acceptable limits was

ta combine cornpletc (independent) replication of the trials with partial or intemal duplication

(i.e., sub-sampling) within trials. To echieve satisfactory prccision in the ekctrophorctic analyses

reponed in Chapters 6 and 7 (only two cornplete rcpctitions of the basic trials), it was decided to

duplicate sampling et the different maction times in addition to subjccting each sample twice to

electrophoresis as dcscrikd in this chapter. Alro, to minimize potential problems related to

mixed using a vortex

sampling afier the addition of NaOH, the (gelkd) samples wem c ~ f u l l y

kfore sampling and pmparing fot elctrophomsis.

 Liquid chmmatography usually is a method of choice (sensitive and rcadily automated) for

quantiwing the action of remet in milk [e.g., van Hooydonk & Olieman. 1982; Shanna, 1992;

Hyldig, 1993 (sirc-exclusion highgerfomance liquid chmmatography of the glyco-

macropeptide); Léonil & MollC, 1991 (cation cxchange fist protein liquid chromatography of the

glycomacropeptide); Dalgleish, 1986; Davies & Law, 1987 (anion exchange FPLC of K-casein);

overview in Strange et al., 19921. Originally it was pluined to implement the FPLC method
described by Dalgleish [1986] and preliminiuy experiments were conducted using sampks

prepmd from 9% RSM, ficeze-dried caseinate, and U-casein. with and without renneting at

amund neutral pH. Sarnple pre-treatment, and equipment and procedure for FPLC were

essentially as described by Dalgleish but difficulties were encountered in obtiiining satisfactory

(consistent) separation profiles. Attempts at nfining the chromatographie procedure were

discontinued because of time constraints and it was decided to rely on SDS-PAGEinstead.

 6. SMALL STRAIN DYNAMK RHEOLOGICAL ANALYSES OF GEL
DEVELOPMENT FROM CULTüRED AND RENNETED MILK
1. Prrctical Aspects

To the Mellco~ofSumiu A. Kkulil

The main intetest in Chapten 6 and 7 is on the process of combined enzyrnatic and lactic
acid coagulation of milk in dynarnic envimnments of pH. In Chapter 6, considentions are given

ta practical aspects of experimentation; tesults of experimcnts proper are discussed and

tentatively interpreted in Chapter 7. To facilitate reading, only key illustrations arc included with

these two chaptets. Deteilcd graphic nfemces are appended as a separate volume and will k

refend ta specifically herein using the prefix 'A' (as in Appertdix).

61.1. ~ e r i m e n t aPlan,
î aud Refennce Systemc and Cond&ios

Figure 6.1 provides a synopsis of gelling systems and gelation conditions investigated.

Expcriments were conducted along the lines of hctional design, with a broad range of

concentrations of cicidiQing starter cultures (Ch j = l * 8) vs. rcnnet enzymes ( R x j , ,1, ,+.,
16,

ofariodly 64 md la)coveisd initially to get a feel for the gelation khaviour of the samples,
as analyzcd mainly by dynamic rhcometry. Measurements of viscoclasticity werc carricd out

primarily with the Namctrc rhcometec wcondajy measurcmcnts w m perfonned with the Carri-

Med thcorneter (Chapter 3, Section 3.4 for theoteticai considerations).Gels were fonned within

the rhcometer systm starting from standard tcconstiaited skim milk under standard conditions

of pH at renneting (pH 6.4) and temperature (40°C), with no calcium chloride (CaC12) addcd.

Control experiments wcrc carricd out on milk coaplated exclusively by remet (mnnet contiols)

or by bactcriological acidification (lactic acid controls).

 occasionally fresh (pasteurized/homogenized) whole milk
- Preheating at QOCfor 1 min
occasionally 62C for 30 min and 11% for 10 min
- Proconcentration by ultrafiltration to 1x to 4x by volume
with or without (pre) heating et QOCfor 1 min

C
O
- Verious additions to milk (CaC12 or NaCI)
- pH at renneting adjusted to 6.4,6.0, or 5.8
- pH cycling 6.7 -> 5.8 -> 6.4 (direct or overnight)
- Temperature of gelation 40C, 30C, 25C, or 20C
C

CO R x ~ Rx8 Rxl6 , (Rx64) (Rxl60)

RO
I I I I I I
1
I

" Cheddarca.che
1 I I

30F
e
lncreaeing concentration of rennet -rn

Figure 6.1. Synopsis of gelling systems and gelation conditions

for srnell straln dynamic rhedogical testing.
 In cornparison, typical conditions for coagulating pasteur ized milk for Cheddar cheese at

amund 30°C comspond to about experimental levels Cf4 (1% v/v lactic starter, typically

Streptococcus lactis ancUor crernoris),and ktween Rx64 and Rx 160 (Rx86r 0.02% vfv single

strength rennet). Standard conditions used in cottage checse-making are near C/1 (5% mixed

culture) at 32OC for the widely uscd short-set method and about C/8-C/4 (0.5- 1%) at 2S°C for the

long-set method, and Rx 1 (2.2~104%single strength rennet) or less. For standardization of the

renneting process, values of p H at nnneting around 6.4 (mainly soft cheeses) and 6.6 (mainly

hard cheeses) are common. The fermentation of pre-heateâ milk to yoghurt products in the

temperature range 304S0C (preferentially 42-43OC) is commonly canied out with levels of

culture organisms between Cl8 and C/ 1 ( 1-5% yoghurt-related starters, typically Streptococcur

salivarius subsp. thermophiius and Lactobucillus delbrueckii subsp. bulgmicur) [Tamime &

Robinson, 1988; Hill, 1994, 199SaJ.

Later studies w m conducted at selected concentrations of culture and rcnnet to by to define

the efiects of various pre-treatments of milk, most importantly heating md incrcasing protcin

concentration, on the pmgress of gel developmmt in cultured and renneted milk.

6.2. Experimeatal Detaib

62.I. Mil&Sanpes and R e - l k e a t ~ ~ ~ n t s

Standard monstihitcd skim milk (MM) was prcparcd fiom a commercial low-heat skim

milk powder free of antibiotics supplkd by Ault Foods Ltd. (Mitchell, Ontario, Canada). Two

batches of powder were used for all the assays. The powder contained 97% total solids and had

an undenatured whey protein nitmgen index (WPNI) above 6.0 mg.gI. For this type of powder,

the milk is usually heated a 63°C for 30 min befon evaporation/spray drying. Typical

approximate composition (in wt. %) is:

 Total soli&
Tme protein
Casein
Se- protein
Non-protein nitmgen (NPw
Fut

The milk was reconstituted to 9% (i.e., total solids content similar to that in h s h skim milk)

using partially deminenlized tap water. The solutions were stimd at mom temperature for about

30 min and stored ovemight (15-20 h) at 4OC to rllow dispersion of the powder and some

revenible changes which occur during drying (e.g., distribution of salts among c w i n particles

and serum. and size and structure of the particles) to be reversed. Typical protein and lactose

analyses of RSM by infra-red (IR) specbophotometry gave 3.2 and 4.8% (W. basis),

respectively. Zoon et al. [1988a] commented on the importance of reconstituting m i k in a

standard way in particular to minimize variations in renneting behaviour brought about by

diffennces in temperature history. Temperature history con also influence the fermentation of

lactic acid bacteria (LAB), e.g., by rnodifjhg the arnount of diswlved oxygen in the milk

[Driessen & Puhan, 19881.

Fresh whole milk was obtained fiom the dairy herd of Holstein cows of the university and

used within a week. Separation into skim milk and cream by centrihipion was as described in

Section 4.2.1. Homogenization of pasteurized whole milk was pcrforrned in a two-stage process

using a standard valve 'Golden' (Manton-Gaulin, Everctt, MA, USA) homogenizer operating at

pressures of about 2413.4 MPa (3,5001500 psi) and about 40T.Commercial homogenized milk

(3.5% fat, 'pure filtered') was also purchascd at a local store. For this type of p d u c t , milk fat is

generally homogenized at rclatively high pressure in two stages also.

The use of sodium aide (NaNj; 0.02% w/v) to delay undesircd microbial growth was

rcstricted to milk samplcs intcnded for coagulation by remet in the absence of starter cultures.

NaN3 was added to prc-heated m p l w <rperthermal matment (Section 62.2).

 All milko were tcmpered at the temperature of coagulation (20-40°C) for ca. 30 min with

modente stimng before culhving and/or renneting. Occasional additions (CaC12, 0.02% wlw

ulculated as anhydrous = 1.8 mM; NaCI, 0.6% wlv 100 mM) wem just kforc inaculating,

adjusting the pH 16 6.4, and renneting (Sections 6.2.5 and 6.2.6).

62.2. Heating R u c e d ~ m

Milk was pre-wanned to room temperature for 15-30 min before heating (that is, for RSM,

heat treatment Mer reconstitution). The conditions for heating were as outlined in Table 6.1. A

level of whey protein denaturation of 7O-95% is usually considered beneficial fiom technological

and nutritional viewpoints. This likely comsponded to experimental thermal treatment of milk

of standard concentration at 90°C for 1 min in the laboratory (defined as standard heat treatment

in this study). Unless othewise noted hcrcafter, 'heated milk' will nfer to this treatment.

Tabk 6.1. Experimental conditions for heat treatment of milk and approximate extent of
denaturation of whev ~roteins.

Heat treatment Temperature/tim@ Approximate % denaturation

combination of whey protein&

Pasteurkation (;butchheating) 62OC for 30 min < 10%

Laborutory heating (Section 4.2.2) 90°C fot 1 min 7Ow90%
Sterilkation in autoclave 115°C for 10 min > 95%

aHolding time a the desircd temperature, Le., not including pre-hcating tirne.
b w i c & Kurmann, 1978; Dannenbcrg & Kesslw, 1988a-c; Femn-Baumy et d.,19911.

After heating, the milk WLP brought directly to the temperature of coagulation. Except for

sterilizcd milk (which was storcd for wvcral days at ambient temperature), and for concentnted

and chemically acidified milks (kept ovemight at 4T), analyses on heated milk were al1 begun

within 30-60 min of heat treatment.

 Protein concentration was varied by ultrafiltration (UF) of RSM (unheated and heated) at

about 40°C using a spiral-wound membrane cartridge (Amiconm. model S 1Y 10; Amicon

Canada Ltd., Oakville, Ontario, Canada). A relatively high feed temperature was used to improve

UF peifonnance. The low-adsorptive, cellulose-based membrane had the following

characteristics [Amicon, Inc., 1995):

Membrane moi. wt. c u t d = 10,000 Da

Total membrane area * 0.09 rn2
Fill volume of ccpnidge and headers J 60 mL
Maximum inlet and drop pressures a 0.4J0.03 MPa (60/5 psi)

The 9.1 x 23.8 cm cartridge was used with a variable-speed peristaltic pump and appropriate
tubing. lt was opcrated, cleaned in place, and stored according to the recommendations of the

manufacturer [Amicon, Inc., 19953. System schematic is shown in Figure 6.2. Milk (2 L) was

pumped from a tempered ùeaker and directed through the feed channels behueen the layen of the

membrane. As it passed over the surface of the membrane, back pressure forced material of low

molecular weight through the membrane layers to a collection tube at the center of the cartridge.

This material (milk ultrafiltrate or permeatc) then exited the cartridge h m a port on the inlet

header. Species with molecular weight above the cutoff of the membrane (proteins and protein-

bound salts) were selectively ntaincd and dimted back to the beaker. concentration of these

materials incresxd as the operation continued.

Figure. 6.2. Schematic of u l ~ l t t a t i o nsyaem with the mic con@ spiral-wound membrane
caddge SIYIO.

Duhg concentration, the rcduction in volume causa a gradua1 increase in milk viscosity.

To compensate for this, the spced of the pump (i.e., the recirculation rate) was d u c e d to

mainiain appmxirnatcly constant inla pressure and to avoid cxcecding the maximum diffemntial

pressure of the cartridge. By adjusting the speed of the pump togahet widi the sctting of the

back-pmsw valve, propcr vclocity of mik tbrough the membrane w u maintaincd throughout

the m.Adequate fluid vclocity is essential to minirnizc concentrationpola7uationandfiuling

(i.e., the accumulation of retained macrosolutcs at the surface of the membrane), two relateci
phenornena which can duce membrane flux (i.e., rate of ultnfilûation) and I
d to the rctention

of nonnally permeating solutes. To takc îhe limiting characteristics of the mirculating pump

into account, operating pressure was kept klow 0.1 MPa (20 psi). Residencc time of mik in the

concenttatot did not e x c d two hours.

 Volumetric concentration factor (VCF = volume of rnilk initially 1 volume of ntentatc) for

miik concentrateci dinctly (standard concentration procedure) ranged h m about 1 to 4, with

concentration factor 1 ( l x ) refemng to non-prc-concentratcd RSM (control). To check for the

possible effect of changes in ionic balance on concmtration, a limited numkr of samples were

pnpared by concentrating unheatcd RSM to about 4x and diluting back to 2 x or 3x the original

concentration by adding the penneate obtained during ultrafilbation. A few samples were also

preparcd by heating concentrated milk (2x and 3x retcntatcs) Mer concentration and cold

stonge ovemight.

Milk concentrates were cooled and storcd ovemight at 4OC before further treabnent/analysis.

Protein, fat, and lactose contents (Table 6.2) were estimated by i n h d spectrophotometry using

a Dairy Lab 2 IR analyser (Multispec, UK). The instrument was setup for quality control of dairy

prducts such as cottage chccse at the Guelph Central Milk Testing Laboratory. For protein

analyses, the instrument was calibnted using the Kjcldahl method (Nx6.38). The approximately

linear relationship ktwccn protein concentration factor (or PCF, i.e., the extcnt to which the

protein was concentrated as compared to unconcentnted nconstituted skim milk) and VCF for

standard unheated and hcatcd UF retmtatcs is illusbated in Figure A6.2. Little information was

available on the analytical pciformancc of the IR rnethod. Repeatability (standard deviation)

a p p c d to k satisfactory, but the merpurcd PCF were substantially lower than expected, the

more so the higher the concentration factor. Prcsumably, chcfking out the Dairy Lab by diluting

UF retentates with pcnneatc andlor specific calibration of the spectrophotometer for retentates
would have bcen necessary to cnsurc accurate measunmcnts.
Table 6.2. ~ v c r a ~ &composition
b (in wt. %) of ultrafiltration retentates prepared fiom skim
milk reconstituted to 9%.

Volumetric concentration factor Truc protein Lactose

Concentrates fiom unheated skim milk

I x unconcentrated control 3.19 f 0.07(4) 4.78 f 0.06 (4)
2x 4.92 f 0.02 (2) 4.59 < 0.01 (2)
2x (dituted back) 5.03 f 0.01 (2) 4.61 < 0.01 (2)
3x 6.87f 0.25 (5) 4.59 f 0.02 (5)
3 x (diluted bac&) 6.96 f 0.02 (2) *
4.6; 0.01 (2)
4x 8.12 IO21 (2) 4.32 f O.19 (2)

Concentrates fiom heated (90°C-1 min) skim milk

1x unconcentmted control 3.28 f 0.03 (4) 4.87 f 0.10 (4)
2x 5.06 î 0.17 (2) 4.57 f 0.07 (2)
2x fieated ujser concentration) 5.33 f 0.01 (2) 4.62 & 0.01 (2)
3x 6.91 i 0.30 (5) 4.58 î 0.13 (5)
3 x (heated after concenirution) 7.62 f 0.03 (2) 4.63 < 0.01 (2)
4x 9.07î 0.50 (3) 4.57k 0.04 (3)

*
aResults are given as arithmetic mcan standard deviation (number of independent replications).
hrace amounts of milk fat arc not reported.

For tentative estimations of elastic and viscous moduli h m consistency measurements with

the Narnetrc rhcometer (Section 6.2.8), we assumed similar values of density @) for unheated

and heated retentates as estimated by picnometry by S h m r [1992] at mund neuapl pH and

30T, VU., 1.035 (VCF lx), 1.044(2x), 1.058 (3x) g.mL-1.

tL2.4. Lactic Ac# Bacteda (W)

and Ropagation CondUons

The starter culture consisted of a 1:3 (vlv) mixture containing (0 a non-ropy single strain
mesophil ic starter of Lactococctls 1afi.ssubsp. factis (fonaerly Streptucoccus luais subsp. lacth;

a commercial fieeze-dricd conccntrated cultute coded 'Dti-Va& 188' obtaincd h m Christian

Hansen's Laboratory, Inc., Milwaukee, WI, USA) and (Y) a non-mpy mixcd thennophilic

yoghurt starter (a biditional liquid starter coded 'S W' kindly supplicd by Dr. C. Duitschacver).
 nie yoghurt starter was a co-culture of ~ o b o c i 1 J t l sdelbmeckii subsp. bulgarims with

Sneptocuccus salivarius subsp. thcrnophilw in a 1 :1 ratio. In mixed cultures in mil4 the coccus

generally gmws fsster than the rod and is primsrily responsiblc for acid production whenos the

md adds flavour and aroma [Pette & Lolkema, 1Hoa,b; Marshall & Law, 19841. Single starters

of S. thermophih are usually unable to decrease the pH lower than CU. 5.2, and for this reason

they an commonly mixed with other starter strains such as Iactobacilli [Shahbal et al., 19911.

The associative (symbiotic or prote-cooperative) growth of the two organisms mults in lactic
acid production et a rate greatcr than that produced by either when growing alone, and more

glutaraldehyde (an important volatile flavour component) is produced by L. bulgmicur when

growing in association [Juillard et al., 1987; Dellaglio, 1988; Sdoff-Coste, 19941. The

combination o f S. thermuphilus and L. lactis is commonly used for acid production in casein

curds (such as cottage cheese curd) that nceive an inennediate cook [Jay, 19921.

The readicd 188 mothcr culture and the SW yoghurt culture w m propagated in sterilized

(1 1SOC- 1O min) 10% RSM following standard sub-culturing pnctices. Propagation conditions

188 1% (v/v) inoculum acrobic incubation at 23OC for 24 h daily

SW 3% (vlv) inoculum aerobic incubation at 43OC for 2 h 45 min every other day

The cultures were stored at 4"C kforc use. The pH of the incubation media was tested

regularly to ensure that it was about constant (CU. 4.9 or less for 188, and 4.5 for SW), le., thst

the net bacterial growth had endcd and that the rate of acid devclopment had stabilitod.

62. S. Bacteriofogicaland Ckemicul Acidifiatio~~

o/MIk

Temperd milk was inoculated with a mined culture of 188 and SW at levels betwccn O and

5% by volume. Relative inoculation rates of 188 and SW were in a 1:3 ratio as this particular

combination proved to have a satisfrtory (acidifying) activity under the expcrimental conditions
uscd [Hill, pers. communication, 199SbJ.Coding for each lcvel of starter inoculum and actual

starter volumes added are s h o w below.

Coding % (v/v) Mixed culture (C) 188 (mUL milk) SW (mUL milk)

The active starters were combined by dispasing in a smaII volume of milk and subsequently

added to the rest of the sample with gentle stirring to avoid excessive incorporation of oxygen.

The pH of the admixture was then standardized to 6.4 (Accumet pH-Meter 9 15, Fisher Scientific,

Unionville, Ontario, Canada) with lactic acid [1040% (vfv), a mixture of D(-) and L(+) isomen

obtained from Sigma Chemical Co., St. Louis, MO, USA].

Some samples (unheated and heated) w e n given a pH cycle h m eu. 6.7 -r 5.8 + 6.7
beforc inoculating with LAB, standardking to pH 6.4, and nnneting. Chemical acidification

(Iactic acid) at around 30°C was either followed by direct neutralization (NaOH) or the samples

were kept ovemight at 4OC at low pH.

For the analyses of rennet gel development at constant pH between pH 5.5 and 6.4, the

procedure for direct chernical acidification of milk (unheated and heated) was similar to that

descrikd in Section 4.2.4, cxccpt for the use of Iactic acid.

62.6 Rennding

Commercial single strcngth Ennet (Christian Hansen's Laboratory Ltd.. Mississauga,

Ontario, Canada) was k s h l y dilutcd to 5% (v/v) in distilled water and added to pre-warmed

(culhued) milk at concentrations betwttn O and 704 pLA (occasionally 2,816 and 7,040 w).
Comspondence ktwecn codcd and explicit values of rcnnet concentrations is shown below.
 Ming Volume of remet 5% Comsponding concentration of
added (CuIL, milk) single strcngth rennet (% vlv)

Analyses were started within 5 min of rennet addition and mixing. The moment at which

measurements with the Nametre and Carri-Med theometers began was taken as zero time.

Continuous monitoring of the changes in milk pH during bacteriological fermentation and

concomitant gel development was carried out in parallel with rheological rneasurements with the

Nametre (Section 6.2.8) on an aliquot of the readied milk held at constant coagulation

temperature. A combined glass electrode (Radiometer, mode1 PHC-GK2701; Bach Simpson,

London, Ontario, Canada) was used in conjunction with a Radiometer pHorneter (mode1 PHM84;

Radiometer, Copenhagen, Denmark). Standardization of the pHmeter before each trial was

cartied out with particular carc ensuring that stable signals were obtained before completing the

calibration procedure as outlined by the manufacturer. The two bufFers (pH 4.0 and pH 7.0) used

were prc-wmed et the appropriate temperature and measund with no stirring as wen the milk

Maintenance of a pH electrode in milk media for a prolonged time can be problematic.

Sporadic rcadings and drifting of the instrument cm occur which can k rclated to fouling of the

electrodc membrane, apecially in high proteidfat environments. We hieâ to minimize these

pmblems by thomughly cleaning the probe foi 5-15 hours after each tun. Cleaning was by

sequentially soaking in a wami enzyme solution (Terg-A-Z ymen", Fisher Labontory Supplieq
Unionville, Ontario, Canada) and in dilute sodium hypochlorite (Renovo.X?, Bach Simpson);

the ekctmde was dso disinfatecl with 8(»C ethanol to minimizc micmbiological cross-

contamination. This was followed by quilibration (several houro) in a saturatcd solution of

potassium chloride. Occasionally, the electrodc was cleancd by immersing in a solution of

K2Cr207 in H2SO4.

(a) Data Acauisition and Treatmg& Voltage signals h m the pHorneter were digitized via an

analog to digital converter and fed into a cornputer. The main steps for data acquisition and

trcatment were as follow. (1) Primary pH data were sarnpled evcry min (60 S. i.e., maximum time

interval allowed by the program written for automated recording) and s t o d . This was achieved

by running the procedure 'atodsamp' with input parameten 1, 1, and 0; and thcn, n (number of

measurements to take), 60, and t (total timt rquired = n x 60). (11) The data set generated was

thcn 'filtemd' ta give mtasumments of pH every 5 or 15 min, which is quivalent to running a 5

or 15-point moving average thmugh the original measunments. This was carried out by running

the pmcedure 'cornpress' with parameten 5 (or 15) and 1. (IU) The output data set was

subscquently importcd into the Quattroa ~ro/Microsoft@

Excel spreadsheets used to analyze

rheological data fiom the Nametre and converted to pH values by multiplying by 20.

The instantancous rate of acidification or first time-derivative of pH [dpH(r)/df, or more

exactly, QH(r/At, Le., graphically, the value of the dope of the pH-time curve at time t,

cxprcsscd as pH unitdmin or ni] was calcuktd as (pH at timc t+lS min - pH at timc t)/lS, ie.,
2-point moving dcrivatization Lhrough the data, and plotted as a funnion of time.

42.b. Rheologicol Meeru~cmen~s

wÙllr the Nametrt Rhmmetr

(a) Instrument se tu^ and Run Conditioap. Changes in consistency (apparent viscosity-density

pmduct, qwxp) during the course of gel development were rnonitored continuously using a

Nunetrc Rhcolina RhcometeP (Namctrc Co., Metuchen, NJ, USA; sec Section 3.4.2 for
instrument description and details of its wodcing). Calibntion of the heometer was checked

initially by messuring samples of known viscosity (minera1 oil standards S20 and N100) at 20,

25, and 40°C. A circulating water bath (fO.l°C) was used to maintain appmximately constant

coagulation temperature in the 400-mLinsulatcd W e r containing the sample (commented on

in Section 6.3.26).

The rheometer was zcroed in air and the sensor sphcn was conditioned in water to the

temperature of the expriment to prevent large temperature differentials when the sensor was

immersed in milk. Test sampks were covered with an insulated lid to limit surface drying and

energy losses. The Iid had a small hole in its center to allow the driving shaft of the sensor to go

through; it did not disturb the masurements because the maximum amplitude of the oscillations

is so small that an object would have to actually touch the sensing probe to offset the readings.

Acquisition of the data commenced simultancously with reaâinp of pH (Section 6.2.7) and other

analyses (Section 6.2.10),approximately 1-2 min after transfer of the probe ta the milk (Le., after

turbulence had stopped and temperature had stabilized). Qualitative visual and tactile

observations about gel state/texturc were also rccorded during and after rheological

measurements, rcspectively. Each run was followcd by carchil washing and disinfecting

(ethanol) of the equipmcnt, c m king taken to avoid damaging the sensing element.

(b) Data Acauisition anâ Trcatment. The output of the Namem was digitized and fed to a

cornputer. Consistency, viscosities (p,

q', and q"), moduli (Ge,
G ', and G"),and temperature
werc calculated and rccorded automatically at 5-min intenmls for up to 15 h using the proprietary

software 'visco23' provided with the rheomcter. This frrqucncy of sampling was found to bc

suitable to cvaluate the rhco-kinctics of gel dcvelopment. QuattmQDPro versions 4.0 and 5.0

(Borland International, Inc., 1992, 19931, and ~ i c r o s o f lExccl 97 were used for fiirther

analyses.
 These included: (4 calculating the first tirne-derivative of consistency, dC(t)/dt (actually,
AC(r)/At), that is, a measure of the instantaneous rate of gelation or timing, as difference

quotients of (consistency at time t+10 min - consistency at time t)/lO (in cPxg.cm-Vmin or /h,
Le., Pa.sxkg.m-Vmin or /h; where 10 = 2xtime interval for data collection), and (il) plotting

(derivative) consistency vs. time and pH curves. Coagulation tirne (CT)was defined as the tirne

when the consistency first exceeded the noise level of the messurements, which typically

comsponded to the second reading of consistency in a series of consecutive rcadings with

positive values of dC(Z)/<t.(Details for curve analysis are given in Section 7.1 .)

62.9. Rkeological Me~~uremcnb

wlth the Carri-Med Rheowwter

(a) Instrument se tu^ and Run Conditions. nie dynamic behaviour of sating milk at small

deformation was also followcd by oscillatory messurements within the coaxial cylindrical

fixnircs (Mooney-Ewart geometry) of a controllcd stress Carri-Med CSLlOO Rheometer~(TA

Instruments, New Castle, DE, USA; described in Section 3.4.3). The samples tested wem either

sub-samples of the milk measuted with the Nametre or, in most expcriments, independent

samples.

The rheometer was operated in the connolled straid'time swccp' made [CaniMed Ltd.,

1989a.b fm details] with the following standard nn parameters: set struin, 0.05; jiequency, 0.1

Hz; sturf twque, 1 W.m; sertie tirne, 1 s; sample fime, 3 S. Effective thermostating of the

measuring system within O.l°C was achieved using a duid jacket and a circulating water bath.

nie main steps for setting up werc as follows. (1) The instrument was allowed to initialize and

'bias' ((Le., stabilize against the windmill efiect of the air karing), without and with the inner

measuring cylinder in position, rcspectively. (U) When the systcm h d rcached thermal

equilibrium a the desircd temperature, the gap for the measuring cell w u set to 77 )un. (iiu The

instrument was subsquently calibrateci for inertia (ovenll, machine, and geometry). (Iv) The cell

' was thcn filled with about 7 mL of trcated milk, the annular space ktween the cyiindea was
c o v e d with a thin layer of light mineral oil @ - 0.849 g.cm-3; Sigma Chernical Co.) to

m i n i m i ~dehyhtion of the sampler. and the instrument was ICA to continuously record

viscalastic pmpaiies during gel development for as long as desid.

Series of pnliminary oscillatory tests w m conducted to get an idea for the mponse of

standard samples of milk and to ensun that the measurcments were taken as close to the region

of linear viscoelasticity (LVE) as possible. In the toque (stress) sweeps (Le., scans) illusaoted in

Figure A6.3, elastic modulus (G3, dynamic viscosity (q3, tun G, and strain (fi of gelling

recanstituted skim rnilks (C14-Rx4) at diffemnt stages of coagulation wete monitored over a

range of stresses at 0.05 Hz and 40°C. The fact that G ' and q' nmained approximately constant

as stress, thus strain, were i n c r c d helped to confirm that teding was in or close to the linear

rcgion of the sctting gels.

In proper cxperiments, the amplitude of the input oscillatory m i n was limited to 5%. A

value for oscillation frcquency of 0.1 Hz was regardcd as a safe choice, a compromise betwcen
measuring too slowly that not enough data are obtained and too f s t . This corresponds closely to

the estimated limit of LVE icported by van Dijk [1982] (3%), Lee [1986] (S 6%), Dcjmek [1981]

(SN, 0.1-10 Hz),Zoon et al. [198&sb](3%. = 0 2 Hz),and Lbpez et al. [1998] (1%, 1 Hz) for
remet gels; by Roefs [19861(S 3.5%, = 0.2 Hz), Stevcnton et al. [1988, 1990) (3%, r 0.02 Hz),
Xiong & Kinsclla [1991alb] (2%, 0.1 Hz), Biliadcris et al. 119921 (1.8%- 1 Hz),Rtlnncgud &
kjmok [1993] (s5%. 0.1 Hz), ARhd et d [1993u,b] (2%, 1 Hz), Rohm [1993] (3%, 0.2

Hz), Rohm & Kovac [1994] (2%, ,

i 0.2 Hz),van Mule & Zwn [1995a] (1%. 0.1 Hz), and Lucey

et al. [1997a,b] (1%. O. 1 Hz) for yoghurt-like gels; and by van Hooydonk et al. [198661(0.2 Hz)

and Noël et al. [l989; 19911 (0.1 Hz) for combined remet and acid gels. In the prcscnt study, the

same set of operational parameters was used for tcsting diffemit samples and conditions of gel
formation as it was reasonable to assume t h a these parameters also appmximatcd the

rcquircments of linear khaviour for such systems.

That the measumnents were perfonned under appropriate conditions was checked further by

observing the quality of the sine cuwes for applied (controlled) m i n and measured stress dunng

runs (discussed further under Section 633a). These were displayed automatically each time an

oscillation step was pecfonned. The output stress wave ought to be well-resolved (Le.. high

enough strain so that readings may &e taken within the detection iimits of the rheometer) and

smoathly sinusoidal (Le., low enough strain so that the sarnple responds in a linear way), as

idealized in Figure 3.4. In a typical experiment, the computer-controlled rheometer was

automatically taken through about five cycles of oscillation and provided that a stability criterion

was met during the last few cycles, the last cycle was stored and used for analysis [Carri-Med

Ltd., 1989a,b]. Together with the fhquency of oscillation and the number of measurements

taken over a spccified period of timc, this defined the (somewhat variable) tirne intervals

between individual rneasurcments (5- 10 min).

(b) Data Acauisition and Treatment. The panmeters calculated and recorded by the oscillation

software version 5.0 of the fieorneter included elastic and viscous moduli, viscosities, loss

tangent (fun 4,strain (displacement), stress (torque), and temperature. Treatment and evaluation

of the primary data were essentially as describeci for analyses with the Nametre rheomcter

(Section 6.2.86).

dt 1û. ConilplementoryAnu&ses

(a) SDS-Polvacrvlamidc Gel Electrodioresis. The extent of K-cwin proteolysis during the time

course of gdation was estimated by SDS-PAGE using an automated PhastSystemTM(Pharmacia

LKB Ltd., Baie d'UrfC, QuCkc, Canada). Dimtly d e r addition of mnnct, milk (an aliquot of

the sunplc that was used for rhcological measurements with the Namctrc thcorneter) was divided
into 16 sub-samples (8x2 repliertes) of 5 mL each that were placed in test tubes, topped with

parafilm@, and incubated at 40°C with no stimng. Sampling at eight pdetermined rcaction

times was carrieci out in duplicate (intemal duplication). Termination of the mzymatic reaction,

sample preparation, experimental conditions for clectmphoretic sepmtion, and subsequent

relative quantification of @mu)u-casein wen as outlined in Sections 5.2 thmugh 5.4 of Chapter

5.

(b) ANS-Fiuorimeûy. Attempts were made to see if additional information on gel formation

could be derived by monitoring the binding and distribution of the hydrophobie marker anilino-

8-naphthalene-1-sulphonate (ANS) between ' k e ' and 'aggregated' protein fractions according

to the fluorimetric method devised by Bonomi et al. [1988] and Peri et al. [tg901 (set Chapter 3,

Section 3.2). Reconstituted skim milk (2 L) was trcated with 0.2 m M ANS (Sigma Chernical

Co.), inoculated with starter (level C/8) with or without nnnet (level Rx8), and divided into two

portions: one was used for measurements of pH and consistency at 40°C (Sections 6.2.7 and

6.2.8) and the second was used for determinations of fluorescence. The latter was tùrther divided

into 10-20 portions (a 50 mL) which were placed in centrifuge tubes and incubated at 40°C.

ANS does not appear to affect coagulation of milk by rennet at the concentration used [Peri et

al., 19901. For milk coagulated by combincd bacteriological acidification and renneting (Cl4-Rx

4), variations between the traces of consistency vs. time obtained with and without ANS (not

shown) were within experimental variation (sec Section 6.3.2~).

Samples were removed (in duplicate) h m the incubation bath at various intervals and

immediately cooled in ice to cffectively stop bacteriological and enzymatic activiiy. Cwled

simples were then centrihiged at 9,950xg (8,500 rpm) and 2°C for 30 min (JI-MCcentrifuge

with JA-17 rotor; Beckrnan Instruments, Inc., Polo Alto, CA, USA). This resulted in their

separation into a 'frce' (supernatant) phase and an ' a g g r e ~(precipitate)

~' phase.
 To asoess the partition of the fluorophorc betwcen supernatant and piecipitate, samples of

supernatant and precipitate were dilutcd appmximately 1: 100 (v:v) with a solution of 1% Triton

X-lûû (Sigma Chemical Co.) in Milli-Q water (MiIlipote purification systcm, MiIlipore Canada

Ltd., Missiaswga, Ontario, Canada). Undct these conditions, the ANS prcscnt in the sarnples,

eithcr fm or bound to protein hydrophobie sites, b m e s adsorbecl by detergent molecules and

is nndeted fluorescent. Rcadings of fluorescence intensity (FI) were taken at m m temperature

with a Shimadzu spectmfluorimetcr, model RF440 (Shimadzu Corp., Kyoto, Japon) at

excitation and emission wavelengths of 390 and 480 nm, respectively; sensitivity was set to 1.

The response of the insüment was standardized with a solution of 0.2 mM ANS in 1% Triton

X-100. Measumnents on the precipitates wete found to k less ptecise (not shown) than those on

the supernatant, prcsumably because of incomplete re-dissolution of the coagula, even aftcr

vorttxing.

(c) Isothennal Micnrcilorimetw. Tentative isothermal analyses of milk gelation phmornena

were pe~omcdusing an Omep Ultrasensitive Isothennal Titistion Calotimetcrm (MicroCal,

Inc., Northampton, MA, USA). The instrument measutes heat (Q) evolved or absorkd. It

contains two identical cells (one for the sample and one for the reference), each of working

volume about 1.7 mL, enclod in an adiabatic jacket. During an isothetmal expetiment, a small

arnount of power (heat) is continuously supplied to the reference cell. The diffetencc in

temperature khmen sample and rcfercncc cells is constantly monitomi and the heat supplied to

the wmple cell is conxpumtly increascd or rcduced to kecp the temperanite difference close to

zero. A signal pmponional to the feedôack power applied to the ample cell is obtained as ACp

(diffcmntial power betwccn samplc and inert rcfercnce, or rate of heat production AQ, in pcal.s-

1) WicmCal, Inc., 19931. With time and temperature, this constitutcs the mw data obtained from

the calorimeier. Thus, a -ion or pmccsr which mults in (net) pmduction of hcat within the

trmple cell (Le, exothermic teaction) causes a negative change in ACp because the hcat evolved
provides heat that the heater of the sample cell is not requircd to pmvide. ACp having units of

power, time integral of the peak in the heat flow curve yields a rneasurement of thermal energy,

AH(heat of rcaction or cnthalpy, in pal).

Before use, the calorirneter was equilibrated (several hours) at gelation temperature of about

40°C, nwring that a stable ACp baseline was obtained. Readied milk samples (Ievels C/8 andlor

Rx8) were degasseâ for eu. 5 min using a vacuum pump. Because of the risk for microbial

deterioration of unacidified milk, most samples were run using Milli-Q water as the (non-sterile)

control system in the refetence cell instead of milk with no culture and no rennet. Data on

isothermal heat flow during coagulation wete collected and analyzed using the software of the

calorirneter (Originw, MicroCal, Inc.), and subsequently plotted using ~ i c r o s o f Excel

l 97.

Betwecn mns, botb cells were thoroughly washcd with w m water plus detergent and rinscd

exhaustively with MilliQ water, as recommended by the manufacturer.

62.I l . SfatbticalAnalyses

All trials were repeated at least two times. The number of replications for rheological

measurements was usually limitcd to strictly two because testing frcquently was over long

periods (8-10 hours). Unless stated othcrwise, and cxcept for the display of timeîurva for

consistency, moduli, and pH, numerical results arc presented as atithmetic means h m replicates

(me und intemal rcplicates when appropriate) f standard deviation (SD) or coefficient of

variation about the mean (CV = SDImean).

63,Pre-Tcdts -Results and Discussion

Before tuming to propcr expcriments and examining their conclusiveness or othenvise, it is

usehl to review some of the practical aspects and assumptions which underlie the analyses, so
that we may ôe better positioned to appreciatc the bearing and limitations of the studies and to

understand the origin of the phenornena o b s e d .

63.1. The Use of Skim MUk Reconsthtedfi'i Povlcr

Most experimcnts wem pcrformed with reconstituted skim milk. To check whether the

course of gel development in RSM was comparable with that in fksh whole milk (checse milk),

the following different types o f milk were treated with culture (level C/4) and rennet (Ievel Rx 1

.or Rx8) and their setting into a gel at 40°C was followed in the Nametre rheometer: (I) standard

9% low-heat D M ,(ii) (parrwized)Pesh whole milk, and (iii) pmrcurized and homogenhed

fiesh/commercial milk. Comspnding time-profiles for consistency and pH are conhasted in

Figures 6.3a&b (Appendix 64a-c).

Important variability of the coagulation profiles was apparent (and probably contributed by

syneresis), in particular with sunples prcpared from hrsh rnilk. The average absolute values of

instantaneous consistency also differed depending on the nature of the milk, but common

features could bc identifed in the traces of consistency vs. tirne, particularly the reproducible

shoulder at about 130460% of the coagulation time necu pH 5.2-5.4 at low concentration of

rennet (Figure 6.3a), and the distinct 'hump' at about 250-2704; of CT near pH 5.7-6.0 at higher

concentration of enzymes (Figure 6.36). The small initial incnasc in consistency pmeding the

coagulation of fnsh unhomogenizcd milk at concentration Rxl, and to a lesser extent Rx8

(Figure A6.4~).was attributcd to the conspicuous rise of milk fat to the top of the samples (i.e.,

creaming). (Details of the gclation curves will k dealt with in the ncxt chapta.)

Thus, the basic cffccts or relations found for eel development starting h m reconstituted

(skim) milk are pmbably al= valid for hrsh (whole) milk, although the prestncc of partially

denatureâ whcy proteins in (evm low-hcat) RSM does modifL the functional propcnia of milk

WcKenna & Anema, 1993; Lucey et al., 199&].

 - !AAAA
Standard 9% recooatitutd
4,
A~ ikim milk (RSM), Cl4-Rx1
(nplicatti 1& 2)
A
A pH
-, -

Y A A A A
GA
2 (Pasteurized) f m b whole milk, .
: pHof A C/CRxl (replicata 1 & 2) -
: p a s t e h d miUc -4

i
A n A
A , Puteorized & homgenized -
A wbole milk, CI4-Rs1

Figure 6 . 3 ~ .Time-courses of consistcncy devclopmcnt and ôacteriological

acidification for a % BSM M. brsh yybpk mik and
bomapcnizsd ma
culhvcd and renncted at C/4-Rxl
at 40°C. Results are shown for comsponding mcasurementsof eonsistency C
and pH carricd out in duplicatc with the Nametrc rheomcter (symbols/lines of
differcnt sizthhickness). Anows point to the region of apparent local slowing
down in C developmcnt and to the comsponding(appoximate) values of pH.
 1
1
- Standard 9% rccoa~titutd ;
aldm mUk (RSM), C/eRrs C

-
Pasteurized dk hoiogeaized
(commercial) wbok milk,

Figure 6.36. Time-courses of consistency development and bacteriological

acidification for QPPdYP S BSM us. && YYhPlt and
lcommercipll- cultured and renncted at
g 4 - u at 40%. Raults are shown for comsponding mcasumnents of
consistency C and pH d o d out in dupliaite with the Nametm iheometer
(symbols/lines of diffcmt sidthickness). h w s point to the regions of
maximum and minimum C and to the comsponding (appron) values of pH.
van Vliet & Dentener-Kikkert [1982] and Zoon et al. [198ki] rcporied linle diffemnce in the

absolute value of rnoduli betwcen gels made by aciditication or renneting of skim vs. whole

milk. Most likely, the cvolution of the gel with respect to time and pH for these systems, and

more specifiully the 'bimodal' types of responses (gel consistency and viscoelastic moduli) of

main interest hercin, entai1 basicaily the same type of phenomena and interaction forces.

63.2. Dyuamic TeMing w# the N a m Rheometer

(a) Sensitivitv and Rcpmducibil itv. Ln principle, well-defined viscoelastic parameten such as
dynamic viscositics and moduli can be derived fiom consistency measurernents with the

Nametre RheolinerfM2010. In practice, however, the values of viscosities and moduli could not

be measured reliably (unlike the values of consistency), especially in unconcenüated setting

milk. On occasions, apparcntly satisfactory patterns of developmcnt of moduli with time were

obtained but in most cases the rcadings appeared Iargely enatic (unreliable), even afier formation

of mature gels (not shown, nor systematically investigated). Similar observations have k e n

made previously [Sharma & Hill, unpublished]. Only for gelling concmtrated milk (VCF 2-4)

and cultured pre-heatcd milk did the shape of the curvcs for moduli vs. time compare rcasonably

well with that for moduli mcasured with the Carri-Mcd rheometer (not shown). The problem

may originate h m a lack of sensitivity of the Nametre. In samples particularly prone ta (micro)

syneresis, it may be accentuated by the developrnent amund the vibrating sphcre of

environments of fluctuating density @sci # -4 ,,,hW), which is expccted to precludr

diable calculations of viscosities and moduli h m mcasurcments of consistency (i.e., the

product of qWxp). The use of the Namctrc instrument (primarily a viscorneter) was

subscquently limited to determinations of consistency, the values of elastic and viscous moduli

and loss tangent reportcd hemftct rcfemn~to complementary mcasurcrnents with the Carri-

Med theorneter.
 The reproducibility of consistency mewmments with the Nmetre rheometer was estirnateci

by testing b e c independent amples of standard RSM identically prepmd (Cl4-Rx4) and

allowed to set at 40°C. 'Comsponding geletion profiles are plotted in Figure A6.5. Means,

standard deviations, and cocficicnts of variation for m e characteristic parameters of combined

coagulation kinetics under this set of experimental conditions are summuized in Table 6.3. (The

possibility of differential reproducibility under different conditions of coagulation was not

investigated.)

Tabk 63. Reproducibility of experimentation witb the Narneüe rheometer: mean, standard
deviation (SD), and coefficient of variation (CV) for characteristic parameters of combined
enzymatic and Iactic acid coagulation kinetics. 9% RSM, C/4-Rx4, 40°C.

Characteristic wintl~armeter Meana SD CV%

Coagulation point (P,)

Coagulation time CT @)b
PH,
Point of consistency maximum (PM&
Time t~ fi) 3 A0 O. 13 3.82
ConsUrency CM (cPxg.cm-3) 1 62.3 7.51 4.63
PHM 5 S8 0.04 0.72

Point of local consistency minimum (Pnin)

Time t,,, 4.08 0.26 6.37
Consistenq Cm(cPxg.cm3) 83.3 16.1 19.3
~Hni 4.93 0.04 0.8 1

6 Hours after rcnnet addition (P6)

Consistency c6 (cPxg.cm-3)
pH6
Maximum nte of firming (dC/dz)mrx
(cPxgsrn-Vh) 246.7 63.7 25.8
Time t- (h) 2.88 0.20 6.94
Consistency C, (cPxg.cm3) 101.7 18.9 18.6
PH~ZX 5.80 0.04 0.69
ahithmetic mean of thme replicatcs.
AS defined under Section 6.2.8.
 Part of the variability observed likcly originated fiom variations in the patterns of milk

acidification by lactic acid bacteria (sce also the illustrations under Section 6.3.4u6b). It is

notcworthy that consistency redings before the point Pmh of local consistency minimum

following coagulation tended to vary less than later d i n g s . The rune trend was observed for

most systems and coagulation conditions investigated, the variability (disparities) at/after Pmin

king largely related it seems to the disturbing effects of syneresis (see also discussion under

Section 7.1.3). Most reproducible and diable quantitative information can therefore be expected

fiom consistency measurements pnor to P i , especially in situations most conducive of

syncrcsis.

Overall, the repeatability of consistency experiments with the Nametre viscometer was

satisfactory in relation to the variations introduced by varying the relative concentrations of

acidifying starters and rennet enzymes. The viscometer did not enable elastic and viscous

phenomcna to be studied separately, but the effects detected in terms of consistency ('bimodal'

responses) compared well with the effects observed in terms of the fundamental viscoelastic

parameters reliably measured with the Carri-Med rhmeter (Sections 6.3.3b and 7.1.1a). The

two instruments were complementary although it should be noted that the values of consistency

and moduli cannot be comparcd dircctly because there is a difference in dimension (tirne or

fhquency) bctween them. The response of the Narneûe is more rdated to the viscosity of gelling

simple, whereas that of the Carri-Med is more relatcâ to the rigidity of the gel.

(b) Ternociahin Fluctuations Accommviqg Gel Dcvelo~men?.A consistent ferhirc of dynamic

measurements with the Nametre viscometer was that the temperature of the milk dmpped (up to

Ca. 3OC) during the early stages of pl formation (Figures A6.64&b), even though the

temperatun of the circulating water bath was kept constant and the samples were covered during

routine experiments. This was obseived largely irrespective of the type and concentration (Mx)

of milk gels, and to a lesser e m t at coagulation temperatures klow 40°C. Heat transfer h m
the walls of the insulatcd W e r to its centcr, where temperature measurcrnents were taken, is by

nanuil convection in the kginaing, and by conduction during transition h m fluid milk to gel.

Most probably the heat transfer propcrties decrewd (i.e., mistance to heat flow increased) as

the consistency of milk i n c r e d . (Measumnents based on heat transfer coefficient are the basis

for the 'hot wire' method describcd by Hori [1985] for monitoring gel development.) Changes in

heat transfer likely tcsultcd in a lower rate of heat transfer and lcss efficient temperature

redistribution within gclling milk, and k a t transfer was no longer mough to offset heat losses.

In some experirnents, the temperature incnased almost ta its initial value aAer some time

(e.g., Figures 7.1.10 and A7.1.24d under Chapter 7). In most cases this was linked to the onset of

appteciable macroscopic syneresis in the beaker (i.e., superficial cracks in the curd, andfor

apparent separation of the gels into a supernatant semm phase and a somewhat sinking coagulum

phase, with concomitant decrase of the contact behueen curd and the consistency/tcmpcrature

probe). Combined with visual observations, this second chatacteristic proved to k a usehl clue

for checking that measuments of consistency were taken without serious perturbations due to

syneresis (sce further Section 7.1.3).

It may be suggested that endothermic (denaturation) processes accompanying gel formation

contributcd to the initial drop in tempcraturc. A suich of the literature tevealed very few

mentions of pmious calorimeeic examinations of gelation phenomena in milk [Caule & Coffn,

1950; Phipps, 19581. In fact, it wems that the coagulation of milk by rcnnet is exothennic

phipps, 19581, as is aggrcgation of pmtcin systcms in general [Jackson & Brandts, 1970;

Privalov et al., 1971;Privalov & Kbeehinashvili, 1974; Stanky B Yada, 19921, and that the net

heat evolved during gel fonnation/synercsis is small (about or l e s than 0.1 caV40 mL of fksh

raw milk [Wipps, 19581). This is also the impression w t got h m isothermal microcalotim*ric

investigations (discussed under Section 63.4~).

 Diffennces ktwcen the development of gel consistency over time with and without

adjusting the temperature (not shown) did not nrceed experimental variation, and so, no attempt

was made to compensate for temperature effccts in subxquent routine expriments. The

similarity bnwecn progrcss curves for Nametre consistency and coagulation profiles obtained

fiom essentially isothermal measurements with the Carri-Med rheometer confirmed that the

influence of temperature fluctuations of the order of 2'C was marginal.

63.3. Dynarnic Testing with the Carni-Med Rheometer

(a) The A~~roximation

of Linear Viscoelasticitv of Gels. Apparent departure from strictly linear

v iscoelasticity of gels in advanced stages of development was noticeable in typical osci1latory

experiments with the Carri-Med rheometer. The non-linearity was observed in the output

diagram of wave hinctions [applied strain (5%) and rneasured stress] in which, as reproduced

schematically in Figure A6.7, the wave fom for the memurcd stress was no longer sinusoidal,

but slightly distorted (shoulder). (Contrast to the theoretical cutves for viscalastic semi-solid

shown in Figure 3.4.) The disymmetry (imgularity) appeamd within few hours of gelation

(typically 15-30 min) and seemed to be more pronounced for gelling concentrated milks. This

-
suggests that the dynamics of the responsc of milk gels to shear became progressively non 1'~near

during the course of gelation and that higher order (odd) hannonics were generated [Granick &

Hsuan-Wei, 19941. What repercussions this non-lineuity had in tems of the values of gel

moduli and loss tangent would k difficult to quanti@; pahaps biascd (downward) estimates of

moduli resulted.

This concuts with the observations of Lee [1986]: the author showed that for renncted milk

of standarâ concentration, the regimc of LVE may cxtend up to 6% deformation amplitude in the

vicinity of the gelation point and that this limit is progrtssivcly reduced to 4%, or kss, as setting

progresses. Gcwais und CO-workers 1198261 alro commented briefly on the non4inear

viscoclastic chanetet of milk gels obtained by rcnneting (7.S0 oscillatory m i n amplitude at

0.033 Hz and 30°C), although they did not clearly statc et what stage of coagulation onset of

non-lincar effccts b e r n e prominent [also Gewais 8 Vermeire, 19831. Presumably, modification

of the conncctivity of the gels at the micro level (e.g., changes of orientation, shortening, or

intemal rupture of structures) when they become too stiff (Le., less compliant) and appmach

long-term pseudo-equilibrium pmpcrtics provokes the onsct of non-lincar behaviour. Synercsis

phenornena may complicate the rheological behaviour fùrther.

Overall, the approximation of linearity held satisfactory, however. No attempts were made to

separste the linear (fundamental) part of the response signal fiom non-linear (hamonic) parts

(e.g., by means of Fourier analysis), partly because of methodological difficulties, and partly

ôecause it seemed reasonable to assume that theoretical data analysis and reduction to

fundamental viscoelastic parameters within the framcwork of lincar viscoelasticity was in fact

largely adequate.

(b) Sensitivitv and Re~ducibility.Of the viscoelastic parameten effectively derived h m

dynarnic measurements with the Carri-Med rheometer, only the moduli and loss tangent (loss

angle) were systematically evaluated in the present study. Coefficients of variation for elastic

modulus and loss tangent-time coordinates at characteristic points of gel development for

standard RSM (C/4-Rx4, 40°C) wcm estimatcd over t h m independent trials. Gelation profiles

are show in Figure A6.8; details are summarized in Tabk 6.4.

As for measurcmcnts with the Nametre viscometer under comparable experimental

conditions, earlier rcadings for moduli tcnded to vary less and later readings more than the

readings at the point Pminof local minimum in moduli. Over the range of conditions of

coagulation investigiteci, measurcments for C h - M e d moduli and loss tangent appcarcâ mon

precise than those for Nametre consistency, perhaps, in part, because the measuring geomeûy of

the conrrollcd s t m s rheometcr minimid the cffects of synncsis.

Tabk 6.4. Rcproducibility of experimentation with the Carri-Med rhcometer: mean, standarâ
deviation (SD), and coeficicnt of variation (CV) for characteristic parameters of combined
enzymatic and lactic acid coagulation kinetics. Ph RSM,CM-Rx4, 40°C, 5% strain, 0.1 Hz.

Characteristic pointfparameter Mean* SD CV%

Coagulation point (P,)

Coagulationtime CT fi)b
toss angle i& (degreeslc

Point of elastic modulus maximum (PM&

Time IM f i ) 2.88 0.13 4.5 1
Elastic moàulus G 'M(Pa) 38.3 3.2 1 8.38
Loss angle & (degroes) 27.8 0.66 2.37

Point of elastic modulus minimum (Pmi&

Time t,,, (%) 3.58 0.16 4.47
Elustic modulus G ',,, (Po) 10.7 1.53 14.3
Lms angle & (degees) 3 1.2 2.00 6.4 1

6 Hours after rennet addition (P6)

Elastic moàrllus G'6 (Pa)
Loss angle 4 (degrees)

Maximum rate of firming (dG'/di),,,,

(pam 72.7 4.93 6.78
Time tma fi) 2.53 0.10 3.95
Elmie moduIus G ' , ,
(Pa) 24.7 0.58 2.3 5
Loss angie 4, (degrecs) 26.4 1.32 5 .O0
aArithmetic mean of three replicates.
AS dcfined under Section 6.2.8.
CTm S= G"/G'.

(c) Gel Develooment at D i f b n t Freau~cies of Oscillatio~. By running prciiminary

cxperiments with gelling RSM (C14-Rx4 and 40°C) at a ftxcd fkquency ktween 0.1 Hz

(standard mcasuring frrquency) and 10 Hz at 5% m i n , it was confinnecl that the= was a

fiequency dependence of die instantancous elastic and viscous moduli (Figures A6.9a-c).

(Frequency dcpcndency was not investigatcd furthcr, Le., no fmquency swtfps were conducted.)

The miti~atingeffect of expwimental fiquency must obvioualy k allowed for when the data
 The trend of incmasing moduli (G'in particular because tun 6 i.e., G ' ï G ' tended to

decrase) with fiquency is consistent with earlier obseivations for acid and mnet milk gels

[e.g., Tokita et d., 1983; Roefs, 1986; Dejmek, 1987; Zoon et al., 1988~;van Vliet et al., 1991~;

Rohm, 1993; Rohm & Kovac, 1994; Lucey et al., 19970, 199w. Figure A6.9~has feahires

analogous to those of the mechanical spectra of ro-called weak gels (e.g., formcd by xanthan)

[Clark & Ross-Murphy, 19871, albeit over a more nanow frequency range: both elastic and

viscous moduli tend to increase with increasing fkquency (Le., decniising time-scale of the

applied deformation), with G ' > G" (Le., tan 6< 1.O, indiciting substantial elastic contribution

to viscoelastic behaviour over the range of frequencies tcsted). This points to a relaxation of the

physical bonds involved in building up the gels within the time-sales of the measurements and

to the existence of a range of relaxation times [sce also Roefs, 19861. It rnay k rationalized that

at higher frcquencies (shorter time-scales) more bonds are 'seen' as non-relaxing (Le., permanent

or clastic in chanrcter), hence the relatively pronounced increase of G '.

Most important for the p w p o ~of following the course of pl development is the close

similarity in the shape of the coagulation profiles of Figure A6.9~. Since the moduli at al1

fiequencies investigated were approximately proportional to each other during coagulation, al1

curves yield the same temporal information. To keep in line with expcrimental conditions in the

literatum mentioned in Section 6.2.9a, measurements with the Carri-Med fieorneter reportcd in

the mst of the prcscnt study werc obtained at 0.1 Hz.

63.4. pH and CniorimctricMcarcrenm~s,and Adhi@ of Bucterùû Cultures

(a) Acidification Kinetics. Exampks of acidification (pH) profiles obtained during coagulation

of standard RSM by strictly fermentation (inoculum level Ci8 to CIl) and by combincd

fermentation (Ci8-CII ) and nnneting at 40°C ue sbown in Figures 6.4 md A6.10-12.

 Incubation time at 40°C (h)

Figure 6 . b . Typical evolution of the pH of milk with time during the incubation
of a)
opipuats pf a p m of 1:3 IAC~OCOCC~LT lactis subsp. iacfis
with Lactobacillus delbrueckii subsp. bulgariinu/treptproccus solivarius subsp.
thermophilus in standard RSM (pp et 40°C (counterpart of the .data for
standard RSM renneted at Rx8 shown in Figure A6.11). Profiles of pH for cach
level of inoculum arc shown for experimcnts replicatcd twa (occasionally four)
times (sarne syrnbols, diffcrent site).
 Incubation time at 40°C (h)

Figure 6A6. Typical evolution of the ptIpf and & fPtC Pf a fimc
PPfllPt u. -9f with time during the incubation of a CO-cultureof
1:3 Luctococcw Iuctis subsp. lu& with Luctobacih delbrueckii subsp.
buIguricus/Streptucuccussafivwitls subsp. thcrmophiIus at level ç14 (pp IfPPO) in
standad RSM at 40°C. Primary and derivative profiles of pH arc shown for
experiments teplicated two times (same symbols, differcnt size; same data as in
Figures 6.40, and A6.10~4,A6.11b , A6.I-b ,and A6.13a). Amws point to
regions of local maxima (Pb! and Pwul) and minimum (PmiJ in the average rate
of bacteriologifal acidification (absolute values of dpWdt).
 The acidifying khaviour of the mixed culture w u largely typical of the activity and growth
characteristics of common lactic acid starters. Typical curves of pH vs. time of incubation (e.g.,

Figures 6.4a, and A6.11-121) showed a limited decrcase in the pH of the milk at first (a period

cornsponding to the initial lag phase of bactenal growth), followed by a rapid quasi linear, albeit

sigmoidal, decreiue in the pH range 6.2-4.7 (loganthmic or exponentisl phase of growth), which

tapeml off as the level of acidity became limiting between pH 4.5-4.0 (stationary phase of

growth) [Tramer, 1973; RaSic & Kurmann, 19781. Curves of this kind cannot be compared easily

without sophisticated modeling [Ross & McMeekin, 1995; Whiting, 1995; BoignC & Dantigny,

l998].

(4 nie fiequency of pH measurements in coagulation experirnents was sufficient to allow

satisfactory estimation of the instantancous rates of change o f pH, Le., acidification kinetics

throughout gel development (Figures 6.46, and A6.l0c&d, 1 lc&d, and 12b&c). Results in terms

of pH gradient [fimtime-derivative of pH, dpH(r)/dt] can be interpreted mon readily than actual

pH data and can aloo be related to the rheo-kinetics of the coagulation process.

By and large, the maximum rates of acidification (ie., the values of @HM at point PM-

unlike those at PM-]) in the quasi linear region of pH decrease appeared to be independent of

the level of milk inoculation in the range Ci8-Cf1 when other growth conditions were kept

constant. (Only a limited increase in rate at P b 2 with increasing starter cultures was observed

in, e.g, Figures A6.1Ocdtd.) This concurs with the observations of Spinnler & Comeu (19891,

and of Julliud [1991] and Dcmaripy et al. [1994] for acidification of low-heat RSM by strains

of Sireptococcus thermophilw at 40°C and by sûains of I;~C~OCOCC~(S

lacris at 30°C, respectively.

Most noticeably, the pH-time curves were shifkd toward shorter times (accelerating effect) with

increasing the concentration of bacterial cultures, as expccted.

Derivative plots also enhance the rcsolution of minor featurcs in the primary curves and can

be upeful to distinpish k o m s n physiological states of bzcterial growth (an approlrh analogous

to the one used to charactctize coagulation behaviour o f milk in Chapter 7). Metabolic and

growth pmperties of differcnt specis or strains within spccies may be characterized and

conveniently compmed using this procedure [Spinnler & Corrieu. 1989; Zanatta & Basso, 19921.

In the present studies, tirne-derivative us. time (or pH) curves of pH data consistently revealed

two relatively distinct peaks of maximum acidification rate in regions referred to as P M ~and
I

[i.e., minimum value of dpH(i)/dt, since dpH(il/dt was < O; e.g., Figure 6.461, rather than
P,+fCU12

just one as was anticipated. F e one expected peak defines the infiection point, Pi, in the dope

in the region of logarithmic growth, which would be characteristic of a sigmoidal (biphasic)

curve with a convex course before Pi and a concave, and ultimately linear, course afier Pi (sa

Figure A7.1.13 under Section 7.1 .Za).] The values of acidification rate at P,44rn2 betwecn pH 5.5

and 5.0 were of the same magnitude as those measund by Spinnla & Corrieu [1989], Le.,

amund 0.6-0.8 unit of pWh (10-14 milliunits of pi-ümin) at 40°C, also with IdpHldt at P ~ ~ 2 1 >

IdpH/dt at P ~ ~ l l .

In non-pre-heated RSM at 40T,acidification temponrily slowed down noticeably after the

fwst peak at PM,], about midway through the logarithmic phase of growth at pH ktwcen 6.0

and 5.5. In pn-heated RSM at 40°C (Figures A6.14~-c),this scemed to occur earlier, Le., at

slightly higher pH.

(U) Possible explanations for this incidental finding may ôc sought in terms of

nutritionaVphysiological limitation(s) a n d h (and most likely) the prduction of ammonia (MIj)

by starter bacteria, which would ncutralia the lactic acid. The phenomcnon is unlikely to stem

h m changes in the acid-base buffering properties of milkicud since maximum buffning of

milk occua in a mgion of lower pH uound S. 1 (5.5-4.5) (Dolby, 1941;Walstra & Jenness, 1984;

Mistry & Kosikowski, 1985; Lucey, 1992; Lucey & Fox, 1993; Gastaldi et al., 1997; Singh et

al., 19971.
 Bacterial production of NH3 in cultured dairy pmducts has ken sunnised to result h m the

deamination of some amino acids (e.g., arginine) [Groux, 1973; Pettersson, 19881; most results,

however, point to the activity of the enzyme urcase, which hydiolyzes urca prescrit in milk

(about 024.4 g.L-1) into ammonia plus carbon dioxide Miller & Kandlet, 1967; Tinson et al.,

1982a.b; Julliard et ai., 1988; Spinnler & Corrieu, 19891. Although Farrow k Collins [1984]

reported that S. thermophilus does not degrade urea, urease activity seems to characterize of a

large num ber of strains of S. thermophiius examincd until now [Tinson et al., 1W h ,b; Spinnler

et al., 1987; Julliard et al., 1988; Zourari et al., 19921. Julliard et ai. [1988] and Spinnler &

Corrieu [1989] showed that urcase activity was maximum between pH 5.5-5.8, dropped during

advanced stationary phase, and increased with temperature in the range 20-70°C. Apparently,

lactococci do not posscss this activity [Miller & Kandler, 1967; Zourari et al., 19921. The initial

lowering of the rate of pH decrcase of cultured milk mcasurcd in this work may k indicative of

the prexnce of an urcase activity in the streptococci-containing SW starter (Section 6.2.4),

thermophilic strcptococci king the main micro-organisms of the mixed culture involved in

initial acidifcation of milk. No effort was made to confinn this point experimentolly or to

furiher quantify the effect.

The effect of urease activity in ternis of relative alkalinization of the growth medium is of

particulu technologifil interest in relation to evaluation/quantificrtion of the activity of

sîrcptococci in milk by pH mcasurements Famelui & Maubois, 1988; Spinnler & Comeu,

1989; Zounri et al., 19911. Because NH3 neuüalizes the acid produced by bacteria, changes in

pH or titratable acidity (Le., active acidity) unnot k used in practice to accurately follow the

production of acid in milk cultures of such organisms. The decrcase in rate of biological

acidification occumd in a range of pH that brackets the points around P M (demarcation

~ in milk

gel consistency) in the coagulation curver at levels of rcnnct abovc Rxl (i.e., amund pH 6.0-5.5;
 Consistency C ( c ~ . ~ . c m " ) Consistency C ( c ~ . ~ . c m * ~ )
and dC/dt (c~.gcrn-~/h) and dC/dt ( c ~ . ~ . c m * ~ / h )
4
- - W W W W P
E; tn u r O u i O u , O V i
0 0 0 0 0 0 0 0 0 0 8

oo--yyyw+,p""PP
ouloLiou,ouiou,ouioul
pH and dpWdt (pH unitdh)
 RSM, C/4-Brp
(repliCates 1& 2)

Figure 6.W. Contrastcd cvolution of the pH Ipp C pf müL. W Pf

Lie. PofllPt with time during the incubation of a CO-cultureof
1:3 Loctucaict~s luch subsp. /lotis with hcto&cillus delbnreckii subsp.
bulgwictCF/Streptococ~tl~ salivarius subsp. themophilus at level in
standard RSM at 40%. Comsponding primary and derivative profiles of pH and
consistency (Nametrc rheomcter) for each lcvsl of minet enzymes are s h o w for
experiments iepl icated two times (sym bolsnines of differcnt siP/thickness; same pH data
in part as in Fi- A6.12udIb). Regions of local maxima (Pm, & Pb(u3 and minimum
(Pmiain the average rate of buctcriologicrl ridification (IdpWdq) uc indicated.
Figures 6.5, and A6.17-19), but the influence on milk gel development was expected to be

marginal (in any case, not easy to take into account).

(b) Effccts of Gmwth (Gelation) Conditions. What is more important to k a r in mind in

comparative evaluations of gel formation in variously treated milks is that the same amounts C/i

of starter cultures did not produce strictly identical envimnments of continuously decmsing pH

depending on pre-tteatment of milk (most notably heating and protein concentration) and

conditions of gelation (e.g., temperatun). Essentially, the pH of cultutecl milk is detemincd by

the arnount of acid produced by the starters (which is related to the level of inoculation and to the

biochemical performance of the bacteria under the growth conditions used, including culture

medium) and by the acid-base buffer capacity of the milk [see review by Singh et al.. 19971.

(0 Typical variations in pH for cultured and renneted unheated us. pre-heated RSM at 40°C
are contrasted in Figures A6.14~-c. It is well established that the development of acidity is

accelerated in pre-heated cultured milk; the application of relatively high heat to milk used for

cultivation of lactic starters and manufacture of fermented milks is standard practice to promote

the growth of starter micro-organisms. Favourable effects of thermal pn-treatment in relation to

the development of starter populations include teduction of the numbers of undesirable

(competing) organisms inactivation of antimicrobial substances present in raw milk, lowering of

redox potential (teduction of the amount of soluble oxygen), ilteration of the structure of milk

proteins making them more rcedily utilizable by the starters, and nlease of stimulatory (SH-)

compounds [Puhan, 19881.

Concentrating RSM by ultrafiltration, by contrast, tcnded to delay lowering of the pH by

(i%)

lactic fermentation ( F i p m A6.15 and 16), as expccted. This can k related dimtly to greatcr

buffeting effe* (highet protein plus minemls) in concentratcd retentates than in unconcentrated

milk Bnilf et al., 1974; Covacevich & Kosikowski, 1979; Mistry & Kosikowski. 1985; Gastaldi

et al.. 19971and to the notable difficuity in attaining optimum pH for quality in UF pmcessing of
dairy foods [Kosikowski, 1986; Mistry & Maubois, 1993; Rosenberg, 19951. High buffa

capacity places more demands on the m e r cultures since large amounts of lactic acid arc

mquircd to produce pH changes. In general, starter bacteria grow to large numbers in retentates

bwrence, 19891. Non-starter bacteria are also concentrated by membrane processes and the

dificulty to rapidly reduce the pH increases further the risk of growth of microbial contaminanis

in unheated tetentates.

(c) Heat Production Dutinn Rennetinn and Bacterial Grom. Tentative microcalorimctric

measurements were conducted to scc whether any heat effects could ôe detected and quantified

dunng isothermal coagulation of differently tteated milks. The thermogtams for standard RSM

renneted ai level Rx8 at pH 6.4 and 40°C with no addition of stPner cultures (Figures 6.60 and

A6.200) suggest that there was little heat evolved (negative change in ACp over time) in rcnneted

milk under the coagulation conditions considered, that is before about 10 h. Precise interpretation

o f the size of the exotherms was problematic (important experimental variability; Figure

A6.20~).Besides given the time-scale of the runs and the telatively elevated temperature of

renneting, there was a definite possibly that part of the exothetmicity measured arose from non-

preventable misrobial growth, evcn though milk had been treated with sodium azide. A crude

estimate of net heat produced over 20 h of renneting of milk at 40°C gave 2 ca111.7 mL of RSM.

If one considers only the small (but rcpduciblc) cxothennic heat effect mund 5 hours of

incubation, net heat produccd would amount ta about 0.08 caV1.7 rnL o f RSM.This would be in

keeping with the w l y estimation of thermal effects in rcnneted fksh milk by Phipps [1958] (m.

0.1 caIf40 mL at 32OC).

In cornparison, sharp and i m p o ~ nchanges

t in A Q with time were observed in RSM culturcd

at levcl C/8 a 40°C, with or without rennct (level Rx8), as shown in Figures 6.66&c and

A6.20b&c (sec alpo Figure 6.6d).

 ---

--
175 - - a*
125 - - RSM, (irmtt
75 +. control), one replicate
25 - -
-25
-7s - -
-125 - -
-175 -: AQ
-22s b - -
.-
--"
200

Incubation timc at 40°C (h)

Figure 6.Q. Contrasted time-cowses of (a),

QlIumdm ridificuion (bX lssaadn bvdmlvsis and
(c) for renncted ai a
at and 40°C. Rimary and derivative thermal. pH,
hydrolysis, and consi~tencyprofiles w show for single
rcprcsmtative experiments, themai data k i n g obtained
indcpcndcntly fmn othcr data.
 Incubation time at 40°C (h)

Figure 6.U. Contrasted time-courses of hCpt (a),

..
aeiditication (b), and GlQuihwskY&ma (cl for Etuidud
culturcd at C/&RxO at 40°C. Primary and derivative
thermal, pH, and consistcncy profiles are show for single
repnsentative expcriments, thermal data k i n g obîained
indepcndcntly h m other data.
Consistency C (c~.~.crn-') Exotherrnic heat flow
and % K-casein hydrolyzed

u
Rate of change in C Rate of change in pH Rate of change in AQ
dC1dt ( c ~ . ~ . c m - ~ / h ) dpWdt (pH unitfi) dAQldt (pc.ai.s"h)
 Incuôation time at 40°C (h)

Figrre 6.6d. Contrastai time-courses of rate of heat production

AQ for standard RSM differentlv imnetcd at 40°C.
Primary and derivative thennognms am shown for single
nprescntativc irothem.d expcriments (same data as in Figures
6 . b to 6 . 6 ~ ) .
 The production of heat in fermented systems is expected to be contributed to large extents by

the metabolism and p w i n g of starter bacteria. That most of the heat measured was indeed

praduced by microbial fennentation was suggested experimentally by the parallel evolution with

time of heat flux and pH curves (Figures 6.6b%c, and to some extent, Figure 6.6~).

Micmcalorimeüy has in fact proven useful in studying microbiological systems [&ezer, 1977;

Monk et al., 1977; Belaich, 19801 and heat production cm be related quantitatively to the
biomass grown [Schaarschmidtel al., 1977; Gram & Sagaard, 19851.

This raises the question as ta whether and how gelation mechanisms in biologically acidifed

milk may be compounded by the integration of starter culture material ( m e r biomass and

metabolic by-pmducts such as carbon dioxide and capsular or ropy extracellular

polysaccharides) into the protein mabix. Detailed accounts for such efiects are not easily at hand

but it appears that the inclusion of large numbers of metabolically active, and possibly

interacting, bacterial cells (CU. 0.5-1 pm) may indeed modi@ (disrupt) pmtein structures and

their continuity in milk gels (as evidenced, e.g., by apparent weakening of the gels) and the

propensity of the gels to synercse [Tamime et al., 1984; Vlahopoulou et al., 1994; Vlahopoulou

& Bell, 1995; Hassan & Frank, 1997; Hess et al., 19971. (This is notwithstanding the general
stabilizing effect of bacterial exopolysaccharides against syneresis of milk gels.)

Overall, important experimental dificulties were encountered with the use of the calorimeter

and since the appmach did not allow differentiation

,
between heat effects attributable to

coagulation reactions and those attributable to micmbial activity, it was discontinued.

63.5. Micmbial Deterioration of Unacidifled Renneted Milk

A major ciifference betwecn acidifying (cultured) and unaciditied renneted milks is that for

acidified milk, bacterial pmblems (and more spccitically spontamous fermentation processes by

endogenous andlot contaminating micro-organisms) are less likely to intetfere with the process

of gel formation. In unprcscrvd mik r e ~ e t e dat pH 6.4 and 400C. unwuitcd micmbial
acidification usually started to h o m e notable within 5- 10 hours o f renneting (not show). With

addition of NaN3 ta these systems and careful clcanin~isinfectingof the rneasuring equipments

between mns, no imporîant drop of the measured pH occumd for CU. 15 hours or more of mnnet

addition. This is beyond the duration of rnost experiments reported in the following chapter.
 7, SMALL STRAIN DYNAMIC RHEOLOGICAL ANALYSES OF GEL
DEVELOPMENT FROM CULTURED AND RENNETED MILK
II. Resulta and Dlscuasion

7.1. Phenomenology of Gel Deve!opment

This chapter begins with a phenomenological description of rhco-kinetic events during the

setting of renneted milk under variable conditions of decreasing pH on the basis of-

essentially-resuhs of dynamic rheological measurements. The effects of pre-treatment of milk

on gel development are exsmined more closely in a second part, followcd by a general discussion

of salient points.

7mImIm Ekamlpies of Diflerent îjpcs of Ge/a?ion Profles Resulting fmm Vatying the

Concenhafionsof Rennet and Starter Cultures

To illustrate the diversity of gelation profiles resulting h m varying the relative

concentrations of rennet enzymes and cultures of lactic acid bacteria, characteristic progrcss

curves of consistency-pH and viscoelastic moduli-loss tangent against incubation time for

standard reconstitutcd skim milk cultud and renneted ai 400C are contrasted in Figures 7.1.1 to

7.1.4 (and A7.1.1 to A7.1.10). (Typically, the illustrations in the Appendjx are displayed as series

of gelation profiles following a low-high xheme with respect to the concentrations C/i and Rxj of

coagulating agents. Data are also shown for rcplicatcd expriments.) Issues pcrtaining to the

analysis proper of gelation profiles shall be addresscd sepamtely in Sections 7.1.2 and 7.1.3.

Of the several combinations of tcnnet and lactic acidification tried, the conditions of

coagulation ktwcen Rx4 and Rr 16 in the prescnce of bacterial starter scemcd to approximate

those reportcd by van Hwydonk et al. [1986b], N d i et d. [1989, 199 11, Dalgleish & Home

[199la.bl, and Schulz et al. [1999]. [Note the differences in, inter dia, coagulation temperatures

(25-34T)and pH at the moment of mu^ aâdition (6.66.0) investigated in these hidies.]

 ~ ~ - q m q n q ~ q n q ~ q e q o q ~ y o
- m r n * v , \ o r - 00 m -
Incubation time at 40°C (h)

F i 7 1 1 Set of typical consistcncy development curves for standard RSM

cultured at level and at 40°C. Piofiles of consistency C us.
tirne for each level Rxj of rcnnct enzymes are shown for single repnsentative
experiments carried out with the Nametre rheometer; representative pH data are
shown only for the milk coagulated at C/4-Rx4 for clority. (Profiles for replicated
and comsponding mcasurcments of milk consistcncy and pH for each lcvel of
rcnnct are displaycd individually and contnsted to profiles obtained at highcr level
of acidiming starter cultures in Figures A7.1.3a-e .)
Figure 7.1.2. Set of typical consistency developmcnt curvcs for
standard RSM rcnneted at lcvcl B& at 40°C. Profils of consistency C us. time for
each kvcl Cli of acidifying starter cultures are shown for single rcpmsentative
experiments c h e d out with the Nametrc rheometcr, representativc pH data king
shown rlcctively for the milks coagulatecl at CIL, C/4-, and CI8-Rx4. For the
combinations Cf40 and CJl-Rx4,same consistency (and pH) data as in Figures 7.1.1
and A'l.l.la&b. (Rofiles for replicated and comsponding measurcments of milk
consistency and pH for each level of cultures are displaycd individwlly and
conûasted to profiles obtaincd a lowcr level of nnnet enzymes in Figures A7.1.44-
e *)
 -

- 9
0
-
M M ,W b !

(milk sample cultwtd

(Nametrcsamptes,

/ RxO [lactic acid controll

Incubrtion time at 40°C (h)

Figure 7.1.3. Set of typical elastic modulus development curves for standard RSM
cultuicd at levcl ç14 and at 40°C. Profiles of elastic modulus.G'
vs. time for each level Rxj of remet enzymes arc show for singk reprcsentative
experiments curied out with the Carri-Med rheometet. Loss angle S data (tan8 =
Gw/G')are shown only for the milk coagulatcd at C14-Rx4 for clwity; pH data for
this combination were obtaind in independent replicated experiments with the
Namctrc fieorneter. (Profiles for replicated and comsponding measurements of
milk viscous and elastic moduli and 10% angle for u e h level of r m ~arct displaycd
individually in Figures A7.1.9~-b.) Compuc with the counterpart tirne-profiles of
consistcncy (Nametre rheometer) and pH show in Figures 7.1.1 and A7.1.3a-d.
 * 0

pics culturcd &/orremetrd

. (Nameire simples,

Q) (remet control at pH 6.4)

, .

Incubation time at 40°C (h)

Figure 7.1.4. Set of typical elastic modulus development curves for diffemitlv
standard RSM renneted at level at 40°C. Profiles of elastic modulus 0'
vs. time for each level Cli of acidifying slaiter cultures are shown for single
representative experimcnts carried out with the Carri-Med rhcometer. Loss angle S
data (tan5 = Gw/G')are shown oelectively for the milks coagulated at C/2-, C/4-, and
CO-Rx4; pH data for the combinations C/2- and Cl4-Rx4 w m obtained in
independent replicatcd expcriments with the Nametre heometer. (Profiles for
mplicatcd and comsponding measurernents of milk viscous and elastic moduli and
loss angle for each level of cultures arc displayed individiully in Figures Al. 1. lOo-
b .) Compare with the counteqwt timc-profiles of consistency (Nametrc theorneter)
and pH shown in Figures 7.1.2 and A7.1.4a-e.
 In particular, the conditions at C/8-Rx4, 40°C, and ttmeting pH rn 6.4 in the pment work

msulted in gelation profiles vety similar to the oncs reportal by Noël et al. [1989, 19911 at about

C / 4 - a 8 (in tmns of apparent concentrations that is, because diffennt pnparations of cultures

and rennet were likely used), mund 30°C, and nnneting pH 6.6-6.0. A central festure of the

rheological time-profiles obtained under such conditions is the distinctly 'bimoâal' (hump

shaped) chamcter of the response measured over the course of coagulation. In the prescrit

experiments, the rheograms for consistency and moduli of gelling milk vs. time showed two local

maxima with variable resolution and amplitude depending, it seemed, on the mode of coagulation
(to be developed in Sections 7.1.4 and 7.1 S).

On combined enymatic-acid coagulation at rennet concentration Rx 1, that is, the lowest

level of rennet addition investigated in this work, the consistency and-to some extent-moduli

increased essentialiy monotonously with time beforc approaching asymptotic pseudo-equili brium

values, or slightly decreasing ultimately. An interesting phenornenon consistently encountered at

this concentration of rennet was the existence of a shoulder in the coagulation curves about

midway through gel development (Le., Ca. 30 min- 1 h aAer the onsct of measurable coagulation).

The controi cuves for milk coagulated exclusively by rennet action at pH 6.4 or by lactic

acidification were characterized by an apparently steady (sigmoid-like, i.e., S-shaped) rise in gel

consistency and rnoduli over time before the viscoelastic parameten leveled off or decreased

slightly, as expectcd [e.g., Scott-Blair & Bumett, 19584.6; Tokita et al., 1982; Johnston, 1984;

Bi liadcris et al., 1992; Mpez et al., 19981.

(a) C consi .

Figures 7.1.5~-cand 7.1 .&c show that the rheological phenornena rcgistered by the Nametrc

viscorneter and the Carri-Md rheorneter compued well with respect to the variations brought

about by changing the proportion of m u ~(RxO-Rx4)

t and acidifying starter cultures (C/4), at

least for standard low-hcat RSM.

 RSM,C / 4 - m
(Iactic acid coatrol;
rheometer repliCates 1 & 2)

F u 7 . 1 . Conûastcd profiles of gel development obtsined for

cultured at level at 40°C using the (upper panel)
and the Cnm-M.d (lowcr pancl). Primary and derivative profiles of
consistency C (and pH; Nametre hoomcter) and elastic modulus G' (and loss angle 6;
Carri-Med rheometer) of milk arc shown for independent cxperiments replicated
times (symboldincs of different sizelthickncss). The a m w points to the region of
apparent local minimum in the average instantanmus rate of comistency development
dC1dt and to the comsponding (approximative) values of pH.
Figure 7.1.Sb. Contrastcd profiles of gel development obtained for
a
cultured at level and remetcd at levcl Bal at 4 0 " ~usine the
(upper panel) and the (lowcr panel). Phw and derivative profiles
of consistcncy C (and pH; Namette thcorneter) and elastic mdulus G' (and loss angle 4
C h - M e d rheometer) of milk am shown for independent experiments rcplicatd two
times (symbols/lines of different size/thickness). Amws point to the region of local
minimum in the average instantantous rate of consistaicy and elastic modulus
development dC/dt and dG'/dt, and to the comsponding (approximative) values of pH.
Figure 7.1.5~. Contrasteci profiles of gel developmcnt obtaincd for
cultured et lcvel and tcnnetcd at level at 40°C using the
(upper panel) and the (lower panel). Rimary and derivative profiles
of consistency C (and pH; Nam- hcometer) and elastic modulus 0' (and loss angle 6;
Carri-Med heometer) of milk am shown for independent cxperirnents replicated two
times (symboldines of differcnt sidihickness). h w s point the region of local
minimum in the average instantantous rate of consistency and elastic modulus
developmcnt dCIdt and dG1/dt,and to the comsponding (approximative) values of pH.
 L œ

vw Pmheated RSM (90°C-1 min), :-

Cf4-Brp (jactic icid control; :_

a Pm-hritcd RSM
cari%& CI4-rn (Iactic acid control;
thtamefer mplicatcs 1 & 2)

Figure 7.1.6~. Contrastcd profiles of gel development obtained for

cultured at level at 40°C using the (upper panel) and
the (lowet panel). Ptimary and derivative profiles of consistency C
(and pH; Nametre rheometer) and elastic modulus O' (and loss angle 8; Carri-Md
rheomctcr) of mik are shown for indcpndent sxpcriments rcplicated two times
(symbol~inesof diffemnt rizc/thickness). Arrows point to the rcgion of appuent local
minimum in the avmge instantanmus rate of consistency and elastic modulus
developmcnt dC/dt and dGV/dt,and to the corrcsponding(approximative) values of pH.
Figure 7.1.6b. Contrasted profiles of gel development obtaineâ for
cultured at level and renneted at level Bal at 40°C using the
(uppet panel) and the rhn>metp (lower panel). Primary and derivative profiles
of consirtency C (and pH;N a m e rheometer) and elastic modulus G' (and loss angle 6j
Carri-Med rheometer) of milk are show for independent expriments isplicated two
times (symbolsAines of differcnt size/thickness). A m s point to the region of local
minimum in the average instantanmus rate of consistency and elutic modulus
developmcnt dC/dt and dGg/dt,and to the comsponding (approximative) values of pH.
Elastic modulus G' (Pa) Consistency C (cp.gcni3)
and dWdt (Pa&) and dCIdt (c~.~.cm%)
 The apparent perturbation in the experimetltal curves mund midcourse of gel fornation at

the lowest addition of coagulating enzymes seemed amplified (betteddifferently molved) in the

time-pcofilcs for dynamic moduli, however, although no pmnounced local minimum in gel

moduli was found at this nlatively low concentration of muia (Figures 7.1 .Sb and 7.1 66).

Pt is noteworthy also that the appeannce of the time-derivative curves of consistency and

moduli tended to differ with respect to the relative importance (amplitude) of the two peaks in the

derivative cuives. In ternis of derivative consistency dC(i)ldt, the first (major) peak usually

preceded a less prominent secondary peak/shoulder. The trend seemed to be reversed for

derivative curves of moduli (e.g., dG '(r)/dt), the first pealdshoulder preceding a more prominent

peak. As shall be seen in Sections 7.1.4, 7.1.5 (and 7.2.3), this was particularly conspicuous for

(pre-heated) milk samples cultured with no or little (below Rx4) rennet (Figures 7.1.60-c).]

Probably these apparent modulations of the effects messured depending on the rheometer

used reflected the better/differential sensitivity of the Carri-Med cornparcd to the Nametre.

Perhaps the fact that the property determined by the Nametre is more relatcd to the viscosity than

to the rigidity of the gel k i n g formed reduces the sensitivity of the viscorneter to the physical

changes that occur as the sarnple attains more solid-like States.

7. I.2. AnaiysLr of GeIation Profles

(a) Usefulntss of Time-Derivative Cuwes. Data obtained directly h m the curves of consistency

and moduli development are only one facet of the information that may be obtained h m such

curves. Calculation of the fim detivative with respect to time (difference quotients) of

viscoelastic parameten gives a measure of the kinetic aspects of gel development, that is, of the

rate of change with time of the processes involved in coagulation. This is commonly done, in

particular to determine the maximum rate of coagulation or gel firming (i.e., the tangent to the

inflection point of consistency or moduli W. time plots). It is noteworthy that (early) changes in

the rate of gel development may be expectd to k of importance in relation to hter processes of

syneresis of gel.
 What is more, graphical display of time derivative data (Le., gradient of the curves) vs. time is

adapted to emphasize subtle changes in the morphology of the primary curves or to reveal barely

discemible featums along the cwes, as mentioned in Chapta 6, Section 6.3.4a&b for the

evaluation of pH data (e.g., Figure 6.1 t 6). [See also the analyses of coagulation parameters by

Storry & Ford, 1 9 8 2 ~McMahon et al., 1984~;Hardy & Schet, 1988; and Schulz et ai., 1997a.l

As well, derivative plots can assist in the location of characteristic pointdregions along the

original curves and the speciilatkn about the lirnits of underlying tvents.

The usefuinesr of derivative plots of gelation curves in the context of comparative studies of

gelation pmcesses in differently coagulated milks is highlighted in Figures 7.1.7adb (A7.1.11-

12). At low addition of rennet, for example, the 'kink' ('shouldered peak') in the traces of

consistency vs. time stood out in the curves of fint derivative consistency [dC(t)/dt] vs. time as a

transient decrease and minimum in the instantaneous rate of consistency developmmt, Le., the

appearance of a second peak in the fvst derivative curves. (A üuly sigmoidal function for

biphasic kinetics would be charactetized by a single peak comsponding to the acceleration and

deceleration phases, as evidenced in Figure A7.1.13 .)

The nference curves for acid coagulation presented an apparently homologous-less

apparent but reproducible-irreguiarity (apparent 'sinplarity'), which would have escaped

observation if it had not been for inspection of the derivative curves. Admittedly, the possibility

of artifacts (e.g., unevenness of derivative traces and incidence of synensis) ought to ôe borne in

mind. Still, the= is satisfactory evidence which supports the suggestion thrt the effect detccted

was indeed common to the coagulation profiles of most fermentcd sampks, whether or not rennet

had been added, only the second peak in the first derivative cuives came CO more complete

development with adding coagulating enzymes (sec discussions under Sections 7.1.4, 7.1 .S. and

7.3.3).
Figwre 7.1.7~. Typical curves of consistcncy devclopment vs. timc for siandard RSM
cuiturcd at level and at 40°C, and & Pf- . . data
[Le., instantancous rate of change of consistcncy C with time or gradient of consistcncy
curvcs, dC(t)/dt] for defining characteristic poinWrcgions dong the primary curves.
Comsponding primuy and derivative profiles of consistmcy (and pH) of milk for eoch
level Rxj of rennet enzymes are s h o w for cxperiments replicated two times with the
Nametre rheometer (symboldines of d iffercnt sizc/thickness). Tuming points in the
profila of gel consistency [i.e. ,(apparent) positive local minimum in dC(tydt, (dCldt),
arc highlifited, togethet with the comsponding approximative values of pH.
 Consistency C ( c ~ . ~ . c r n - ~ ) Consistency C ( c ~ . ~ . c r n - ~ )
and dCldt ( c ~ . ~ . c m - ~ / h )
# B
i
i L - - W h )
ui
0
O
0
th
0 0
u i O u , O u i
6 6 0 6 0

+PP-rPP?YLP
* , o ~ o m o ~ o m o u i
pH and dpH/dt (pH unitdh)
 Patterns in the formation and dynamic behaviour of milk gels can therefm bc clearly
identified by simple derivative analyses of coagulation curvcs. It is intcicsting to note also that

the (asymmetric) sigrnoid-like naturc of coagulation kinctics can be mdily appmiated using
awtiliary derivative representations (e.g., Figure A7.1.13).

Likewise, replotting viscoelastic date as a fûnction of timc-dependant variables such as pH or

proportion of K-casein hydroiyzed by rennet rather than time cm facilitate visualization and

Vi~rpretationof the results, e.g., to e m p h v i p the deteminant influence of the pH (Figures

7.1.8~-cand A7.1.14-17; al- A6.lOd-1 ld).

(b) Conversion of w-Cwiq. Progress curves for the enzymatic conversion of u-cascin to pmo-w-

casein in standard RSM at 40°C under sorne of the conditions of rennet and decreasing pH just

delineated are shown in Figures A7.1.18-19, along with the comsponding rheological and pH

profiles in Figures A7.1.2û-21 and 7.1.9. As indicated in Chapter 5, SDS-polyaciylamide gel

electrophoresis was a practical alternative to chromatographic techniques, if not for detailed

kinetic analyses of the primary phase of renneting (die sensitivity of the method was not

sufficient to study the reaction during its w l y stages), at l a s i for visualizing the enzymatic

reaction and estimating the extent of hydrolysis of micellar K-cwin at different stages of gel

developrnent (summaries in Table 7.1 and Figures A7.1.6 1a-e; comsponding average values of

pH in Figures A7.1.60~-e).

Essentially, at concentrations beiwan Rx4 and Rx16 of rennet, and betwcen Cl8 and Cl2

(CA not tested) of acidifying culhvcs, an average of about 55-6W of K-casein had k e n
hydrolyzed when the consisiency sîmted to incrcase meawrably at pH ktwcen 6.4 and 6.0. (Ihe

fact that perceotage hydrolysis at coagulation time remained esscntially constant with increasing .

culture andor mnnet concentration ought to be considcd in puillel with the decrase in

coagulation time; Figure A7.1.59a.)

 RSM, C I I - r n
(hctic acid controb)

Consistency us. pH

Smaller dots = C/8

C/4
cn
Largcr dois = C/1

pH of miik cultured at 40°C (values in reverse order)

Figure 7 m l m 8 ~ . Typical profiles of consistency C u,pti for standard

RSM differrntlv &J& at 40°C. Refiles for a c h levcl Cli of acidifying starter
cultures arc shown for single rcprcscntative expenments carriecl out with the
Nametre rheometer ( m e primary data u in Figure 7.1. Ma). The urow points to
the region of pH for which an apparent local minimum in the average instantancous
rate of consistency devclopment dC/dt was obsewed. (ProfIles vs. pH for rcplicated
and corresponding measumments of milk consistency and pH are show in Figure
A7.1.17a.)
 pH of milk culturcd and renneted at 40°C (values in reverse onier) .

Figure 7.1.8b. Typical profiles of consistency C y ~ ptl

, for differrntlv
standard RSM renneted at level at 40°C. Profiles for each level Ch' of
acidifying starter cultures arc show for single npnsentative cxperimcnts carried
out with the Nametre rheometer (same primary data as in Figure 7.1 .l au). The
airow points to the region of pH for which a local minimum in the average
instantaneous rate of consistcncy development dC/dt was observcd. (Profiles vs. pH
for nplicated and comsponding mewrerncnts of milk consistency and pH are
shown in Figures A7.1.16and 17.)
 I
a
ConsUfmcy vs. pH
a
Y
Smailtr dots = C/8 0
cf4 I a

pH of milk cultured and rennetrd at 40°C (values in merse order)

Figure 7.1.û~. Typical profiles of consistency C y~. for diffenntlv

standard RSM renncted at level at 40°C. Profiles for each level C/i of
acidiQing starter cultures am show for single reprcsentaîive expcrirnents &d
out with the Nametrc hcomctcr (same primary data as in Figure 7.1.186). The
arrow points to the region of pH for which a local minimum in the average
instantaneous rate of consistency developmmt dC/& was obscrved. (Rofiles vs. pH
for replicated and comsponding measurements of mik consistency and pH are
shown in Figures A7.l .l6 and 17.)
 *~'mm-=:~rA

m4m2m
RSM, Wh1
4 r (replirrtea 1 & 2)
f .A9.- =

Figure 7.1.9. Contrastcd evoiution of die aeiccnapc

.
p f m Idmvitive)
c, and gIi pf mfi with tirne for standard RSM culturcd at Ievcl and
diffcrrnilv at 40°C. Profiles of % hydrolysis of K-casein(SDS-PAGE) are show
for experimcnts replicated two t h e s (triangles of diffmnt sire, with average % displayed
as the largest filled syrnbols and c w e s only meant to guide the eye through experimental
poinîs, as in prcvious figures), together with comsponding pmfiles of consistency
(Nam- rhcomctcr) and pH (thinnest Iines and small triangles, nspectivcly).
Comsponding C and pH data are also shown for independent expcriments replicated two .
times (symbols/lincs of dincmt sizcHbickness; same data in part as in Figure A7.1 Ac).
Table 7.1. Pemntage hydrolysis of u-casein', as estimatecl by SDS-polyaciylamide gel
eloctrophoresiq at various stages during the coagulation of standard reconstitutcd skim milk at
40°C under different conditions of concentration of acidifying starter cultures (Cli) and rsnnet
enzymes (Rxj) (same data as plottecl in Figures A7.1.61~-e). Diffcrent regions of extent of
hydrolysis are highlightcd, ie., < ca. 55%, [55-ca 60"!], [ c a 60-75%], and > eu. 75%.

Rx1 Rx4 Rx8 Rx16

At the onset of coaplation (Le., fint

memurable i n c w in consistency)

At point of maximum rate of consistency

development dC(t)/dt

At point Pm (or its deemed equivalent,

ie., local minimum in dC(r)/dt)

At point Pmb(or its deemed equivalent,

i.e., focal minimum in dçO/dt)

At maximum consistency after points Pm & Pm

(or its deemed equivalent)

8
Results arc given as arithmetic mean of two independent mplications.
' ~ o dctennincd.
t %ot applicable.
About 70-80% of u-casein had k c n hydrolyzcd at the tuming point Pu, of first local consistency

maximum [Le., a m dC(t)ldt] at pH bctween 6.0 and 5.5, and the cxtent of hydrolysis leveled off

amund 90% plus pACr incubation for about 8 h (pH 4.5-4.0).

At rennet concentration Rxl (regarâkss of C/i, esstntially), K-casein hydrolysis was of the

order of 3O-3S% at the ons* of coagulation at pH 5.&5.7.40% at the point decmed quivalent to

P&PIbt [i.e., local minimum in dC(r)M st about pH 5.2, and 45% after about 8 h.
For control milks r e ~ e t e dat pH 6.4, the proportion of hydrolysis a&coagulation time and

afier about 8 h was less than or mund Ca. 45% at concentration Rxl; and about 50 and 75% on

average at levels ktween Rx4 and Rx 16. This is about 10-2û% less than previous estimations of

conversion ai CT under comparable conditions of pH, but then one ought to fator in the

difference in temperature, i.e., 40°C in this work vs. 25°C [Chaptcr 5 and van Hooydonk et al.,

1986bJ.Kinetic aspects oC~-cascinpmteolysis will k discussed fifier in following sections on

the particulars of gel development for standard RSM and, to a lesrr extent, for RSM pre-hewd

at 90°C-1 min.

(c) Variations in ANS-Fluorescence. There was little to note regarding die variations of ANS-
fluorescence o v s time for milks diffemtly cultured and renneted at 40°C and for that reason this

line of investigation was discontinued. The curves in Figure A7.1.22 show that the relative

intensity of fluorescence in the supernatant obtained by centrifbgation decmased initially and

remained constant within the uncectainty in the measuremcnts aiter the consistmcy started to

increasc. This bchaviour merely reflccta the f a t that an incmsing m i o n of casein-ANS

complcxcs p a s d to the precipitate phase as aggregation and, consequently, coagulation

proceeded .
The rclatively carly decrrw in fluorcscenc~lmdescribed by Peri et al. [1990] for
rmncting at constant pH in the range 6.8-6.3-suggests thrt the @ara)casein particles kgin to

aggrcgatc early in the coagulation pmcepa, more pccisely at ôelow 40-5096 conversion of K-
casein for C/8-Rx8 milk in a region of pH betwecn 6.4-62 at 4 K . For acidifjhg controls at C/8,

it semed chat coagulation (first measurable incmase in consistency) took place k f o n the

readings of supernatant fluorescence reached low limiting values. This may be understood in

terms of the importance of interaction forces other thm hydrophobie effects (specifically,

electrostatic interactions) in the gelling of acidifiai milk [ s e also the conclusions of Lefebvre-

Cases et al,, 19981.

7.1.3. TlCe Problcni o / S ' e m b

As alluded to earlier, Chapter 6 included, the possible contribution of secondary processes of

syneresis to the effeas rneasured tumed out to be an important point in question. It is well known

indeed that the conditions under which the observations were made in this midy (Le., fairly long

run times, decreasing milk pH, and relativcly high temperature) an conducive to the separation of

whey h m the casein gel. The nature of the tests performed may accentuate the problem as even

small shear strains may k sufficient to induce syneresis in the gel, in particular when large

sample volumes (containers) are used. AS mentioned in Chapter 3, the probkm posed by
syneresis in the context of dynamic rheological analyses is that it may hinder proper adhesion

between the geVcurd and the measuring element. This may transfate into biased estimations of the

viscoelasticity of gelling systems and compound the enc*J of changing physico-chernical

environment with tirnc. Except in extrcme (obvious) cases, the magnitude of the problem is

difficult to assess usually, particularly when direct observation of the gelling specimens is not

possible as is the case for continuous monitoring with the Carri-Med rheometer.

(i) Several approaches wcre taktn to check for possibly confounding effects related to (large-

scale) synmsis, in addition to visual checks for the samples musuicd using the Narnetre

viscorneter. In pomc experiments we sought to induce synercsis delibnrtely to get an idea of what

happens in terms of the parameters measund when the gel is allowed to set with showing

(visible) evidence of whey oepamtion. The early incidence of extensive syncrcsis under the
conditions of hi@ rcnnet in Fipres 7.1.10 and A7.1.23adkb probably resulted h m rapid

enzymatic coagulation at slightly acidic pH and mlativcly hi& tmipcratwc.

For such sunples, signs of detachment of the gel h m the sensing sphere of the Nametre at

the centres where the whey accumulated became apparent within about 0.5-1 h of coagulation.

Later, pools of syneretic liquid formed mund the sensor. The pronounced decrease in

consistency registend during this period (and the concomitant increax of the temperature

measured to about initial values, Figure 7.1.10, upper panel; also Figure A7.1.236, lower panel)

could be attributed to the fact that the liquid phase was king measured rather than the coagulum

phase.

Comparativcly, samples such as those whose coagulation curves are shown in Figures

A7.1.24~-cshowcd little appreciable syneresis within the time-fime considered. Only a thin

layer of moistun appeared on the surface of the gel at mund the time at which the consistency

decreaxd slightly beforc stabilizing ultirnately (le., within 3-6 h of measurable coagulation). In

general with standard milk, it seemed that satisfactory conditions of rneasurement resulted in

mon continuous and regular (symrnetric) gelation profiles overall (e.g.. contrast the satisfactory

conditions of memurement in Figure A7.1.24~ to the degrading conditions in Figures

A7.1.24dde). This fcahirc was also readily apparent in the rheological profiles of coagulation of

N&l et al. [1991].

Syneresis manifestcd somewhat differently in some cases. The shape of the later put of the

curvcs illustrated in Figures 7.1.10 (lower panel) and A7.1.24d (upper panel) was also modulated,

it appeamd, by (sporadic) syneretic processes. For these samplcs, the gel scemed to be contracting

around the rneasmgngsphere and the expclled whey tendcd to collcct at the walls of the kaker

holding the mik some time a k r the secondary risc in consistency (Le.. 3-5 h afbr coagulation).

Relatively smooth, apparcntly satisfactory protilcs wcrc obtaind but the Iatcr, incrcasing portion

of the curves pmbably is not vcry mwingfùl (exaggcratcd) since the gels wen not only

kcoming '~tmnger'but also loosing moisturc (shrinking) during the Iater stages of experiment.
Figure 7.1.10. Paralkl evolution of consistcncy C us. time for standard
RSM coagulated at 40°C under conditions of acidity and renneting conducive to
pf O(il. Comsponding primary (and derivative) profiles of
consistency, temperature, and pH of milk for cach combination of the levels C/i-Rxj of
acidifying starter cuihires and rennet enzymes arc shown for experiments rcplicated two
times with the (symboldlincs of differcnt siahhickness; x e Figure
A7.133b for the piofiles of gel dcvclopment obtained at C18-Rx160using the Carri-Med
rheomeier). Rcgions conesponding to the occumnce of apprcciable macroscopic
syneresis of gel are indicatcd.
 (ii) Another way of checking for effects introduced by syneresis was through modifications of

prc-test and/or test conditions. Tmtments such as pre-heating and concentration of milk and

relatively low coagulation temperatures (< 40°C) are known to effectively d u c e syneresis.

lndeed, milk so treatcd and coagulated showed v i ~ l l no

y synemis thioughout gel development

under the conditions of renneting and acidification investigated (Figures A7.1.25-26, A7.1.27-28,

and A7.1.29; refer to Section 7.2 for further results and discussions). This, of course, does not

imply that no syneresis WRS possible in these samples, thougb it mu& have bcen mudi less

marked than in standard milk. As exemplified in Figures A7.1.25-29, particularities of combined

rennet-acid coagulation were also present in the rheologicsl time-profiles of sarnples coagulated

under test conditions that minimized syneresis.

This gave confidence that, unless othenvise specified in the discussion, the rneasurements

report4 reflected changes in the physical characteristics of the systems snidied rather adequately,

and not simply perturbations arising from troublesome syneresis. It is probable, however, that

some of the effects found were intimately related to and perhaps overlapped with (micro)

syneretic events, as discussed under Section 7.3.3. To be sure, as pointed out by NMl et al.

[1991], it is important to k aware of the relatively limited (quantitative) reliability of

(rheological) data collected at long incubation times in geneml.

Descriptions of the particulars of gelation profiles for control reconstituted skim milks

coagulated by m e t enzymes vs. bacteriological acidification are given next as references for

subsequent analyses of gelation in differcntly cultured and renneted milk systems.

7.1.4. Analys& of GeIation hflafor Reference MN& Systems

(a) Evolution Over Time of (Derivative1 Consistencv. Dvnamic Moduli. and Loss Tannent for
Standard Milk Coamilateâ bv Rennet at Constant DY.Gelation profiles characteristic for standard

RSM m e t e d at constant pH of 6.4 and 400C are s h o w in Figures A7.1.30a-c and A7.1.3 1o-c
for experiments with the Nametrc viscorneter and the Carri-Med ihcomctcr, respeaively. (For

RSM pmheated at 90°C- 1 min, sec Figures A7.1.32 and 33.) For cnzymatic controls, consistcncy
and dynamic moduli, and their time-dcrivatives, started to incrcasc measurably Mme time afbr

the addition of remet cotrcsponding to the secondary (non-enzymatic) phase of casein

coagulation. Under standard experimental conditions in this work at rennct levels k t w a n Rxl

and Rx16, appmximately 45-SM4 of micellu u-casein had k c n hydrolyzed at the onset of

coagulation at pH 6.4 and 40°C (sce alw Table 7.1). Note that, as reported initially by Scott-Blair

& Oosthuizen [1961 study of rennet coagulation a mund neutral pH by viscometry], them al=

was widence of thc primary (entymatic) phase of renneting as an early (limited) decrease of the

consistency prior to coagulation (not show nor systematically examineci).

(4 Gel asxmbly and firming were reflected in the development of consistency and moduli
over time, rapidly at first (before inflection) and thcn more slowly before apparent stabilization

uitirnatelpi.e., essentiaîly biphasic (asyrnmctric) sigrnoidal kinetics of gel development, as

evidenced also by, e.g., Scott-Blair & Bumctt [19580,6], Tokita et al. [1982], Bohlin et al.

[1984], Johnston [1984], Nd1 et al. [1989], and ndpa et al. [1998]. (One may think of there

king an analogy with an autocatalytic or self-entcrtained phenornenon.) Unlike in the work of

Storry â Ford [1982a.b] with k s h whok milk at constant pH between 6.6-6.0 at and klow

3S°C, no clear secondary maximum in rate of gel development (tirnaderivative consistency and

elastic modulus) w u discemible over time, at last at and above pH 6.4. The tailing-off of

derivative traces, Le., the rclatively sustained rate of devclopment (firming), may still bc

interpreted as an indication of the gndual intcgntion of îascin particles into the gel structure.

(U) With the Carri-Med theorneter (Figures 7.1. I 1, and A7.1.3 1 and 33), transition fiom fluid

miik (loss tangent, ton 6= G "IG' > 1) to viseoclastic mui* gel was also evident fiom the sudden

decmse of l o s tangent (or loss angle di), which then rcmained about constant during the whole

pmess of pl devclopment. (Note that shvp initial d e c m in fun G was not always obsmred

because of unnliable mcasumnents kfore the gel point.)

 - m

-
œ

I I R=f,*ry
I I ( m ~ i tcontrob
t 8t pH 6.4)
0

œ
œ 9 -0
*- m
*

LOSSangle 6
.
œ
S

m
(samplcs rcnneted at and Rx4)
I

Elastic modulus G'

(at Rx 1:no measurable

Incubation time at 40°C (h)

Figure 7.1.11. Overview of elastic madulus development curves for

standard RSM treated with 0.02% N d 3 (wh) and diffsrantlv a IIpfl at
40°C. Profiles of elastic modulus G' vs. time for each level Rxj of rennet enzymes
are show for single representative expcrimcnts funcd out with the
.- Reprcsentative loss angle 6 data (tan6 = G"/Gt)are show sekctively for
the milks renneted at level Rx8 and Rx4. (Profiles for ieplicated and cornsponding
measurements of milk elastic moduli and loss angle for each level of rennet are
displayeâ individually in Figures A7.1.3 1b&c .) Compare with the counterpart time-
profiles of consistency (Nametrc rheometer) and pH show in Figures A7.1.30a-c.
 In rennet controls, the evolution of ton 6 points to the development of important relative

elasticity within the gel nstwork and the acquisition of characteristic rhcological behaviour very

euly in the sctting of gel. Relatively stable values of tan 6 over time suggest that elastic and

viscous compnents contribute in constant proportions to increasing gel sûength. Similar

observations have been made, e.g., by Bohlin et d. 119841, Walsüa & van Vlid [1986], Dejmek

119871, Noël et al. [1989], and L&pezet d.[1998].

nie suggestion by Dcjmtk and Walstn & van Vliet that the nature of interactions dominating
the rheological behaviour of enzymatic casein gels does not evolve has to be considered with

caution, however. It is also possible that the use of tm 6 t o resolve changes in the interactions

within the gel be limited. Control rennet gels measmd in this work were characterizcd by

asymptotic values of fun 6 at long times mund 0.50-0.53 (6 27029~).nie values of 6 for

mature rennet gels quoted in the literature tend to vary depending on the publications, which

probably refiects different instrumental and essay conditions, including frequency and

temperature: for exampk, around 15-16' according to Bohlin et al. [1984] (0.5 Hz-3 IV),

Dejmek [1987] (0.1 Hz-3 1°C),N d l et al. [19?39] (0.1 Hz-30°C), and L6pu et al. [19981 (1 Hz-
30°C); 28O according to Zoon et al. [1988b] (+ 0.2 Hz-4OT); and 29O according to van Vlict et al.

1199la] ( x 0.2 Hz-400C).

Occasionally, a slight local maximum of tm G(minimum relative elasticity) was measured in

the eady stages of milk coagulation by rennet, befon the point of inflection in the curves of

elastic rnodulus at pH 6.0 and 40°C (Figure 7.1.12). This detail would have gone unnoticed or

interprcted as an artifact had a similar hcological responr not k c n apparent also in the work of

Bohlin et al. 119841 and Dejmek [1987] (standard RSM and k s h whole milk measured at

appmntly unadjusted pH around 30°C in a Bohlin Universal rheometcr).

 RSM,Cû-Br4
(rcnaet coatml i t -5.8;
replicatcs 1 & 2)

o a
.
O
- q
L1
* : m
2 " 2 V)
X w
Incubation time at 40% (h)
Figure 7.1.12. Profiles of elastic modulus G', its rate of change with time dG'Idt, and loss
angle 8 (tan6 = G"/G') us. timc for
..
standard RSM partially Me-acidified lQ
Pincccpt a pf pti éq and rcnneted at levcl W at 40°C. Comsponding
primary (and derivative) profiles of O' and 8 (Cim-Mad for each value of
re~etingpH are s h o w for cxpcrimcnts repliutcd two times (symboldiines of d i f f i t
sizelthickness). Amws point to the region of apparent local maximum in loss angle. No
local minimum in the avenge instantancous rate of elastic modulus development dGQ/dt
was apparent, unlike in Figure 7.1.13.
 Such a responsc may indicate limiteci relaxation (loosening) of the relatively weak para-

casein network fomed initially, possibly facilitating rearrangcment of the gel. This may Ilad

support to the views of gel assembiy as a multiphasic process discussed under Section 2.2.36 of

the literature survey.

(b) Effects of Concentration of Rennet at Constant DH.Increasing the concentration of rennet

enzymes from Rxl to Rx16 at constant pH (6.4) and temperature (40°C) had the anticipated

effects of reducing coagulation tirne, and incmsing ratc of fiming and (apparently) maximum
consistency and rnodulus of gels (summaries in Figum A7.1.59a, 58a,d&J and 62a,d&$). Still,

the chmcter of the response measured thmughout gel development was conxwed, albeit on a

different time-sale (Figures A7.1.30a-c and 3 1u-c).At the onset of measurable coagulation, the

average values of K-casein hydrolysis estimated by SDS-PAGE were comparable within

experimental variation at al1 levels of rennet investigated (around 4540%).

The values of tan Gafter a few hours of aging were similar within experimental variation as

well, which is to k predicteâ if tan 6esxntially nflects the type of the dominant interactions

within the casein nctwork. Mainly the number of interactions is expected to change on increasing

the concentration of rennet.

(c) J3f'cts of Relativelv Acidic DH at Rennetinn at Constant Concentration of Rennet. For

cornpuison, the efkcts of constant, lower than standard pH on gel formation fiom renneteci

standard RSM were investigated in experiments in which milk was partly acidified by direct

addition of lactic acid prior to renneting at Rx4 or Rx8 and 40°C (Figures A7.1.34adb and

7.1.12;Figures A7.1.36a-c for RSM pre-heated at 90°C-1 min). Known e f f ' of partial pre-

acidifcation included duction of corplation tirne by rennet and degree of conversion of

micellu u-casein at coagulation (not tabulateci), and restoration of the mnetability of pre-heatd

RSM.Measumnents of viscalastic panmeters wcrc complicated by syneresis, but acidification

to constant pH ktwcen pH 6.4 and 6.0 at constant level of rcnnct appeated to incrcasc rate of

firming and maximum consistency (and modulus).

Comparable asymptotic values of tan 6 were obtained (m 0.50-0.53, Le., 6 m 26-2g0), in

qualitative agreement with the nsults of Lopez et al. [1998] bctween pH 6.74 and 6.25. Below

pH 6.0, it seemed that maximum gel consistency and modulus decreased, and that tun 6 increased

(10.70, Le., 6=3 3 O et pH 5.5). 'Ibis would also be in line with pnviously published observations

[e.g., Walstra & van Vliet, 19861 and may point to a shift in the type of dominant interactions

within the gel on lowering the pH.

An interesting feature in some profiles of consistency YS. time for partly acidified renneted

RSM was the existence of a shallow xcondary maximum (shoulder) in the time-derivative
(finning) curves mund the time of inflation (Figures 7.1.13 and A7.134aBrb). An apparently

similar fcature was observcd for pre-heated milk renneted at slightly acidic pH (Figures

A7.1.36aûkb. lower panel). This may concur with the observations and intcrpretation of rennet gel

development proposeci by Stony & Ford [1982a,b](summary under Section 2.2.36). No such a

particularity wap apparent it seems in our analyses of gel modulus fiom complementary testing

with the Carri-Med rheometer, however (Figures 7.1.12 and A7.1.36c), although testing with this

rheometer revealed a slight local maximum in loss angle in the early stages of coagulation

(Section 7.1.4a).
(d) Com~arisonwith Milk Coamlated bv tactic Acid. Standard control milks coagulated by
bacteriological acidifiçation at 40°C tcndcd to have similar instrumental consistency and lower

viscoclastic moduli compared to rennct controls, but otherwisc analogous (sigmoid-like) curves

for gel dcvelopmcnt were obtaincd (Figures 7.1.14-1 5a&b; Figures 7.1.16uûéb for RSM pre-

heated at 90°C- 1 min. Appendices 7.1.3 F 3 W and 7.1.39-4ûu-c&d).

 (remet control at

0 q
O
- * H z
C
" 2 " 9 VI
2 w
Incubation time at 40°C (h)
Figure 7.1.13. Profiles of consistcncy C and pH of mik, their rate of change with time
(Le., dC/dt and dpWdt), and temperature vs. time for standard RSM
partially
. . $Q dinmnt a Pf b 69 and rcnneted at level && at
40°C. Comspmding prirnary (and derivative) profiles of consistency (Nametre
rheometcr), pH, and temperature for each value of tenncting pH are shown for
experimcnts rcplicated two tirncs (symbols/iincs of differmt sizdthickness). Arrows
point to the region of appucnt local minimum in the average instantancous rate of
consistency dcvelopmcnt dcldt.
 Incubation time at 40°C (h)

F u 7 . 1 . 1 Ovewiew of consistency development curves for

standard RSM diffnentlv at 40°C. Profiles of consistency and pH vs. time
for each level C/i of acidifying starter cultures are shown for single repre~ntative
experiments carried out with the Nametrc rheometer. (Profiles for replicated and
comsponding measurements of milk consistency and pH for each level of cultures
are displayed individually in Figures A7.1.37c&d .)
F .
u 7.1.14. Ovewiew of time.denvative Pf u>nsistencv (i.e. , rate of
change in consistency C with time, dCldt) for standard RSM
.
et 40°C. Profiles of derivative consistency and of pH vs tirne
for each level C/i of acidifying starter cultures arc show for single representative
experiments carried out with the Nametre theorneter (wne p r i m q data as in Figure
7.1 .Ma).
Amws point to the region of apparent local minimun in the instantaneous rate of
consistency development dC/dt and to the comsponding (approximate) values of
pH. (Profiles for replicricd and comsponding meaourements of milk consistency
and pH for each level of culhires are displayed individually in Figures A7.1.37c&d.)
 m.

Elastic modulus G'

Incubation time at 40°C (h)

Q u m 7.1.1Sa. Ovcrview of elastic modulus development curves for

MM at 40°C. Profiles of elastic modulus G' and loss angk 6
(tan6 = Gf'/G') vs. time for each levd C/i of acidifjmg starter cultures are shown for
single repnsentative expcrimcnts carricd out with the -. Data of
pH were obtained in independent reprcsnitative experiments wiih the Namctrc
rheometer (same data as in Figure 7.1.14~). (Profiles for nplicated and
comsponding mecisumnents of milk viscous and elastic moduli and loss angle for
each level of cultures are displaycd individwlly in Fiprcs A7.1.3&&d .) Compare
with the countcrpart time-profiles of consistcncy (Nametn rhtomctcr) and pH show
in Figure 7.1.140.
 (iictic acM controis) :

(samplcs cultured at
-
0 2 -,U4-,
and Ci89

o g R q ~ q n q a q v , q m q i . . y
m m v r ' u e

Incubation time at 40°C (h)

Fipre 7.1.156. Overview of Pfws (Le. ,rate of

change in elastic modulus O' with time, dG'/dt) for RSM differantly
at 40°C. Profiles of derivative elastic modulus G' and of loss angle S (tans
= Gt'/O') YS. time for each level Cli of acidifying starter cultures are shown for
single representative experiments Earried out with the (same
primary data as in Figure 7.1.1 Sa). Data of pH werc obtained in independent
representative expniments with the Nametrc theorneter (same data as in Figures
7.1. Ma&b).
Arrows point to the region of apparent local maximum in S and to the corresponding
(approximate) values of pH. (Plofiles for mplicated and comsponding
measurements of milk viscous and elastic moduli and loss angle for each level of
cultures arc displaycd individually in Figures Al.l.i&&d.) Compare with the
counterpart tirne-piofiles of derivative consistency (Nametre theorneter) and pH
shown in Figure 7.1.146.
 = PH
(ramplq cularrd at PH-beateà RSM (9û°C-1 min), 1

Incubation time at 40°C (h)

Figure 7.l.IQ. Ovewicw o f consistency development curvcs for

RSM 9O0C4, min and diffmntly a at 40°C. Profiles of
.
consistcncy vs timc for cach levcl C/i of acidifjhg starter cultwes a n show for
single representative experiments carricd out with the Namctn rheometcr. Data of
p H are shown when avaihblc. (Profiles for replicated and comsponding
rneasurements of milk consistency and p H for cach level of cultures are displayed
individually in Figures A7.1.39ctEd.)
 . - PH
(samples~ulturedat Pm-kitcd RSM (9Q0C-1min),

Incubation time at 40°C (h)

Figure 7.1.166. Overview of

. . E
iime.denvaiive Pf
U ~ V ~ (i.e ., rate of
change in consistency C with timc, dC/dt) for RSM 81
1 min and differrntlv at 40°C. Profiles of derivative consistency vs. timc
for each level C/i of acidifying starter cultures are shown for single reprcsentative
experiments carried out with the Namctrt rheometer ( m e primary data as in Figure
7.1.16o). Data of pH am show whcn available. (Profiles for repiicated and
cornsponding measurements of milk consistency and pH for cach lcvcl of cultures
are displayed individually in Figures A7.1.39c&d .)
 0 A distinctive feature for culnid controls was the evolution of #un 6(=G"IG 3 with time
evidenced in investigations with the Ch-Med heometet (Figures 7.1.1 Sa&b and A7.1.38a-d for

standard low-heat RSM,and A7.1.40u-c for pn-heated RSM). A gradua1 rise and pronouncd

local maximum of tun 6 around pH 5 2 (Le., a tnnsient decrease of relative elasticity) were

consistently found just after the onset of measutable rigidity around pH 5.5-5.4, before

(secondary) inflection in the cuwes of elastic modulus.

Maximum values of tan S mund 0.40-0.45 (Le., 6 r 22-23') were rneasured for standard
(low-heat) RSM,but it is possible that these values were undenaimatcd bccaur of experimental

dificulties in measunng the relatively weak gels at this stage of development. (This charactetistic

evolution of loss tangent was in fact mote systematically evidenced on acid coagulation of pre-

heated milk, possibly because gelation of heated milk started at higher pH, Le., earlier, and

'stronger* gels resulted; see Section 1.2.30.) Thes observations are in keeping, qualitatively and

to some extent quantitatively, with earliet teports by Biliaderis et al. [1992] and R6nnegBid &

Dejmek [1993] [acidifying (pre-heatedüF-concenaatcd)yoghurt milk measured at above 40°C

-
and O. 1 I Hz in a Bohlin VOR theorneter], van Marle & Zoon [1995a] [high-pasteurizcd cultuted
milk, 3Z°C.and = 0.2 Hz],Gastaldi et al. [1997] [GDL-acidified RSM, 20°C, and 10 Hz], Lucey

& Singh [1997] and Lucey et al. [1998c,e.d] [GDL-acidified and cultured (pre-heated) milks, 30

and 42OC, and 0.1 Hz], and Ozet et al. [1998] [cultuted UF-milk. 2S°C, and 0.25 Hz].

The evolution of tan 6 indicates that the viscous character of the gel incrases fssicr initially

than its elasticity. The peak in mote viscous-like khaviovr may be relatcd to the obsetvations by
Roefs [1986] and Roefs et al. [19906] in chemically aciditied milk summatized under Section

2.130 of the litciahire review (maximum in T2H-NMR relaxation timc near pH 5.4-5.2 and
maximum in tan Gfor a pH 5.2). Distinction bstwccn the initial stages of the setting of milk by

bactcriological acidifiicrton vs. rcnncting has also been made bascâ on ultiaponic analysa

m n p i p i et al., 19941.
 These observations suggest partial loosening (increasing relaxation) of the acid-set casein

nctwork in the bcginning of wmbly, with progressive acquisition of chuacteristic rhcological

behaviour Iater in the gclation process. Gradwl solubilization of colloiâal Ca phosphate

(danincralization of casein) over the pH range 6.7-5.0, together with xnne (limited) concomitant

dissociation of casein molecules at acidic pH (especially below 6.01, probably is a major

deteminant of the apparent loosening khaviour at 40°C (see Section 7.3.3 for details on possible

interpntation). This may have hindered gel development to some extent and perhaps contributed

to the intermediate deceleration of consistency (rnodulus) development amund pH 5.2 evidenced

in some derivative plots [Le., tcmporarily decreasing (but positive) or leveling off values of

dC(r)ldt and dG '(t)/df].

Markedly lower tan 8cornpared to m e t standards werc reachcd ultimately amund 0.23-

0.25 (6 13-14') (Figures 7.1.lfa&b). Values of 6 of the same order have boen reported for

mature (lactic) acid gels (whether fiom unheated or heated milk): around 15-l P according to

Schulze et al. (19911 (1 Hz-5-43'C), Biliaderis et al. [1992) (1 Hz420C), and Gastaldi et al.

[1996] (10 Hz-20°C); and 12-14' according to R d et al. [1990b] (a 0.2 Hz-20°C), van Vliet et

al. [1991a] (2x10~-2Hz-3W), Rohm [1993], Rohm & Kovac [1994] (a 0.2 Hz-40-45T),

Rannegh-d 8 Dcjmek [1993] (W2-1 Hz-440C), van Made Br Zoon [1995u] (= 0.2 Hz-3Z°C),

Lucey & Singh [1997] and Lucey et al. [1997a, 1998c.d (0.1 Hz40 and 42OC). The values of 2S0

nfemd to by N&l et al. [1989] (original mults of Lehembrc 119861; unspecified fkquency and

temperature) and of 19-26' mported by Vlahopoulou 81 Be11 [1990] (0.5 Hz-2S°C) seem to depart

h m the above data.

(U) Less clear but somewhat consistent wem the sccondary variations in the rate of gel

development klow pH m n d 52-5.0, in the mgion of apparent dccekntion of gel dcvclopment

(rg., Figum 7.1.14b and 7.1.19). As for the (secmingly analogous) observations pettaining to

partly acidifiai rennctcd milk just pnsentcd, the secondary maximum (or shoulder) in the tima
derivative curves tended not to k apparent in the pmfiles of derivative modulus derived h m

analyses with the C h - M e d rheomcter, at lcast for non-pre-heated RSM (Figure 7.1.1 Sb; also

comparative illustrations in Figures 7.1.1 7u&b and A7.1 Alu-c). Actually, the effect was most

conspicuous for pre-heated acidifying milk, w u n d pH 5.2-5.0 (gelation pH was around 5.8). both

in tenns of consistency and elastic modulus curves (Figures 7.1.166, A7.1.406, and 7.1.1 7b; s a

also Section 7.2.3). mote how the appearance of the two p&s (shoulden) in the derivative

curves of consistency and modulus differs as alluded to in Section 7.1 .!a.]

From parallel rneasurements with the Carri-Med rheometer, it appars that the secondary

changes in rate of gel finning were concomitant with the peakinglgndual decline in tm 6 pst-

gelation. Seemingly related (albeit not commented) effects are apparent in the coagulation

profiles for acidifying (pre-heated) yoghurt milk reported by Schulze et al. [1991], Biliaderis et

al. [1992], Mnnegilrd & Dejmek [1993], Kim & Kinsella [1989b] (milk acidified with GDL),

Lucey & Singh il9971 and Lucey et al. [19984 (GDL-acidifkd and cultmd milks), which

appeared to be mostly detertnined by the degree of acidification a h , rather than by the amount of

stuters per se, Le., time. (This can be evidenced in plots of viscoelastic parameters us. pH; e.g.,

Figure 7.1.8a.) It seems that the ratc of acidification did not change enough in this region of pH

for the secondary increase in firming rate to be directly related to acidification kinetics.

(e) Effects of Concentration of Starter Cultures. The acceleration effcctp of increasing the

concentration of starter cultures betwcen C/8 and CI1 were most evident in the shifi of gelation

profiles t o w d shorter times, including shorter gel times (e.g., Figures 7.1.146- 16a and Figure

A7.1.59~). The (maximum) rates of acid gel development were moderately affected (Figures

7.1.146-1 66 and A7.1.58fi, which may be explained in part in ternis of the moderate effect the

ievel of milk inoculation had on the actual rate of pH dccrease QHldt as alluded to in Section

6.3.4a.
 30 *
controls; replicates 1 & 2) - -

6 (lactic acid gels) . -

Figure 7.1.17a. Contrasting of the primary and derivative profiles of consistency C & pH
(upper panel) and elastic modulus G' & loss angle 6 (lower panel) vs. t h e for standard
RSM renneted at level BirP a fi and for standard RSM cultured at
level ÇIIIIlafiif et 40°C. Comsponding profiles of C & pH, and of G' & 6
(tan6 = G ' W ) are shown for expcrimcnts replicated two times with the
and the mcomcter. respectivcly (symboldiines of different
sizelthickness). Amws point to apparently similar featurcs in the evolution of dC/dt
(dG'fdt?) (apparent local minimum), and 6 (apparent local maximum) for Ennet and lactic
acid control gels.
Figure 'l.l.l7b. Contrasting of the primary and derivative profiles of consistency C & pH
(upper panel) and elastic modulus G' & loss angle 6 (lower panel) us. time for
rcnnetcd at levcl I(rr4 a ptl LI1 Lrnipd. and for cultuted at
levela llrclifa at 40°C. Concsponding profiles of C & pH, and of G' & 6
(tan6 = GVG') are shown for cxperimcnts replicated two times with the
and the ibcMacta. mpectively (symbols/liocs of differcnt
sidthickne~s). Anows point to apparcntly similar faturcs in the evolution of dCldt and
dGtldt (apparent local minimum), and 8 (apparent local maximum) for m e t and lactic
acid control gels.
 The main effect of increasing starter concentration was on shonening the Iag time of bacterial

growth, thereby advancing the time at which important lowering of milk pH, hmce gel formation,

occumd. Consistency and modulus of Iactic acid gels reached similar plateau values for starter

concentrations between C/8 and CM.

Notably lower consistency resulted for low-heat RSM at starter concentration C/I (Figures

7.1.140 and A7.1.58d). possibly because of relatively high rate of milk acidification. (No

measurernents of modulus were carried out at level W.) It is known that too high addition of

culture (2 5% dv), Le., excessive rate of acid development at high gelation temperature may

contribute to poor gel fonnation [Kosikowski, 1977; M i e & Kumiann, 19781. Rapid

acidification may hinder efktive organization of acid gel structure, multing in increased

rearrangements in a cocuscr netwotk with less numemus interconnectivity and lower consistency

(see Lucey et al. [1997b,c],

GDL gels, 30°C]. (Possible mitigating influence of bacterial cells and

metabolites on the sûucturing of gel in fennented milk mu* be borne in mind too, as pointed out

in Section 6.3.46,c.) Effects (or absence thereof') of inoculum level on the viscoelastic properties
of yoghurt gels have bcen documented by Arshad et 1[1993a.b] and Vlahopoulou et al. [1994].

It was not clear from experimmtal observations whether changing culture concentration

(through effects on, e.g., rate of milk acidification &or consistency of gel) affected the

secondary vui*ationsin rate of gel development in non-pre-heated and pre-heated milk, but these

seemed to be more discemible at the lowest concentrations (Figures 7.1.Mb, 7.1.166 and

A7.1.40k see also Section 7.3.20). It may be speculated that relatively slow acidification leads to

more gmdual (effkctive) incorporation of material into the asxmbling gel and hence bettcr

rcsolution between the successive stages of coagulation.

7.1.S. Anu&s& of Ge1rili011R

omfor Crrltuced and Renmted Müks

(a) Effects of Concentration of Starter Cultum at C m t Concentration of Renne! . Families of

plation curves obtained for differcnt concentrations of bacterial starter at givm concentrations of
rennet at 40°C are shown in Figures 7.1.1806b (A7.1.42 through 46) [Namette rhtometer] and

7.1.19 (A7.1.47 through 50) [Cd-Med hcometer]. As can be seen. at al1 levels of remet

studied, increasing the concentration of acidifying cultures h m Cf8 to C/l had moderate effccts

on the ovemll progress and dynamics of gel devclopment in standard RSM. Vsrying the

concentration of nnnct enzymes was appmiably more influential in 'shaping' gelation profiles,

as shall be detailed in Section (b).

(I) A conspicuous (expected) effect of i n c r ~ ~ i nconcentration

g of starter culaires was to

reduce the time rcquired for coagulation by combined rennet and acid (espccially at rennet

concentrations strictly below Rx16; summary in Figure A7.1.59~).Shifiing of the coagulation

curves along the time axis with changing acidification regime was most apparent at the lowest

concentration of nnnet relative to cultures, Le., at and klow Rxl, which conesponded to

conditions such that the effects of bacteriological acidification were central to gel setting. (For

cuiture levels in the range CI8-CC2 et rennet level R x 1, the degree of hydrolysis of u-casein at the

onset of messurable coagulation was about 30% at pH a 5.7; Table 7.1, Figures A7.I .6Oa and

6 1a.)

As for the observations pertaining to the cultured controls, the rates of gel development for

'minimally' m t e d milks changed mderately with increasing starter concentration (Figure

A7.1.58f at RxO and Rxl). Higher maximum values of consistency resulted for culture
concentrations betwecn CI8-CR thon for CI1 (e.g., Figures 7.1.18~and A7.1.42a&b). It was not

clear either whether changes in the concentration of acidifying starters (or consistency of gel)

affected the definition of the ~conâaryvariations in rate of gel development (ive., shoulder in the

primary gelation curves).

(io At higher concentrations of rennet between Rx4 and Rx16 (especially ûelow Rx16) and

40°C, the rates of gel developmcnt increried with incming the amount of cultures, as was

evidcnt h m the stccpcr initid portion of consistency curves kfore the tuming point Pm, with
 Incubation time at 40°C (h)

F i 7 . 1 . Overview of consistency development curves for diffcrrntlv

standad RSM rcnneted at level at 40°C. Profiles of consistency C us.
time for each level C/i of acidifying starter cultures are show for single
representative expcriments carricd out with the Nametre rheometer, representative
pH data king show selectively for the milks coagulated at C/L, C/4-, and C/8-
h l . Anows point to the mgion of local minimum in the average instantancous rate
of consistency development dC/dt at Cl1 and C/8. and to the (appmximate) values of
pH. (Profiles for rcplicatcd and corresponding measurcments of milk consistcncy
snd pH for each fevel of cultures arc displayed individually in Figures Al. 1.4- .)
 Incubation time at 40°C (h)

F u 7 . 1 . . Overview of consistency dcvelopment curves for diffcrrntlv

standard RSM rcnneted at level at 40°C. Profiles of consistency C vu.
time for each level Cli of acidifying s m e r cultures are shown for single
representative experiments carricd out with the Nametrc rheometer, representative
pH data king show xlectively for the milks coagulated ai CIL, C/4-, and C/8-
Rx4. Arrows point to the ngions of local maximum and minimum in consistency at
C/l and C/8, and to the comsponding (approximate) values of pH. (Profiles for
replicated and comsponding measurcments of milk consistency and pH for each
level of cultures are displayed individually in Figures A7.1.45a-c .)
 Incubation time at 40°C (h)

Figure 7.1.19. Ovcrvkw of elastic rnodulus dcvelopmcnt c w e s for diffnaitiv

standad RSM renneteci at level at 40°C. M l e s o f clastic modulus G'
vs. time for each level C/i of acidifying starter cultures are shown for single
representative experiments d e d out w Y the m.
Representative loss angk 6 data (tan6 = GV'/G')arc shown selectivcly for the milks
coagulated at Cl2-, C/4-, and CO-Rx4; pH data for the combinations Cl2- and C/4-
Rx4 werc obtained in independent replicated experiments with the Nametre
rheometcr. (Profiles for rcplicrteâ and comsponding measurements of milk viscous
and clastic moduli and loss angle for each level of cultures are displayed
individually in Figures A7.1.49a&b .) Compare with the counterpart time-profiles
of consistcncy (Nametir rhcometcr) and pH shown in F i p n 7.1.18b.
modemte ductions in coagulation time and slight incrrrssp in gel consistency at P D ~(the
, latter
for m e t levcls sîrictly abve Rx4 it sems) (Figures A7.1.58jig and 59a). (In the range of

conditions of hydiolysis between Rx4-Rx 16, percentage conversion of K-casein et mwsurable

coagulation was about 55% at pH ktween 6.4-6.0; Figures A7.1.60~and 610.)

Most of the abovc observations may be explained in the light of the effects highlighted in

Section 7.1.4 for the teference milks, and in particular the kneficial effects of acidifying milk on

the eficiency or the ovemtl renncting process.

(b) Effects of Concentration of Rennet at Constant Concentration of Starter Cultures. The effects

of varying the arnount of rennet enqmes added to cultured RSM can be more readily appreciated
in the contmting of gelation time-profiles obtained for constant concena~tionîof starter cultures
under similar ngimes of acidification Figures 7.1.2Oa&é and A7.1.5 1-54 (Nametre rheometer).

and 7.1.2 la&b and A7. I 5 5 4 7 (Carri-Med rheometer)]. Gradation in the character of the profiles

resulting from varying rennet concentration was rnanifest in al1 series at constant concentration of

starters bctween Cl8 and C/I at 40°C. nim basic types of coagulation curves were evidenced,

including the refeme profiles for milks cultured with no rennet (and milks renneted at constant

slightly acidic pH) as derribed undr Section 7.1.4.

(0 In culturcd m ilk containing the lowest arnount of nnna studied, vu., R x 1, a characteristic
coagulation behaviour was consistentiy observai, apparently intermediate between the

comparativcly simpkr (sigrnoid-like) pattern for contml cultutcd milks and the 'humpshaped'

pattern for milks with higher mnnct concentration at 40°C. Substantial incrase in initial rates of

gel development and (maximum) values of consistency and modulus contributeû to the distinct

shape of gelation c u m at Rxl compared to the pmfiks for acidifiedlrrnneted controls. A

shoulder was systematiully prcsent about midway through consistcncy development, near pH

5.2. In tams of the cvolution of dynamic modulus, this fmre appeared as a transient relative

leveling (hence the stcplike evolution) of cxperimental values of elastic modulus.

 Incubation time at 40°C (h)

Figure 7.1.29o. Ovewiew of consistency development curves for standard RSM

cultured at level and at 40°C. Profiles of consistency C vs.
time for each level Rxj of m u ~ enzymes
t are shown for single representative
experiments carried out with the Nametre rheometer, representative pH data k i n g
shown sclectively for the milk coagulateci at CM-Rx4. Amws point to the regions
of local maximum and minimum in consistency at C/4-Rx4 and to the corresponding
(approximate) values of pH. (Profiles for rcplicated and comsponding
measurements of milk consistency and pH for each level of rennet are displayed
individually in Figures A7.1 S3a-c .)
 - .
m
PH
(miksample culaucd

Incubation time at 40°C (h)

Figure 7.1306. Ovcrview o f consistency developmcnt curvcs for standard RSM

cultured nt levela and diffmntlv at 40°C. Profiles of consistency C vs.
time for each level Rxj of mnnet enzymes arc shown for single rcpm«itative
experiments cmied out with the Nametm rheometer, repremtative pH data being
s h o w sclectively for the milk coagulateci at C/2-Rx4. Arrows point to the regions
of local maximum and minimum in consistency at C/2-Rx4 and to the comsponding
(approximate) valws of pH.
 Incubation time at 40°C (h)

Figure 7.1.21a. ûverview of elastic modulus development curves for standard

RSM cultured at level and at 40°C. Profiles of elastic
modulus G' vs. time for each level Ry of remet enzymes are shown for single
npresentative experiments carricd out with the -.
Repmentative loss angle 6 data (tan6 = GVG') are shown selectively for the milks
coagulated at C/4-Rx4. -Rxl, and -RxO; pH data for the combination C/4-Rx4 were
obtained in independent rcplicated experiments with the Nametre theorneter.
(Profiles for replicated and comsponding measurcments of milk viscous and elastic
moduli and loss angle for each lcvel of rcnnet are displayed individually in Figures
A7.1.56adkb .) Cornpure widi the countcrprrt timc-profiles of consistency (Nametre
rheometer) and pH shown in Figun 7.1.2Oa.
 9
m
0
.

. (Nametrc samples,

Elastic modulus

Incubation time at 40°C (h)

Figwe 7.1316. Overview of elastic modulus development curves for standard

RSM cultured at level ç12 and differrntlv @ at 40°C. Profiles of elastic
modulus G' and loss angle 6 (tans = GW/G')vs. time for each level Rxj of rennet
enzymes are shown for single reprcscntative experiments carricd out with the
m.Data of pH for the combination C/2-Rx4 w e n obtained in
independent expcriments with the Nametre heomcter. (Profiles for replicated and
conesponding measurcmcnts of milk viscous and elastic moduli and loss angle at
each level of m e t are displaycd individually in Figures A7.1 .S7u&b .) Compare
with the counterpart time-profiles of consistency (Nameüc heometer) and pH
shown in Figure 7.1.2Ob.
 The time-derivatives dC(l)ldf and dG'(r)ldr remained cssentially positive throughout gel

differentiation, at least for non-pre-heatd MM. About 30?! of K-casein had been hydrolyzed at

the onset of coagulation mund pH 5.7 (C/8-C/l),

and about 40% at the point of local minimum
in consistency derivative -und pH 5.2 (C18-CII ) (surnmaries in Table 7.1, and Figures A7.1.60~
and 61c). The marked (secondary) strengthening of gel typical for the regimes of renneting-

acidification at low concentration of rennet (see also Figure A7.1.58d) seems to be related to

direct and perhaps some indirect (kinetic) cfTects of acidity devefoprnent on the remethg proccss.

The resernblance between the time-courses of tan 6 (and elastic modulus initially) for

cultured controls (Section 7.1.46) and milks treated with low rennet (see overview in Figure

7.1.22 hereafter) seems to point to the important (direct) contribution of steady acidification fiom

the early stages of gel formation in minirnally renneted milks also. Maximum values of tari S

were around 0.62-0.67 (Le., 6 = 32-34') near pH 5.2. Equilibrium values at long-times wcre

similat to those for control lactic gels at 40°C (a 0.23-0.25, Le., 62 13- Mo). Perhaps the limited

stabilization of tan Sneiu 0.55 (62 28O a Sfor mature control rennet gels at constant pH between

6.4-6.0 at 40°C) that seemed to m u r initially just a h r gel transition (Le., in a rcgion of pH

above about 5.5) may be seen as an indication for the establishment of a gelled structure with

(rheological) propeities related to those of casein gels produced by rennet pmteolysis. Such

modulations in ovedl coagulation behaviour concur in underlining gradua1 transition h m acid-

set to rennct-set gels and the determinant function of remet in minirnally renneted acidifying

milk, in particular with respect to imparting strength ta the gels.

(U) In milk m e t e d at and above Rx4 at 40DC. a distinctly bimodal-like fonn of khaviour

was observed. The evolution of p l consistency and modulus over timc was characterized by a

first maximum, Ph, 0.5-2 h aftcr coagulation time, near pH 5.65.9 (Rx4-Rx 16); and a clear

local minimum, Pmh 0.5-1 h later near pH 5.0-5.4 (Rx4-Rx 16) kfore a pmnounfed secondary

rise (average values of time and pH in Figures A7.1.59c&d and 6ûc&d). For most cuives, the
smwthness of gelation profiles and in particular the relative symmetry of the peak in gel

consistency and modulus stood out. (The latter featun may be viewed as an indication that there

exists some relatbnship between the rate and magnitude of the processes before and afier Pm,

vit., gel fiming and gel softening.) About 55% of u-casein had been hydrolyzed at the point of

coagulation at pH between 6.06.4, 75% et Ph (pH 5.6-5.9), and 80-85% at Pmh (pH 5.0-5.4)

(Rx4-Rx 16) (Figures A7.1.6 lcdid). Experimental evidence was consistent in showing that the

inherent coagulating propatin of remet were essential for initiating gel formation under

experirnental conditions of renneting-acidification for levels of rennet 2 Rx4. 'Background'

reduction of the pH in the range 6.4-6.0 certainly played an indirect part through promoting the

eflïciency of coagulation by rennet (decreasing stabilizing ability of K-casein and increasing

enzymatic activity; Chapters 4 and 5).

Analyses of loss tangent (los angle) provided useful complementary indications as to the

relative contributions of rennet action vs. continuous acidification to gel development under

conditions of important renneting (overview in Figure 7.1.22; also Figures A7.1.66~-e). The

initial part of the curves of tan 6(kfore PM) showed characteristics strongly reminiscent of

those for control rennet gels fomed at constant pH in the range 6.4-6.0 at 40°C (Sections 7.1.4~-

c). Comparable, relatively high values of tan 6 were mersurcd (0.47-0.58, Le., 6 = 25-30').

Evolution of tun 6 kyond Pb was typical of the evolution for cultured milks with no or little

rennet added. The increase in tan Gstarted shortly before Ph and the peaking around 0.62-0.67

(6 u 32034~)occuned in a region of pH betwcen approxlmately 5.3-5.5 (Rx4-Rx16), slightly

ôefore PM it seemed. Note that it is uncertain whether details of the evolution of loss tangent

(e.g., amplitude of change) for acidifying systems can bc mlyzed meaningfùlly, e.g., with views

to quantifying gelation khaviour.

 Incubation time at 40°C (h)

Figure 7.132. Typical evolution of loss angk 6 ( t d = GiG') upon the

coagulation of a [ C / 4 - u ) vs. &GO-w
a constan(pM and a&Q)
.cidified s m at 40°C. Profile
of S vs. time for each set of coagulation conditions are shown for single
rcpresentative expcrimcnts c h e d out with the Carri-Med rheometer. (Profiles for
replicated and corresponding mcasumnents of milk loss angle, viscous and elastic
moduli Gwand G',and approximate pH arc show elsewhere in the dissertation.)
Conaast to the counterpart timc-profiles of 8 for differently pre-treated or
coagulated RSM shown in Figure 7.2.14.
 These obsewations underline the Iatet influence of fiirther, important acidification on gel

development, Le., 11# relative decoupling in time between the effects of renncting vs. stcady

acidification at nîatively high concentration of rennet. Initiakainly enzymatic-

destabilization and coagulation of milk c w i n thus appear to lead to the formation of a prim~ry

nnnet-like gel which then undergoes importent modification andfor destabilization [apparent

loosening and mostly concurrent, gradua1 softening, i.e., dC(r)/dt or dG'(r)/dt c O] when the

acidity reaches a critical level iuound pH 5.5 at 40°C. Differentiation toward increasingiy acidic

gel state seems to lead to secondary finning afler Pm&.

The two h i c tendencies of milk casein to gradually demineralitc and aggregate on

increasing acidification am ccrtainly rcflccted in the evolution of gel viscoelastic propnties,

especially beyond Pm (see Section 7.3.3 for tùrther interpretation of gel developmcnt). Despite

the important vuiability chaiscterizing the experimcntal values for gel consistency and modulus

at long times, it was apparent that stmnger gels multed at around Pm (and long times) as

compared to control gels produced by acidification or by renneting at constant pH ktween 6.4-

6.0.

(di0 There is an obvious similitude between the qualitlitive pattern jus d e r r i k d and the
rheological profiles rcported by van Hooydonk et al. [1986&] [applied kquency 0.2 Hz;

pasteurued skim miU<;25T; unadjustd pHJ, Noël et al. [1989; 19911 [O. 1 Hz; RSM; 0.0 1%

CaC12;32-34°C; pH at rcnneting 6.5-6.0; apparent concentrations of starter (constant) and rennet

C/4 and RxO.5-Rx23, respcctively], and Schulz et d (1999) [experimcntal conditions

unspccifkd]. Somc of the effccts relatai to incmsing the concentration of minet describd by

N&I and c~workcrs[1991] have also been evidenced under the standard conditions in the

prcsent study, primarily through investigations wiîh the Nametre viscorneter. (Se quantitative

summaries in Figures A7.1.58 to 61 for consistency, timc, pH, and hydrolysis data; md Fipm

A7.1.62 and 63 for parameters measured with the Curi-Mcd theorneter.)

 These effects include the limitcd influence at 40°C of high kvels of rennet on: O decreasing

coagulation time (especially above R x 16 for concentration of starters Cl8; and above Rx8-Rx 16

for starters between C14-CIL; Figure A7.1.590), (ii) increasing first maximum rate of gel finning,

before stage Pm that is (above Rx4 for concentrations of starters behmen Cl8-CI!; F i g m

7.1 23a, and A7.1.58fig and 62f&g), (UI) and increasing gel consistency (modulus) at P-

(above Rx4 for concentrations of starters between CI8-CIZ; and above Rx8 for CIl; Figures

7.1.23b. and A7. I .58b&g and 62bdg).

(it is noteworthy that for the gels obtained at Rxl (below CA) the maximum values of

consistency (modulus) attained (afler the shoulder in the primary curve that is) were higher than

the optimum consistency at Ph for the gels formed at Rx4-Rx 16 (Figures A7.1 S8e and 62e).

Certainly, the fact that these values of consistency did not comspond to equivalent (homologous)

stages of gel development at Rx 1 vs. at Rx4-Rx 16 ought to be borne in mind.] At concentrations

of starters sbictly below CI1, the time comsponding to P- tended to decrease (i.e.. the pH at

Pm tended to be higher) with increasing rennet (Figures A7.1.59~and 60c).

(iv) No gradation in the procea of milk gel development with increasing minet concentration

has k e n clearly established in previous works, however, except perhaps (indinctly) in the work

of Dalgleish & Home [1991a,b] (see Section 7.3.2 for attempted detailed cornparison).

Conespondence among the different (rheological) protifes in this work was made dificult in part

because of experimental variability (including in the regimes of bacteriological acidification), but

available evidence suggests that the patterns of coagulation khaviour distinguished are

homologous. Most Iikely these a r i s h m the same h i c physico-chernical processes

of-prrdominantly-ecid-induced progressive demineralization and charge neutralization of

gelling or gellcd @ara) -in. The 'maximum-minimum' patterns of consistency and modulus
development evidenccd at high concentrations of minet may thus be viewed as an amplification

of the shouldcr that cmcrged at the lowest remet.

 oncentration of

Concentration of rennet enzymes

(multiple of R = 2.2xlo4%v/v of single strength remet)

Figure Xl.23~.Average valw s of maximum Pf a

(before point PHU or its deemed equivalent, that is) for standard RSM differcntly
culturcd and renneted at 40°C. Data of average derivative consisteiicy plus
comsponding standard deviations (vertical e m r bars) are shown for independent
experiments nplicated two to three times with the Nametre rheometer. (The same data
are shown in Figure A7.1 S8g in contrast to the average values of consistency C ai
point Pm or its deemed equivalent.)
 Concentration of
acidifLing cultures,

-
GQfmumu
bH C/Q,
CQ, and Cf 1, Le,,
O to 5.00/0 v/v

--
Concentration of nnnet enzymes
(multiple of R = 2.2xlo4% vlv of single strength rennet)

Figure 7.1336. Average values of consistency C ppipt (Le ., fim zero in rate
of consistency development dC/dt, as detined in Figures 7.1.7 and A7.1.12 for minet-
lactic acid gels at Rx4-16) gt & (i.e., apparent positive local
minimum in dCldt for strictly lactic acid gels at RxO and for lactic acid-rennct gels at
h l ) for standard RSM diffcmntly culhucd and rennetcd at 40°C. (For strictly rennet
gels at CO and pH 6.4 for which no local minimum in dCldt was apparent, the values of
consistency shown arc thosc measured atter incubation for 10 h.) Data of averaBe
consistency plus comsponding standard dcviations (vertical error bars) are shown for
independent experiments repiicatcd two to t h m times with the Namette rheometer.
 This argument shall be developed under Section 7.3.3. As alluded to earlier, it cannot k niled

out that fsctors less obvious than-but nlated to-the degree of milk acidity (e.g., gel strength

through some kind of mcunive effect) modutatcd the evolution of the gels.

(v) It is questionable that relative amplitude and resolution (width) of the optima in

experimental consistency and modulus of gels can be evaluated in a meaningful (accurate and

precise) way (e.g., by inteption for more complete characterization), especially at

concenirations of r e ~ c above
t Rx4-Rx8 in part because of compounding effects of syneresis at

long times (Section 7.1.3). Loss tangent (absolute) data ought to be interpreted with prudence as

well, keeping in mind the possible limitations of such measurements (e.g., limited resolution of

the different types of interactions that govem gel properties) and the influence of measuring

fiequency and temperature. Perhaps integration of the peak in fan 6 may yield quantitative

information regarding the degm of reamngement the aggregating caseinlwhey protein particles

likely undergo depending on gelation conditions.

Noël et d. [1989, 19911 did not measure a pronounced difference between the values of G for

rennet-lactic skim milk gels kfore and fier Pb (a 14- 16*, mspectively, compared to 15- 16' for

enzyrnatic controls at pH 6.6, = 30°C, and 0.1 Hz). Refemng to the values of loss angle measured

by Lehembre (19861 for lactic coagula (a 25"; unspecifîed temperature and fiequency), N d 1 and

cbworkers concluded to apparent (structural) similarity between mature rennet-lactic gels and

rennet gels. This xemed to be at variance with the findings in the present work (6for mature

rennet-Iactic gels .r 6 for mature btic gels m 13-14O at 4 0 T and 0.1 Hz) but secondary

investigations (Section 7.2.2; Figure 7.2.3) showed that there was an imporiant effect of gelation

temperature in the range 25-40°C on the experimental values of 6 (specifically those attained on

the development of a primary-mainly rennet-gel initially).

Clearly, howevet, remet-lactic acid gels developcd higher consistency and dynarnic modulus

at long times (Pm) than controi lrtic gels. Our observations Jccm to echo those made by Rocfs
[1986] and Roefs et al. [1990b] in studies of nnnet-acid skim mik gels prcpared by acidificqtion

with HCI in the cold, wiîh or without icnnet, and subsequent warming and mersuring at 2OT: at

and below pH 5.1, the gels obtained by combining acidification and renneting had a rheological

behaviour (including relaxation behaviour of the protein-protein bonds in terms of the values of

tm Gand their dependence on mecisuring hquency) similar to that for strictiy acid gels but with

considembly higher values of dynarnic moduli.

(vi) With respect to defming the conditions that determine global coagulation behaviour, it is

noteworthy that nasoning in ternis of simply the (relative) concenirations of renneting vs.

acidifying agents can k misleading. For the actual concentrations chosen in this midy, it is easy

to see that the following combinations gave equivalent proportions of rennet and s m e r cultures:

Rx 1-C/4 Rx4-CI1 and Rx 1-Cf8 = Rx4-CR a Rx8-Ch. It is unlikely, however, that such a
proportionality existed in terms of the (relative) efleccr~of rennet and acid on coagulation

reactions; certainly the bcneficial influence of acidic environment on the veloçity/coagdating

efficiency of rennet in miik would have to be factored in to define effective concentration of

rennet.

That distinct transitional patterns of development of gel consistency and mdulus resulted for

the combinations at Rxl cornpareci to those at and above Rx4 at 40% may have to do with the

differential efficiency of renneting uoder the conditions of mildly acidic pH dunng the lag phase

of bacterial growth. It may k rationalid that the eficiency of renneting (including the
proteolytic activity of rennet enzymes) can only be substantially potentiated by acidification

provided then is suffcient minet in the milk.

One may draw an analogy with the concepts of 'power curve' and 'upswing point' to

illustnte this idea (explained in Figure A7.l.64 and Hall [1997]). The upswing point in the powcr

cuwe of eficiency of ovcrall renneting as a tùnction of rennet concentration would correspond to

a critical or liminal level of rcnnct whereupon the conditions of minet and acid bcgin to gcnerate
important increase of the influence of nnnet as a result of comparatively small change in enzyme

concentration (under othenvise constant coagulation conditions). Over the range of conditions of

acidification in this study (standard RSM at 40°C), this critical concentration of rennct would be

between Rxl and Rx4. F e specificity of nnnet for K-caseinis expected to be independent of

rennet concentration. The ratio of rennet efilciency over remet concentration may k thought of

as a coefkient of efficiency (a function of coagulation conditions, notably, ionic (pH) conditions,

temperature, and pie-treatrnent of milk)]. The sharp timing u p w d of power cuwes beyond the

upswing point is commonly refemd to as the 'power curve zone'. Only within this zone of

nlatively high eficiency would the e f f a of rennet action distinctly prevail over the effects of

acid production in tenns of initiating milk coagulation as tcntativcly illustrated in panei (cl of

Figures 7.3.2adb (Chapter 7, Section 7.3.3). The points r a i d here may help explaining the

moderate effects of varying the concentration of acidifying cultures on the character of

experimental gelation profiles: presumably the span of effective concentrations for cuhrt% in this

worlc was much namwer cornparrd with that for rennet. Such gradation of enzymatic activity

would concur with the observation that increasing culture concentration had nlatively little eff't

on the rate of milk acidification.

The observations pmsented in this section shed additional light on the gradations in the

coagulation behaviour of standard milk that arise when the specific contributions of renneting and

continuous acidification reactions are varied. It was shown that different patterns of gel

development could be clearly evideneed bascd on small m i n dynamic heological measumnents

and carchil analysis of the profiles of gel fornation obtained. The infonnation conveyed by

derivative plots of the pnmary curvcs of consistency (viscoeiastic rnoduli) over time pmved

usehl in that respect, allowing for characterization of the gelation profiles (cuwes of tun 6

included) on a more detailed level. Indeeâ, this simple methoci of rcfning analysis made it

possible to asscss, h m the primuy and derivative cuves, the conditions under which
coagulation had taken place, and also provided hints for what (common) underlying pmcesses

may be at play.

Over the range of concentrations of rennet and stMer cultures investigated at 40°C, varying

the concentration of rennet was found to have a predominant effect on the evolution of skim milk

gel viscoelastic properties. With regard to the conditions that led to gel formation initial&, two

situations were distinguished, in addition to the limiting cases of stnctly lactic acid and enzymatic

coagulation, vu., (i) ~elationconditions such that the effects o f b~cterioloeicalacidification were

integrnt Cat. and ~resumablvalso bclow rennet concentration Rx 1), and 0 those such that the
effects of rennet action prevailed (above Rxl). Although the inherent coagul~tingproperties of

tennet were of relatively secondary importance in the former situation, the added rennet had an

important directing influence, imparting to the developing gels viscoelastic properties

(consistency and moduli that is) distinctly related to those of met-acid casein gels produced

with little variation of the pH amund pH 6.4-5.7 initially (Le., systems that had undergone more

extensive enzymatic conversion of rnicellar K-casein).This points to a gradation in the regimes of

milk coagulation that was not clear to us before.

All cultured milks undenvent important acidification afier some time. As was made apparent,

the stage of gel development (nnneting) at which this happened, i.e, the extent to which

renneting and acidification overlapped-as determined largely by rennet concentration under the

conditions of coagulation investigated-seemed to be a major determinant of overall coagulation

behaviour. The cornerstone here is that the diffcrent Patterns of coanulation behaviour at and

above rennet concentration Rx 1seem to arise fiom diflercnt mttems of succession of renneting

and continuous acidificatioq (to be elaborated upon under Section 7.3.3).

In the following subsections, we examine experimental evidence more closely for the

influence of milk composition and pre-trcatment, including high heating and increasing pmtein

concentration, on the development of gel on combined renneting and bacteriological acidifcation.

Qualitative arguments for the possible physico-chmical bases for the patterns of coagulation

khaviour obssrved will be discussed more hilly in a later put.

7.2. Gel Dcvclopment from Cultured and Rcnncted M i k as Anected by Pm-

Tiuatment of Milk
In the secondary studies summuized herein we sought to asrss the extent to which the

accounts of combined rennet-acid coagulation pmnining to standad reconstituted skim milk

under standard experimental conditions may k generalized to milks of different compositions

and to differently pre-treated or coagulated milks. Only the mon salient points are dealt with

herein.

Attention was given to a range of milk systems and gelation conditions, as outlined in

Chapter 6, Figure 6.1. Testing was restricted to a nmow range of conditions of renneting and

acidification, with rheological measurements carricd out primarily with the Nametre viscorneter.

and no determination of K-casein hydrolysis except for some pre-heated samples. Fractical

considerations such as the estimated tirne-fiame of gelation experiments (especiaily at

temperatures lower than 40°C) were taken into account for selecting the concentrations o f rcnnct

and starter cultures. Rationalization of the cffkcts observed was attempted in light of the

experimental evidence obtained for standard systems and cumntly available knowledge about

milk coagulation proprties (sec schematic rcprcstntation of coagulation conditions in Figures

7.3.2u&b and 7.3.3, and details for their interpretation in Section 7.3.3).

7.2.1. The Use of Diffennt MUks and the Effects of VadousAddfdlonr

(a) Coanulation of Whole Milk vs. Reconstinited Skim Milk and Effects of Homonenization. As

mentioned in the preliminuy discussion in Section 6.3.1, qualitatively, similar patterns of

developmcnt of gel consistmcy msultcd on coagulation of h s h whde milk (whethet pasttutized

and homogenized or not) and standard RSM at C/4-Rxl, -Rx4, and -Rx8 at 400C (Figures
A7.2.l u&b and A 7 2 2 to A7.2.4). Gel fonnation h m non-homogenized (pasteurized) whole

milk (CM-Rxl and 4 x 8 ; Figures A7.2.2u&b and 73.3a&b)was notably slower and the values of

gel consistency werc lower cornparcd to plling homogmizcd whole milk and standard RSM,

however. Creaming and synemis phenomcna in non-homogenized whok milk probably

interfered with gel dcvelopment and its meuniment to a -ter extent than for homogenized

samples, especially at the lowcst concentrations of rennet (i.e., longest incubation before

c~agulation).

Many casein particles and smaller cwin-containing structures become adsorbed at the

interface be-n senun and ncwly formed fat globules following (valve) homogenization of

whole milk. Appmtly the partition of Ca phosphate in the milk remains largely unaffected

[Mulder & Walstra, 1974; Robson & Dalgleish, 19841. The action of mnet is analogous in

homogenized and non-homogenized milk but the homogenized fatkasein particles do not exactly

behave as large casein particles. Typically, Le coagulation of homogenized (pasteurized) milk

renneted at constant pH around neutrality occurs aftet a slightly shorter time, and gel formation

(fiming) and syneresis (with concomitant hision of gel particles) are slower than for untreated

milk. The diffeicnce in rennet coagulation tirne seems to arise because the critical level of

proteolysis of 'micellu' K-caseinrcquircd for aggregation is less for homogenized milk [Robson

Br Dalgleish, 19841. (Spreading of K-casein around the homogenized pmicks may lead to a

reduction of its stabilizing powcr.) The slowing down of coagulation seems to be contributed by

reduction of the total sudace ucr of the casein particks available for mutual interaction on

aggregation ( M e charge efftcts) and possibly also by reduction of the concentration of casein

particles in the scrum [Greenet d.,1983; Dalgleish, 1984; Robson & Dalgleish, 1984, 19871.

The possible mitigating influence of pre-hcating ought to k borne in mind.

In the pmsent work it is probable that the effccts of homogcnization on icnnet coagulation

wcre mostly ovmidden/countemcted by the cffccts of (sirnultancous) ridification invedigstcd.

This would have contributcd to lcaving the ôalance (successiveness) of the cffécts o f m t i n g

and acidification in homogenized whole mik largely unchanged compared with non-

homogenized nfennce miks (including MM), hence the broad sirnilruity ktween the

qualitative patterns of gel development resulting h m varying the concentration of minet

enzymes (Figures A7.2.4~-e).

m a t the gels formed fiom homogenized milk devclopcd higher apparent consistency than the

gels from unhomogenized whole milk and, to a llesser extent, RSM likely stemmed h m inclusion

of the homogenized fatkasein complexes within the coagulum structure. This would concur with

the reinforcing effect (increase in the numôer of junctions within the network) of fiimly divided

interactive fat globules (so-called 'filler' pcuiicles) reported by, e.g., van Vliet & Dentenerg

Kikkert [1982], van Vliet [1988], Aguilera & Kessler [1988], Xiong el al. [199 11, and Matsumura

et al. 119931 in differently prepared (milk) gel systems. Natuml fat globules in gels fiom non-

homogenized whole milk are merely caught in the casein network; this probably hampers the

formation of a continuous n e ~ o r k ,hence the relatively weak gels. Certainly, it is well

,

established in industrial pnctice that, unlike hard checscs, sane sofi cheeses and yoghurt-likc

fennented products do knefit fkom pre-pasteurization and homogenization of milk in ternis of

the textural characteristics such as consistency and viscosity and the reduced separation o f serum

in the final product [Puhan, 1988; Jana & Upadhyay, 19921.

(b) E f f w of Various Additions. The effca~of changing ion composition/distribution on

consistency devclopment fiom RSM at U4-Rx4 (-Rxl) and 40°C are illustratcd in Figures 72.1

and A7.2.S thmugh A7.29. Modifications included adding CaCI2 (0.02 % w/w rn 1.8 mM) or

NaCl (0.6% w/w rn 100 mM), or cycling the pH of the milk h m ca. 6.7 -) 5.8 (lactic acid) 6.7

(NaOH) rt klow 20°C either directly or a f k ovcrnight storage at pH 5.8 and 4*C, prior to

culturing, standardizingthe pH to 6.4, and renncting i t 40°C.

 0

350 -.
9

-RSR;pH RSM + 0.6% NiCl

.
m
RSM witb various rdditioar,
œ
CICRrQ 3

A
300 - - m 4

3 m 1

E6 250
9
b
1
- *

-r

Incubation time at 40°C (h)

Figure 7.2.1. ûverview of consistcncy dcvelopment curves for (pre-hcated)

a or pcvdipo Pfa&J lp lp prior to culturing and
rcnncting at u4-mat 40°C. Profiles of consistcncy C vs . time for each type of
milk are shown for single rcpnsentative expiments carried out with the Namem
rhcometcr, rcprcsentative pH &ta king shown ~lectivelyfor standard 9% RSM (no
addition or cycling of pH) and for RSM with 0.6% (wiw) NaCl added. A m w s point
to the regions of local maximum and minimum in consistency for such milks and to
the comsponding (appmximate) values of pH. (Plofiles for rcplicatcd and
comsponding measurcments of mik consistcncy and pH arc displaycd individwliy
in Figures A7.2.6 to A7.2.9 h contrast to the profiles obtained with standard RSM.)
 (0 The enrichment of chase milk with small amounts (e.g., 0.01%) of CaCI2 at constant
slightly acidic or mund neutml pH is frquent to pmmote both gel formation (aggregation and

finning) and synemsis. Robrbly calcium ions bind to the cwins in such a way as to reduce the

net (ncgative) surfafe charge of the renncted casein particles, and perhaps enhance tkir

hydrophobicity. ca2' may also participate in specific (bridging) interactions that favour

reticulation of the gel. There are suggestions [e.g., Roefs et al., 1985; van Hwydonk et al.,

198681 that the amount of colloidal Ca phosphate in the casein p h c l e s (which increases on

adding Ca to milk [e.g., van Hooydonk et al., 19866; N&l et al., 19891) is actually more

important for the renneting properties than is ionic Ca.

The addition of CaClt at the maximum level permissible and CM-Rx4, keeping the pH at

renneting constant, did not hindamentally affect the development of gel consistency in this work

(Figure A7.2.6). Most notable effects seemed to concord with those documented by NoZl et al.

[1989], qualitatively at leiut. [Direct (quantitative) comparison of the two sets of results is

dificult in part kcause of differences in experimental conditions, including pH ai rennet addition

(6.4 vs. 6.0) and coagulation temperatun (40 vs. 3OOC).] Experimental coagulation time was

essentially unchanged and so were the times comsponding to P- and *.P compared with

reference RSM with no added CaCI2. The (fint maximum) rate of gel firming increaseû slightly,

especially before Ph. The values of gel consistency were comparable (slightly lower) at P-

near pH 5.8, and slightly lower at Pmknear pH 5.4.

The moderate appreciable cffccts overall of adding CaCll under the conditions of combined

coagulation investigatcd may bc explained in part by considering the concentration of soluble

(rather than aâded) calcium, as emphasized by Noël et

.
al. [1989]. It cm be reasoned that

important soluble Ca was prcsent in the refetence milk in the beginning of renneting owing to, in

part, solubilization of micellar Ca on partial chernical and bacteriological pre-acidification.

Presumably the concentration (activity) of ionic Ca was a l d y mough to effectively pmmote

coagulation reactions-mostly through cffècts on renncting initially since a relatively high

concentration of rennet was used-so that additional ca2+at the level uscd had little influence on

coagulation behaviour (Le., 1ag and intcrplay bctwccn renneting and acidification processes).

Note that it is difncult to explain why sofiening of the gel may k more pronounced [i.e.,

dC(t)ldt more negative and gel consistency at Pm,,,

lower] in rnilk with addcd CaC12. Perhaps the

expected increase in micellar Ca phosphate on addition of Ca plays a part, e.g., through

ampliQing the effcîts of relatively late acid-deminemlization of the @ma) casein gel.

Confounding eflects related to (micro) syneresis are also possible.

The observations reported hem w m not detailcd enough to allow for determination of most

favounble mount of CaCI2for combined remet-acid coagulation as investigated in mis work. In

light of the results of Noël et al, [1989], taking into account the di-nces in pH at renneting, it

may be suggested that addition between 0.004 and 0.02% may be advantageous. Excessive

addition of CaCIfihrough important increase of ionic strcngth (and hence weakening of

electrostatic interactions) and possibly blockage by ca2+

of otherwise reactive miculation sites on

the casein*, would probably be detrimental to gel development, as suggested by the results of

Noël et al. [1989] for rennet and acid coagulation [ a h McMahon et al., 1984~;Patel & Reuter,

1986;and van Hooydonk et al., 1986c for strictly rennet coagulation].

(io The addition of NaCl to RSM at constant pH at renneting resulted in a conspicuous

attenuation of the 'shouldcr' and 'maximum-minimum' features characteristic for standard

coagulation conditions at ~ / 4 - ~ xand

l C/4-Rx4, respectively (Figures A7.2.7u&b). The

resemblance ôetwcen the piofiles obtaincd at C/4-Rx4 with d e d NaCI and those at CM-Rxl

with no added N d suggests that modulation of the coagulation khaviour of trcated us.

untreated samplcs arose (at lcast partially) because of important reduction of the efficiency of

(overall) remeting in the p m a r e of sait. It may bc sumiscd that renneting effkiency under

experimcntal conditions at C k R x 4 with NaCl approximatcd thit under the conditions i t CM-
Rx 1 with no NaCI. Similarly, the conditions at C/4-Rx 1 with NaCl seemed to appcoximate those
at C/4-RxO (acid control) with no salt.

The remcting experirnmb of van Hooydonk et al. [1986c] at constant pH amund 6.8-6.7 and

30°C showed that the addition of mther high levels (> 60 mM) of NaCl decrcases both the tate of

hydrolysis of micellu K-casein and the rate of gel fming [ a h Dalgkish, 1983; Grufferty &

Fox, 19851. Both the aymatic and aggregation stages of renneting arc probably impacted

through large increases in ionic strength (impoitant rreening of charges by electrolytes), Le.,

impairnent of the formation of ionic bonds, despite the reduction of electmstatic repulsion. Some

steric effects and reduction of the amount of micellar Ca (exchange nactions of casein-bound

ca2+with ~ a [Parker
3 & Dalgkish, 1981;van Hooydonk et al., 1986~1
may corne into play.

Zoon et al. [1989] showed that if rennet coagulation time was kept constant, additions of

NaCl up to 200 mM incrcascd gel dynarnic modulus at pH 6.65 and 300C. (At constant

concentration of rennet this happened only up to 100 mM NaCI.) The effect was partiy ascribed

to the increase in concentration and activity of Ca ions a e r salt addition. At pH 6.25, more

rennet was needed to kecp the coagulation time constant; rennet gel formation and aging wete

retarded at and especially above 100 mM NaCi, with no substantial change of ultimate gel

modulus. These observations were accounted for by considering the already quite high activity of

Ca ions at pH 6.25 (hence the limited influence of a further incrcase in Ca activity on decreasing

coagulation time and incrcasing modulus) and the more negative effect of shieiding of charges at

more acidic pH.

Acid Na caseinate gels made with 0.4-0.5% GDL by Lucey et al. (19976tc] had longer time

of formation, pH at fonnation lowcr by ca 0.1 pH unit, and Iower initial rate of increase of elastic

modulus when NaCl was added (120 mM) at 20,30, and 40°C.

The effcçts of dding NaCl undcr the conditions of expiments hetcin seem to involvc
modification of the relative coagulating efficiency of rcnneting vs. acidification, tesulting in
greatcr overlap of renneting and acidification. This would allow for the dmlopment of acidity at

wlier stages of gel fonnation (renneting) cornpucd with untreated milks, thereby limiting the

effects of latc acidification (rearrangement and soflening) of gel. [It seems that bacteriological

acidification was slowed down slightly in the prrsence of NaCI (LAB are relatively salt tolcrant

[R.Sic & Kumann, 1978)). but not enough to make up for the apparent slowing down of

renneting, Le., to keep the balance between the effects of re~etingand aciditication as in

reference miiks.]
It might bc that reduction in micellar Ca content contributed to limiting the apparent

softening of gel aftcr Ph by lessening the effects of acid-demineralization. The distinct

strengthening of gel at Rx4 for milk with added NaCl may reflect the combined effects of limited

hydrolysis of cc-casein and neutralization (including some shielding) of neptively charged groups

of the caseins by acidifcation and salt. For the conditions at Rxl with added NaCI (Le., little

renneting eEciency apparently), lower rates of firming and lower consistency resulted compared

to strictly acid gels with no salt. This probably nflects important inhibition of ionic bond

fonnation due to ionic eflects (screening of charges). These were piobably compcnsated for at

highet rennet concentration by the introduction of supplementary reactive (hydrophobic) sites on

the partly renneted casein particles.

(UI) Chemical pr-acidification of milk to about pH 5.8 followed by immediate neutralizntion

and standardization of the pH at rennet addition had littk (attenustion) effects on the development

of gel consistency from both standard and pre-hcated (90°C-1 min) RSM at Cl4-Rx4 (Figures

A7.2.k and A72.9~).Holding the m p l e s at the low pH and 4 O C for 15-20 h before

neutralization, howevet, rcsulted in a distinctly more shallow maximum-minimum consistency

pattern in both standard and prc-hcated milk (Figures A7.2.86 and A7.2.96). The development of

gel consistency was slightly rctardd and skwer ovcrall cornparcd to control RSM, suggcsting
that the indirect acidification/neutralization p d u m had adverse effccts on the effkicncy of the

nnneting proccss.

Lowering the pH of milk at nlatively low temperature solubilizes a substantiel amount of the

indigenous (and heat-precipitateâ) micellar Ca phosphate as well as c a s c i d u s dismpting the

micellar s û u c m u t without leading to coagulation. Subsequent neutralization apparently

leads to (partial) reformation of casein-Ca phosphate complexes although it is unlikely that the

original micellar characteristics are restoml. The cxperiments perfomed by Lucey and CO-

workers 11992, 19961, for instance, showed that the casein particles in acidified and directly

neutralized ('re-fomed') unheated milk have renneting and buffering properties distinct 6mn

those in the original milk. Elevatod ca2+activity (which pmbably occun at the expense of the

content of Ca phosphate-and casein?-assaciated with the reformed particles) appws to be

central to the impmved rcnnet coagulability at constant pH around 6.7 and 30°C of unheated and

hi&-heated refomed milks [van Hooydonk et al., 1986b; Singh et al., 1988; Lucey et al.,

1993a.c; Lucey et al., 19961. (The improvemcnt is gcnenlly most pmnounced for indirectly

neuûalized rnilks.)

Interpretation of the findings in this work, and in particular the differences in minet-acid

coagulation behaviour following direct us. indirect cycling of the pH, is not easily at hand. In

directly neutralized milks, it may be envisaged that not too dns<i; disruption of the micellar

systern occurted on temporary aicidification and/or that casein-Ca phosphate piuticlcs with (rennet

coagulation) pmperties not too different fkom those of the original cisein particks emerged on

neutralization. It may k that the expcctcd incrcase in ca2' in the nformed m i b had limited

effects on coagulation fatum for rcasons similar to those put fmard prcviously for acidifying

and renneting milks supplemented with CaCb.

Proôably the severity of the prc-ocidifcation step was amplificd by pmlongeâ storage at low

temperature. [It is well establishcâ thrt the coolin8 of milk (at physiological pH) under 1 O T leads
to important tirnadependent solubilization of micellu Ca phosphate and dissociation of casein.

These chanp arc lugely mversed by nising the temperature but then is an irrevenible increase

of the pH after cooling.] It may k suggestd that the apparent los of rennctability of indimtly

neuûalized milk under the coagulation conditions in this study originated in part fiom excessive

amounts of ionic Ca in the milk (as contributed by indirect neutralization and subsequent m i a l

acidification). At suboptimal levels of ionic Ca, molecular fxstion of cal*may have hindered

gel formation and organization initially by blocking potential reticulation sites on the @ma)
caseins and Iater by limiting (counteracting) acid-demineralization of the caseins, i.e., by

preventing the likration of othenuir reactive reticulation sites et al., 19891. Reduced

content of Ca phosphate in the ckccin particles and alteration of the enzymatic activity of rennet

may have contributed to decreasing n~etability.This would account (in part) for the delay of

coagulation, the reâuction of (first maximum) rate of finning and consistency of gel at Pm, and

the relatively limited sofiening subsequently.

Perhaps coagulation characteristics mon like those of d i r d y neutralized and reference

milks would have been measured (Le., more favourable repartition of ions and caseins attained in

the system) had longer equilibration times (> 30 min) at the coagulation temperature ken

allowed between indirect neutditition and coagulation. Cold storage of milk may also cause

retardation of acid production by yoghurt bacteria [Mit & Kunnann, 19781. It is not clar

occumd in our cxpcriments but if them was such a delay in acid production
whether this ac~ally

it proôably w u not sufiicient to maintain the relative eficiency between renneting and

acidification as in rcference milks.

Z2.2. Eflects of Gelutibn Temlpcrciturc

nie influence of temperature on the coagulation khviour of standard RSM was investigated
o v r the range 20.40°C at concentrations of starter culhucs and iennet Cf4, CE, and U1,and
Rx8 and Rx16,nspectively (pH at mnet addition 6.4). Samples pre-heated at 900C for 1 min

were also tested at concentrations Ca-Rx8.

In al1 the temperature series for both types of milk, decreasing the temperature of coagulation
resultcd in distinct mtardation and genaal slowing down of consistency (modulus) development,

as expcted, with attenuation of the maximum-minimum pattern characteristic for standard

coagulation conditions at relatively high concentrations of rennet and 40°C (Figures 7.2.2a&b and

A7.2.10 through A7.2.13). nie effects were most ~onspicuousbeiow 30°C: at 25 and 20°C there
was no clear maximum/minimum in gel consistency (modulus) Lie., the values of dC(i)/dt and

dG '(t)/dt remained essentially positive throughout gel differentiation and only a local (non-zero)

minimum in dC(r)ldt and dG '(Wdt was observable in the region deemed equivalent to P&P,I.

(i) The time-profiles of elastic modulus obtained fiom standard RSM at 25°C and Ca-Rx8
using the Carri-Med rheometer (Figure A7.2.12c, upper panel) compared well (qualitative\y) with

the profile of elastic modulus obtained by van Hooydonk et al. [1986b]at the same temperature

using an Instron Universal Testing Instrument [applied fkquency 0.2 Hz, pasteurid skim miUc,

apparent concentration of rennet bctwcen Rx4-Rx8,unspecified concentration of lactic acid

starter (aCl4-Cf2 judging by the profile of pH), and pH at renneting a 6.6). In the work of van

Hooydonk, a shallow maximum-minimum pattern of development is apparent with values of G '

(arbitrary units), pH, and % hydiolysis of K-casein mund 20, 5.7,95 at tuming point Pr*,about

12.5 h after rennet addition; and around 15, 5.3, 2 95 at point hi,about 13.5 h aAer rennet

addition. (About 90% of K-casein had been hydrolyzed at the onset of measurable coagulation

around pH 6.3, afkr about 10 h of incubation at 2S°C.)

In the present work at 2S°C, the point decmed equivalent to PMnr/Pmin occuncd amund pH

5.5-5.2. Ovcrall, simiP values of pH wete mersureci at the characteristic points in the cwves at

al1 the temperatures studied. The ralatively stocp secondary incrase in gel madulus (consistency)
 Incubation time at different temperatuns (h)

Figure 72-20. Ovewiew of consistency development curves for standard RSM

cultured and remeted at W - M ai a
tsmDerahius bmvtcn pad &e
Profiles of consistency C and pH vs. time at each temperature am shown for single
representative experiments carricd out with the Namette rhcometer. Arrows point
to tuming point(s) in the curves of consistency and to the comsponding
(appmximate) values of pH. (Profiles for replicated and comsponding
measurements of milk consistency and pH are displayed individually in Figures
A7.2.lh&b .)
 Incubation time at different temperatures (h)

Figure 7.2.26. Ovewiew of consistency development curves for

90°C-1 min and cultured and renneted at Q 7 - r n at betwm
4 a. Profiles of consistency C (and pH when available) vs. time at each
temperature are shown for single rcpresentative experiments carried out with the
Nametrc rheometer. Amws point to tuming point@) in the curves of consistency
and to the comsponding (approximate) values of pH. (Profiles for replicated and
comsponding measurernents of milk consistency and pH are displayed individually
in Figures A l S . l3a&b .)
ùeyond (apparent) Pw,in the region of pH ôetween 5.5/5.0 and 4.5, s t d out in both the pmfik

reportcd by van ~ k ~ d o et k [1986b] and the profiles obtaincd at 25 and 20°C in our work.
n al.

The gsneral impression was that the coagulation profiles at the lower temperatures could k
derived fiom the ones at 40°C by stretching ('unfolding') the curve in a mostly horizontal

direction (i.e., increasing the the-=ale of coagulation nattions). with an upward twist in the part

of the cuwe comsponding to more acidic stage of gel evolution below pH s 5.5-5.0.

In the profiles of gel modulus at 2S°C in our çnidy, the zone of important acidification below

pH = 5.6 was apparent also from the characteristic transient increase of tan Gfiom badine values

around 0.23-0.25 (i.e., 6 = 13- 14') and peaking at values around 0.32-0.36 (i.e., 6 18-20')

(Figure A7.2.12~).The maximum in fun doccuned shortly afier the tuming point equivalent to

P&PLIk in the curve of elastic modulus. Stabilization of tm 6 in the later stages of gel

developmcnt occuncd mund 0.23-0.25 (6 = 13-14O), Le., at comparable values as for manire

acid and rennet-acid gels a 40°C and 0.1 Hz (Sections 7.1.4 and 7.1.5, and Figure 7.2.3).

(io Elements of explmation for L e observed gradation in the character of coagulation

profiles on lowering coagulation temperature may be found in the (differential) effect of

temperature on the rate of the mctions at play. Over the range of temperatures studied, the

hydrolysis of K-casein by remet enzym-d rnost likely also the production of acid during

fennentation by L w considcrably less affected by changing the temperature than the

aggngation and gelation of (para)casein. Undcr the conditions of experimmtation in the present

study, one may reasonably assume temperature coefficients (Q,3 of the ordet of 2 for both the

enzymatic naction of rcnneting and the growth of LAB (Le., the biological acidification of milk),

and 16 for the aggtegation mctions of rennaing [e.g., van Hooydonk & van den Berg, 1988; Jay,
19921. Below 300C the aggregation stage of mnneting kcomes rate-limiting and bth the time at

the onset of corgulation and the time at which gel 'stm>gth' is suficient to start cuning for

mriking ch- increcisc markcdly with decreasing the temperature.

 Incubation time at 25 or 40°C (h)

Figure 7.2.3. Typical evolution of l o s angle 6 (tan6 = Gt'/G') upon the coagulation
of cultured and remeted standard RSM (C/2-Rx8) vs. renneted (CO-Rx8 at pH 6.4)
and biologically acidificd (C/2-RxO) standard RSM at 2 a a. Profiles of 6 us.
time for each set of coagulation conditions are show for single representative
experiments carried out with the Carri-Mcd rheometer. (Profiles for replicated and
corresponding measurements of milk loss angle, viscous and elastic moduli G" and
G', and approximative pH are shown elscwhere in the dissertation.)
 It is Iikely, thenfore, that part of the obxrved mitigating influence of temperature at the

levels of rennet used occurred through modification of the patterns of succession of renneting and

biological acidification. It may be reasoned that the icnneting and acidification processes became

increasingly concurrent (overlapping) due to important slowing down of renneting as the

coagulation temperature was lowered (especially in the range 30-20°C). n i e largely monotonous

development of gel strength at temperatures strictly below 30°C and rennet concentrations Rx8-

Rx 16 may thuç be seen as an indication that the relative coagulating eficiency of renneting under

these conditions approximated the (iimited) efliciency of renneting at concentrations around R x 1

at 40°C. It is also probable that the 'smoothing' of coagulation cuwes below 30°C was

contributed by important slowing down of the ceactions involved in gel softening a higher

temperatures. lncrekpcd initial concentration of soluble Ca phosphate in the milks incubated at

lower temperatures may have played a mle also. Gels may be stronger andor of a different type.

(Ui) M e r notable featurcs in the experimental profiles at different temperatures may be

related to differential effects of temperature on the characteristics of rennet vs. acid milk gels.

Conflicting results exist in the literature about the temperature-dependence of rennet gel

'strength' at constant pH m n et al., 19886 for a review]. To be sure, cornparisons are seldom

straightforward partly kcause the measumnents reporteâ are often carried out within variable

times of gel fomatiodaging, at temperatures that may or may not be those of gelation. Home

[1998] reported an approximately four-fold linear increase of rennet gel modulus (independent

from enzyme activity) in the range 20 to 35-40°C.

For typical acid (yoghurt) gels, high rate of acid development a high incubation temperature

genetally contributcs to poor (CO-) gel formation and incrcased synensis. L-ng the

temperature h m Ca. 44OC to S 38OC is actually recommended to improve gel finnness,

consistency, and (apparent) viscosity [Kosikowski, 19771. For acid gels resulting fiwn the

hydrolysis of GDL, low gelation temperatures (e-g., 20°C) alro go along with higher elastic
rnodulus at long times [Arshad et al., 1993a,b; Cobos et al., 1995; Lucey et al., 1997b. 1 9 9 q .

Low temperatures (< 30°C) afùr completcd formation of acid gels tend to favour higher

consistency and moduli [Rriic & K m a n n , 1978; Roefs, 1986; Schulze et al., 1991; Lucey et al.,

1997a,b].

From the profiles of remet-acid coagulation obtained in this work, it may bc suggested that
then was a different temperature-dependence of the magnitude of gel consistency (modulus) in

the poflions of the cuwes before and afkr the point (equivalent ?O) Pd Le., at values of pH

above and below 5.5-5.2. In control experiments at CO-Rx8 and CI2-RxO, respectively, no

wbstantial effcct of coagulation temperature (25 vs. 400C) on the long-terni values of consistency

of remet and acid gels was observed (Figures A72.14aûlb and A7.2.l5a&b). [Note the variations

in experimental rate of gel finning (as dC/di) around pH 5.0-4.6 for the acid controls at 25T in
Figure A7.2.15~. It is not clear whether these variations reflected actual evolution of gel

characteristics and whether the secondary variations observed for the acid controlr at 400C in the

same region of pH werc of an allied nature (Section 7.1.4d-e).] Certainly, one has to be cautious

in the interpretation of the variations in gel consistency (modulus) on changing coagulation

temperature. In particular it is difficult to assess the cxtent to which the strengthening of rennet-

acid gel beyond the tuming point in the coagulation profiles at 20 and 2S°C may be contributed

by interaction effects of minimal renneting and acidification. effects of temperature, andlor

confounding effccts of time (Le, c~v~y-over

of the slow incomplete setting of primary rcnnet-like

gel).
(iv) More unambiguous effects of varying coagulation tempmture were noticcable in the

profiles of tan 6(= G'IG'), as alluded to in Section 7.1.5 (Figures 7.2.3 and A7.2.12~). In the

portion of the curves cornsponding to the setting of a mainly rcnnet milk gel above pH = 5.6, the

(essentidly constant) values of fun Gdccrcwd mukcdly on lowering coagulation temperature. In

terms of the values of Gmeasurcd at 0.1 Hz, the decreasc was fiom about 25-30° at 40°C to about
13-14' at 2SOC. In contrast, the asymptotic values of 6in the latter (acidic) part of the curves did

not change appreciably with temperature, nor did the amplitude of the increase in G(relative to

the 'rennet bsseline' that is) in the apparent transition zone. This means that the maximum value

of Galso decreased with decreasing temperature, fiom about 30-32O at 40T to 18-20' at 2 S C [A

similar fmre is apparent in the profiles of lois tangent pmented by Lucey & Singh [1997] for

pre-heated milk acidified with GDL (i.e., gelation conditions akin to control conditions in our

work), with Gmaxirnum decreasing fiom ca. 270 ai JO°C to CU. 2 2 O at 30°C.J

Globally. the above observations concur with earlier reports on the temperature-dependence

of t m Gfor (mature) rennet- and acid-induced skim milk gels. Zoon et al. [1988b] and van Vliet

et al. [1991u] documented important e f f m of coagulationltest temperature (especidiy in the

range 40-30°C at 0.1 Hz) on the value of tan Gfor rennet gels prepared around neutral pH (6%29'

at 4WC, = 17' at 30°C, and a 14' at 25T). This contrasted with the moderate temperature

-
dependence of tan G for acid (GDL) gels in the range 20-400C at O. 1 and 1 Hz (6a 1 1 14O w f s ,

1986; Arshad et al.. l993a,b; Lucey et d, 1997b]). Schulze et al. [199 11 further showed that the

value of tun 6 for set yoghurt gels (fiom pre-heated skim milk) measured at 1 Hz was largely

unaf5ected (Ga ISO) by changing the temperature of measurnent in the range S43T. It may be

envisaged that the distinct liquid-like (dynmic) character of (mainly) rennet gels (as estimated by

the relatively high value of tun 6) at the considered fkquency and 40°C makes them more

susceptible to tempcraturc than acid gels: lower rates of relaxation of the interactions within

rennet gels at lower temperatures (Le., lower rates of thermal motion) would imply that a larger

proportion of the total number of interactions is 'seen' as elastically effective, hence the lower

values of tan S.

In the contcxt of the pmmt study, the differcntial tcmpcraturc-dependence of tm Gfor rennet

and acid gels certainly highlightcd the danamation khveen mostly cnzymatic and acidic stages

of gel formation. &tta overall distinction betwan the processes of gel devclopment at
temperabms above 30°C actudly was one of the w o n s fw standardizing gelation temperature

at 40°C in the study, despite Le incrcaseâ likelihood of gel syneresis and undesirable bacterial

growth. From a practical standpoint, this mcant that the dumtion of coagulation cxpdments

could ôe kept within a reasonablc range using reasonable arnounts of rennet and starter cultures.

7.2.3. Effectsof Pn-Heaîing M.&

Typical investigations of the cffects of pre-heating milk at high temperatures on gel

development wcre canitd out using -dard (low-heat) RSM heated at 90°C for 1 min in a water

bath afler reconstitution. The pH at renneting was standardized to 6.4. Series of coagulation

profiles for such pre-heated milk were obtained at 40°C (4) at constant concentration of rennet

enzymes (Rx 1 and Rx4) for concentrations of acidifjing cultures over the range Cl8-Cl1 (Figures

7.2.4a&b and A7.2.16 to A7.2.20), and pi) at constant concentration of starter cultures (Cn,
C/4,

and occasionally C/8) for concentrations of rennet over the range Rx 1-Rx 16 [Figures 7.2.5 and

A7.2.2 1-A7.224 (Nametre rheometer), and 7.2.6 and A7.2.25-A7.2.28 (Carri-Med rheometer)].

Additional combinations of the levcls of starter cultures and rennet were testcd when

pertinent, including control conditions with cultures only (Cf8-C/l), as in yoghuit-making, and

rennet only (Rx 1-Rx 16) at pH 6.4. To hirther the cornparisons and dernonstrate differences in

coagulation bchaviour arnong diffetently pn-heatcd milks, few experiments were conducted in

which RSM had been s u b j d to more intense pre-heat tteatmcnt mounting to sterilization

undet retort-style conditions by autociaving 8t 1 15% for 10 min.

(a) Gelation Profiles for (Derivativel Coosistcncv. Dvnamic Modulus. and L o s T m .As can
k appmiated in Figures 7.2.4 and A U . 16 and 18, the characteristic patterns of instrumental gel
consistency development evidenced for bacteriologically acidified low-heat RSM renneted at Rx 1

and abovc werc also rocognizable (with some differences) for RSM prc-hcated at 900C-f min.

(Gloldly dso, the cffects of cycling the pH and lowering gelation tcmpmtute below 40°C were

similu in both types of milk; sec discussion in Sections 7 . U b and 73.2, isspcctively.)
 1 8.:-

- 9 i
PH Prc-heated RSM (!Mec-1min),
- ,,(sarnples culturrd C/8-Rx
Fm
rcnnetcd
at C/1-, C/2-, and
&
1)
cii-m
1
0 r

Consistency .

Incubation time at 40°C (h)

Figure 7 3 . k . Overvicw of consistency development curves for differrntlv

RSM pt 90°C-4 a
and rcnneted at Ievel at 40°C. Profiles
.
of consistency C and pH vs time for each lcvel Cli of acidifying starter cultures are
shown for single representative experiments carried out with the Nametre rheometer.
Anows point to the regions of local mwimum/minimum in consistency and to the
corresponding (approximate) values of pH. (Profiles for replicated and
comsponding measurements of milk consistency and pH at each level of cultures
are displaycd individually in Figures A7.2.l8a&b .) Compan with the counterpart
tirnaprofiles of consistency and pH for non prc-heated standard RSM shown in
Figure 7.1.18~.
 s 6.5
mm. - œ
m m rn
pH Pmbcitcd RSM (!M°C-1 min), 1

Consistency

Incubation time at 40°C (h)

Figure 7.2.46. Overview of consistency development cutvcs for differrntlv

RSM 81 90°C-1 and rennetcd at level Ba4 at 40°C. Profiles of
consistency C vs. time for each level Cli of acidifying starter cultures are shown for
single representative experiments camied out with the Namette rheometer,
representative pH data k i n g shown selectively for the milks coaplated at CIL, Cl4-,
and C18-Rx4. Arrows point to the regions of local maximum and minimum in
consistency at Cl1 and Cl8, and to the corresponding (appmnimate) values of pH.
(Profiles for replicated and corresponding measurements of milk consistency and pH
for each levcl of cultures am displayed individually in Figures A7.2.19u&b.)
Compare with the counterpmt time-profila of consistency and pH for non pre-heated
standard RSM shown in Figure 7.1 ,186 .
 9 PH
Pm-brtd RSM (90°C-1 min), :
(mik samplc cultured WRr/
.
I

& rcnneted at a4-m

'

-
I' -
Consistency
1

Incubation time at 40°C (h)

Figure 73.5. Overview of consistency development curves for RSM pt

90°C:-1 & cultured at level and diffmntlv & et 40°C. Profiles of
consistency C us. time for each level Rq' of rennet enzymes are shown for single
representative cxperiments carried out with the Nametre rheometer, representative
pH data king shown selectivcly for the milk coagulated at Cl4-Rx4. h w s point
to the regions o f local maximum and minimum in consistcncy at C14-RxQ and to the
comsponding (approximate) values of pH. (Pmfilcs for replicated and
correspondhg messurements of milk consistency and pH for cach lcvel of rennet are
displayed individually in Figures A7.2.23adb .) Compare with the counterpart time-
profiles of consistency and pH for non pre-heatd standard RSM s h o w in Figure
7.1.200.
 I

Elastic modulus G'

Incubation time at 40°C(h)

Figure 7.2.6. Overview of elastic modulus dcvelopment curves for RSM

a 90°C-L culturcd at level and at 40°C. Profiles of
clastic modulus G' vs. time for each level Rxj of rennet enzymes are show for
single representative experiments camed out with the .-
Representative loss angle S data (tan6 = G"/Gr)are show selectively for the milks
coagulated at C/4-Rx4, -Rxi,and -RxO; pH data for the combination C/4-Rx4 were
obtained in independent replicated expcriments with the Nametre rheometer.
(Profiles for replicated and comsponding masurement. of milk viscous and elastic
moduli and loss angk for each level of rcnnet are displaycd individually in Figures
A7.2.26u&b .) Compare with the counterpart timc-profiles of consistency (Narnetre
rheometer) and pH (for pre-heated RSM)shown in Figure 7.2.5 and with the profiles
of G' and 6 for non pre-heated standard RSM in Figure 7.1.21~.
 The time-scaie of gel development was comparable to that for low-heat milk at

concentrations of remet Rx4 and above, and slightly reduced at concentrations Rxl and below

(contrast Figures 7.2.4a&b and 7.2.5 to Figures 7.1. lladtb and 7.1.2Oa; sec dso Figures A7.2.16-

24). The average values of pH md K-caseinhydrolysis at distinct stages of gel formation h m

heated and standarâ RSM were similar within experimental variations: about pH 5.7 and 30-35%

at the onset of measurabk coagulation, and pH 5.2 and 40% around P&Pmki at concentration of

rennet Rxl; and about pH 6.4-6.0and 55% at the onset of coagulation, pH 5.5-5.7 and 75% at

P*, and pH 5.0 and 80% at Pmh at concentrations of rennet above Rxl. (Estimates of the
conversion of u-casein for pre-heated milk were obtained at 40°C for the combinations of cultures

and rennet CO- and CI8-Rx 1, -Rx8,and -Rx 16; and Cl2-Rx 1 and -Rx8.)

(i) A notable difference concemed the magnitude of gel consistency (and absolute rate of

development thereof) which was consistently lower during remet-acid coagulation of pre-hcated

RSM compared to non-pre-heated RSM. Consistency optima at Pm and P,,,h, for example, were

markedly Iower for heated milk than for untreated milk.

It was aiso noted that for the conditions of combined coagulation at concentration of rennet

Rx 1, the shoulder in the traces of consistency for standard gelling milk (Section 7.1 Sb) tended to
take the fom of a better nsolved, cilbeit shallow, maximum-minimum in the profiles of

consistency for pre-hcatcd milk over the region of pH between 5.5 and 5.0 [i.e., unlikc in

unheated milk, gel consistency developmnit from minimally m e t c d heatcd milk was
characterizcd by the existence of nul1 or negative values of dC(r)ldt; contrast Figures 7.2.4~and

A7.2.I8a&b to Figures 7.1.180 and A7.1 .#Mc].

For the conditions of coagulation at above Rxl, it seemed that the humpshaped p r o f k of

gel consistency for pre-heated milk wac not as mproducible and symmetrical as for standard

milk, even though then did not ~ c c mto be important synercsis in rcnnetcd hcat-ecmd systems

[sec Section (b) for a possible explmation]. The abovc modulations in the cvolution (magnitude)
of Nametre consistency for acidified and nnneted pmheated vs. non-prc-heated RSM wcre al1

the mon intriguing in vicw of the contrastcd cvolution of Carri-Med modulus for such milks, and

the developmcnt of both consistency and dynunic modulus for bacteriologically acidified pre-

heatod vs. non-pre-heated controls, as discussed bslow.

(U) Gelation profiles for cu~tundcontrds fiom RSM pre-heatcd at 90°C-1 min are s h o w in

Figures A7.2.16~and A7.2.17a&b. As expected lactic acid coagulation of heated milk occurnd

at slightly higher pH around 5.8-5.1 (Le.. sharier times) and more nipidly than for untreated milk
(Section 7.1.44 Figures 7.1.14- 16). m e effects of thermal processing on the coagulability of

milk by acid have been reviewed by Mulvihill & Gnifferty, 1995.) Of paiticular interest in the

context of the prcscnt study wcre the highet values of experimental gel consistency (and elastic

modulus) which resulted on strictly acid coagulation of pre-heated milk compared to standard

milk. (A similar impression was derived fiom subjective evaluation of gel 'fimness' by the touch

following the completion of instrumental mcasuments.) Certainly thex rcsults are consistent

with the well-recognized beneficial influence of optimal pre-heating of milk (Le.. optimal

denaturation and integration of the whey pmteins) on the fimness of acid (yoghurt) gels [e.g..

Rdic & Kmann, 1978; Tamime & Robinson, 1985; Dannenberg & Kesskr, 1988a.bJ.Net as

clear an effect of increasing the concentration of acidifying starters (Cf!?-C/1)on the consistency

of mature acid gels from prc-heated RSM was notd cornparcd their non-pre-heated counterparts
(Section 7.1.4e).

Another notable festure for bacteriologically acidified pre-heated RSM evidenced in this

study concemed the relatively well-nsolved secondary variations in the rate of consistency (and

modulus) devclopment in the region of pH h a n 5.2 and 4 . M e more so it seemed, the

lower the concentration of starter ôacteria, as was described in Section 1.1.4e (Figum 7.1.16udtb

and Af.l.40adib). This concurs with the observations of Lucey et al. [199w at values of pH

ktwecn 5.2-5.0 [mwwments of G ' and tm 6at 0.1 Hz at 30 and 42OC for RSM pre-hcatcd at
8S°C-30 min and inoculateci with 2% (wlw) starter cuitun or 1.3% (wlw) GDL]and of Kelly &
O'Kennedy [20ûû; GDL, 40°C]. Expenmcntal cvidcnce suggcsts that this panicularity was an

amplificatioion of the effect more 'latent' in cultured low-heat milk. Perhaps this is related ta

incnwd apparent efficicncy of coagulation piocesses in acidified heated milk. (Expwimentation

wilh prc-heated systcms actually proved quitc usefil, adding confidence that the observations

pertaining to standard millcs wcre not merely instrumental artifacts.) Note the continuity between

the latter observation and the apparent modulation (better molution) of the maximum-minimum

in the consistency of minimally renneted aciditied milk brought about by pm-heating.

It rnay be envisaged, in light of the results of Law [1996] and Singh et al. [1996] (Section

2.2.6u), that the particular behaviour of acidified pre-heated milk reflects distinct solubilization

andlor associative pmperties of the heat-modified caseins-whey proteins v i s - b i s interchange (ce-

association) nations with the m m upon acidification. Promotion of overall protein association,
for exarnple, may promotc the coagulating efficiency of acidification in heated milk. The

(specific) role of heat-denatureû whey proteins and K-casein in the destabilization and

aggregation of such systems surely remains elusive. There are suggestions that the complexation

between Plactoglobulin and micellar u-casein reduces the ability of the K-casein to stabilise the

casein particles against acid-induced coagulation [reg.,Home & Davidson, 1 9 9 3 and

~ ~ results in

Section 4.31 and reduces the tendency of the awgated particles to tùse into larger clusten

during fermentation [e.g., Dannenôerg & Kessler, 1988bl (the latter reaction would contribute to

impmving overall hydiophilidtcxhue popntin of yoghurt gels made fiorn optirnally pre-heatcd

milk). Perhaps active participation of the whey proteins in acid gel assembly increases the

effective concentration of gelling protein md/or initiates culy aggregation owing to the iclatively

high isoelectnc pH of PLg (= 5.3) [e.g., Lucey et d., 1997u, 1998c,e]. (Apparently, the colloidal

Ca phosphates in pre-heated and unhcatcd milks have m e basic behaviour on acidification with

about completc solubilkation arnind pH 5 2 pdglcish & Law, 1989; Singh et al., 19961.)As put

forward in the discussion of the cffefo of adding CiCl,, it may be that elevated content (and
somewhat different properties) of micellar Ca phosphate (and related reduction of soluble c ~ H in
)

heated milk play a mle in modulating the npercussions of subsequent acid-demineralization of

the caseins with respect to the finning ( s o k i n g ) of the gel phase.

In tenns of the evolution of loss tangent (tun 6 = G"/G'; Figures 7.2.6 and A7.2.25-28, and

Figure 7.2.7 us. 7.1.22), comparable profiles were obtained for biologically acidified heated us.

non-pre-heated RSM, with the exception that for heated acid controls the values of tan 6(4over

the region of local maximum devclopment wcrc higher by about SO,with peak values around 27-

29'. In light of the results obtained for acidified and renneted millcs (see klow), it can not be

excluded that part of this difference reflects instrumental limitations in molving viscoelastic

parameten for low-heat acidifying milks in the eatly stages of gel formation because of the

relatively weak gels which resulted in such systems (see Section 7.1.4.d). Ultimately, loss angle

for pre-heated and non-pre-heated milk gels tended toward similsi, lower values around 13- 14 O .

The data for pre-heated milk a g m well with the values of Grcpotted by Schulze et al. [1991]

and Ronnegaid & Dejmek [1993] for the formation of acid gels by acidification of heated milk

with yoghurt bacteria (or glucon~&lactonein the work reportcd by Lucey & Singh [1997] and

Lucey et al. [1998c,d]). The results o f Lucey et al. [1998c,dj also pointed to a differential

evolution of tan 6 for high heat-treated milk (maximum in ton6 around pH 5.1 at 30T) us.

unheated milk (no maximum). For pre-heated milk (80/8S°C-30 min), the substantial increase in

ton 6shortly after gel formation w u tentatively related to an incrcaseâ susceptibility of the bonds

to breaklrelax and, hence, to an increased propensity for structural tcamngements (including

visible cracking) [Lucey et d.,1998~~4.

Covalent interactions of dcnaturcd whey proteins with

wasein appeared to play a key mle in the occurrence of the phenornenon and it was suggested

that maximum in ton 6 may tcflect a transition h m an acid gel initialîy dominated by

interactions contributcd by denaaucd whey pmtcins to a network dominated by casein-casein

interactions at values of pH below 5 .O bucey et al., 1998~1.

 Incubation timc at 40°C (h)

Figure 7.2.7. Typical evolution of loss angle 6 (tan6 = G"/G') upon the coagulation
of cultured and renneted RSM (CM-Rxj) pt 90°C-Lmin (or J 1Sac-1
O
vs. renneted (CO-Rxj at constant pH) and biologically acidified (C/i-RxO)
.
at 40°C. Profiks of 6 vs time for each set of coagulation conditions are shown
for singk representative experiments carcicd out with the C h - M e d rheometer.
(Profiles for nplicated and comsponding measurements of loss angle, milk viscous
and elastic moduli O" and G', and approximate pH arc show elsewhere in the
dissertation.) Contrast to the counterpart time-profiles of 6 for non pre-heated
standard RSM shown in Figure 7.1.22.
 @hi) Typical profiles of dynamic modulus development for cultumi and renneted milks as

mcasured with the Carri-Med rheometer are illustrated in Figures 72.6 and A7.2.25 to 28. n i e

impression h m the profiles of G '(0 that rcsultcd at constant level of starter cultures ((214) was

that the positive effect of pic-heating RSM at 90°C-1 min on increasing the modulus of strictly

acid milk gels (and its rate of change over tirne) was still much in evidence in acidified milks

renneted at concentrations between Rxl and Rx8. (Similar impressions were derived fiom

subjective assessments of gel finnness.) At concentration of rennet Rxl, this was apparent

throughout the course of gel formation, while at concentrations above Rxl, this was particularly

manifest in the stages of development beyond P&PIIa, that is, during the mainly acidic stages of

gel development. Long-tem values of elastic modulus did not vary substantially with increasing

rennet concentration in the range R x 1-Rx8.

These obsewations were at variance with the effcets of pre-heating and combined renneting-

acidification (mon precirly, the apparent deleterious cffcct of renneting) as estimated through

measurernents of gel consistency with the Nametre viscorneter (e.g.. F i p m 7.2.5 and A7.2.2 1 to

24). The overall shape of modulus coagulation vs. consistency cuwes also appeared to differ: at

concentrations of rennet above Rx 1, in particular, the 'hurnpy' character of modulus development

for pre-heated gelling milk appeared distinctly shallower [Le., less negative values of dG '(r)ldt

minima wen measured with a l e s clear minimum in G '(0 at P.*] than for non-pre-heated mik

(Figure 7.2.6 vs. 7.1.21~;Figure A7.2.266 vs. A7.1.566; and A7.2.27 vs. A7.1.57b). The latter

observation actually seems to concur with the general reduction in absolute rate of gel

development [including rate of gel softening, ie., less negative values of dC(t)ldr minima]

evidenced in terms of the development of consistency for acidified and renneted pre-heated milk.

It may k that additional cross-links withinlarnong the gel particles through hydmphobic

interactions with denaturd whcy proteins countcracted the effects of acid-induced

demineralization (gel sofkening) to some extcnt.

 In put, the abve observations about gel modulus development seem to concur with the

anecdotal observations of van Hooydonk et al. [19866] regarding the setting of high-heatcd

(yoghurt) skim milk vs. pasteurized skim rnilk. (Their expcnments were perfonned at 2ST and

0.2 Hz with an Instron Universal Testing Instrument, apparent concentrations of cultures and

rennet between C/4-CI2 and Rx4-Rx8, nspectively, and pH at rcnneting = 6.6.) van Hooydonk

and CO-workersalso reported a similar pattern of elastic modulus development in milks subjected

to hi* and low heat intensity, and noted that the minimum in p
l modulus for hi&-heated milk

was not as distinct as for pasteurized milk. The apparent retardation in the development of

modulus for heated milk gel they mentioned in the region of Pm* around pH 5.3 was not observed

in the present study, however.

Qualitatively, the overall evolution of loss tangent (and its concurrence with the evolution of

elastic modulus) for pre-heated gelling milk resembled closely that for standard milk under

similar conditions of rennet and acid coagulation (Figures 7.2.6 vs. 7.1.2 la; 7.2.7 vs. 7.1.22; and

b). Most apparent differences concemed the evolution (magnitude) of

A72.26u&b vs. A7.1 .Sh&

tan 6 over the region of local maximum development (i.e., the region that corresponds to the

development of gel modulus up to about P,h and likely encompasses the setting of mainly rennet

gel and its early differentiation toward more acidic state, as discusxd in Section 7.1.5 for

standarâ milk.) In pre-heatcd milk the values of 6 over these initial stages of rennet-acid

coagulation were systematically lower by about S0 ( i e , the relative elasticity of the developing

gel higher) thon in non-pre-huted milk (but similat to the values for the acidified controls fiom

heated milk). Even lower values of 6were memred for autoclaved milk. (Whether this apparent

increase in solid-like character for pre-heated gelling milks had to do with the apparent lespening

of gel softening ktwcen Pm and Pik is an open question.) Surely, one would expcct

modification of the pmtcin (and mineral) fictions to modifL the nature of dominant interactions

in pre-heated gclling milk [sec also Lucey et al., 1997a, 1998~1.Perhaps the .relativelylow values
of Gfor pre-heatcd milk rcflect enhancement of (hydmphobic) interactions within and among gel

particles.

It was also notd that at nnnet concentration Rx4 (and to a lesser extent Rx8), tan 6 for

cultured heated milk tendcd to increase slightly following the onset of measunble coagulation, in

con- with the relatively constant values of fun 6 measud for unheated milk (Figures

A7.2.26b vs. A7.1.566). (Recall that the initial stabilization of rm 6 in standard cultured milk

containing reiativciy high ieveis of remet was taken as an indication for the setting of a gel with a

predominant ensymatic chamter.) The early upward drift of tm G for pre-heated milk may point

to increased (direct) contribution of acidification vs. renneting to coagulation processes initially.

This would concur with the idea of better coaplating eficiency of acidification in heited mi&.

(nie acceleration of bacctcriological acidification in heated milk may bc influential in that

respect-by making the renneting and acidification processes more overlapping, even though this

did not appear to be sufficient to substantially modiQ the patterns of succession of renneting and

acidification; see below.) Incrcased coagulating eficiency of acidification would contribute to

rnaking up for the reduced efficiency of renncting reactions in high-heated milk.

(b) Possible Intemretation of the Coagulation Behaviour of Hinh-Heated Milk and Cornparison

with that of Ultra-Hi~hHeated Milk. It secms reasonable to suggest that the similarities between

the coagulation of prc-heated (90°C-1 min) and non-prc-heated RSM under the conditions of

renneting and acidification investigated rcflect similar synchronicitia betwan the renneting and

acidification processes in both types of milks. Pic-heating to temperatutes-times such that most

denanucd whey pmteins becorne associatcd with the casein markedly impairs the coagulability of

milk by rcnnet enzymes but it is well-documentcd t h t the detrimental effects can be (partly)

rectified, provided the heating w u not tao rrcverc, by modemte acidification and/or incteasing the

concentration of soluble ionic Ca. Such favourablt conditions result on the simultaneous

culnuing rnd renneting of milk as investigated in this study. It is Iikely thmefore that the ovenll
coagulating eficiency of remet in pm-heated milks did not differ substantially fiom that in

standard milh, hence the gencral tescmblance between the patterns of gel development

evidenced on varying rennet concentration in pr-heated and standard milks.

(4The coa8ulation khaviour of RSM autaclaved at temperatures in excess of 1 10°C seems

to fit with explanations of this nature. As can be seen in Figures 7.2.8 and 7.2.9 (A7.2.29-300 and

A7.2.3 1-32a), the profIles of consistency (and modulus) development obtained for autoclaved

milk et C/4-Rx4 (-Rx8) and CI8-Rxl6 displayed chmcteristics typical of the setting of mainly
acid gels fiom milks subjected to less extreme conditions of pre-heating (Figures A7.2.30c&e and

A7.2.32cd;d). (Note the distinctly shallower evolution of trn G for autoclaved milic, however.)

Only at much higher concentrations of rennet (Rx64 and Rxl6O) did patterns more

reminiscent of coagulation by combined renneting and aciditication emerge (Figures A7.2.33 and

A7.2.340; contrait to Figures A7.2.346&c). It seems logical to explain the behaviour of ultra-high

heated milk by considering the important (Iargely imversible) reduction of the effîciency of

renneting [e.g., Leaver et al., 19951, as well as the increased eficiency of acidification, that is, in

ternis of the net efkct, the substantial overlap ktween (limited) renneting and acidification.

The predominantly acid character of the gels abtained h m acidified autoclaved milk

renneted at and k l o w Rx16 seems to bc consistent with the observation that comparable (or

slightly higher) values of gel consistency (and modulus) and rate of development thereof were

rneasured for these systems as compamd to less intensely pre-hcatcd milks containing minimum

or no rennet. [RemIl that only for lactic acid controls did pre-heating at 90°C-1 min issult in

increased values of instrumental gel consistency, i.e., the acid us. rennet chmctcr of gels scemed

to have an important effcet on the magnitude of consistency sensed by the Nametre viscorneter,

which contrasted with the rcsults obtained in ternis of gel modulus using the Carri-Med rheometet

(to k discussed fiuther).]

 Incubation time at 40°C (h)

Figure 7.2.8. ûverview of consistency development curvcs for

culturcd and muieted at g4-B;aB at 40°C. Profiles of consistency C
and pH us. timc for cach type of milk are shown for single reprexntative
experiments carricd out with the Namem iheomctcr. Arrows point to the regions of
local maximum and minimum in consistency (or its deemcd quivalent) and to the
cornsponding (approximatc) values of pH. (Profiles for rcplicated and
comsponding measumnents of milk consistency and pH are displayed individually
and differently contrastecl in Figures A7.2.30a-e .)
 Incubation time at 40°C (h)

Figure 7.2.9. Overview of elastic modulus development cuwes for diffcmiilv

cultured and renneted et CI4-m at 40°C. Profiles of elastic modulus
G' and loss angle 6 (tan6 = GVG') vs. time for each type of milk are shown for
single npresentative experimcnts carried out with the .-
(Profiles for replicated and comsponding rneasurements of milk .viscous and elastic
moduli and loss angle are displaycd individually and differently contrasted in
Figures A7.2.3îu-d.) Compare with the counterpart time-profiles of consistcncy
(Nameûc rheomcter) and pH shown in Figure 7.2.8.
 The marked reduction in consistency that resuited for acidified autoclaved milk renneted at
Rx64 and Rxl60 may k viewed as the hallmark for the setting of gels with a more prominent

enzyrnatic charactet, in line with pevious observations about the effccts of pn-heating at 90°C-1

min and combined renneting-acidificationon instrumental gel consistency.

(io It is noteworthy that mature rennet-acid gels obtained h m autoclaved milk typically

lacked the 'body' of the gels h m less intensively pre-heated milks as was evident (subjectively)

ftom the liquefaction of the former gels on pouring. Along with the apparent smoothness

imparted to the gels, the former charactetistic would certainly be desirable for the production of

cultuted milk products of the beverage type (e.g., yoghun drinks) with a liquid texture or 'thin'

consistency. ('Weak' body or low fimness of gel for drinking yo&urt is commonly achieved by

mechanically breaking (e.g., hornogenizing) the coagulum after fermentation [Morley, 1979;

Kurmann & W i c , 1988; Tamime & Robinson, 1988; Driesrn & Loones, 19921.) Pte-heating

milk at ultra-hieh temperatuns may be urful (although perhaps no<so sound energetically and

nutntionally) for minimizing thickening or re-bodying of the coagulum after mechanical

processing. Such secondary increase in the viscosity or 'shear-thickening'-i.e.. thickening

through (pseudo) thixotmpy; to be commentcd further-generslly plays a part in the production

of stirreû yoghurt w i c & Kurmann, 19781. There have actually ken suggestions about the

potential of processing of milk at ultra-high temperature (149'C-3.3 s) prior to culhuing for the

manufacture of fluid yoghua [Labropoulos et al., 19841, and numerous reports about the

relatively weak yoghurt gels that mult h m pre-heating milk unda UHT-style conditions [e.g.,

Parnell-Clunies et al., 1986; Dannenbcrg & Kessler, 19883; Mottar et al., 1989; Savello &

Dargan, 1995, for milk first pn-concentrated by UF].

The effwts of ultra-high temperatures appcar not to k simply nlated to the degree of heat

denaturation of the whey protein but the mechanisms involved in determining gel properiies

remain poarly understd. Explanations have k e n attemptcd (ofien loosely) based on, inter dia,
reâuced complex formation khmcn denatureâ whey proteins and casein particles (and more

spccifically, d u c e d amount of complexed ~lactoglobulinrelative to a-lactalbumin), decreased

integrity of the casein particles, and i n c d dcgm of puticle aggrcgation upon acid

coagulation (the comsponding duction in micro-porosity would contribute to less efficient

physical mention of milk s

em within the gel). Polyrners of whey pmteins, if non-interacting

with micellar casein, may also disrupt casein aggngation. Thinning behaviour of autoclaved milk

tended not to be nflected in the (mlatively high) values of instrumenta! gel consistcncy and

dynamic modulus in the prcsent work (especially at concentration of remet Rx16 and below),

possibly because of the diffemnt conditions of, e.g., shearing underlying the subjective vs.

instrumental assessments of FI physical properties.

(U1) Continuing in this vein of msoning. one may suggest that the apparent discrepancy

between the effects of pre-heating in acidified and renneted milks estimated with the Nametre

viscometer vs. the Carri-Med rheometer had to do (in part) with differences in operating

conditions related to shear. (Diffemices in the rate and ftequcncy of shear m e n the two

rheomcters immcdiately corne to mind, viz., of the ordcr of 4000 S.' and 650 Hz for the Nametre

viscometer. and approximately 500 '.S and 0.1 Hz for the Carri-Md rheometer.) The fact thrt the

property detennincd by the Nametn is more related to the (apparent) viscosity than to the rigidity

of the gelling samples may have contributed. Also, the weight of gelling material uscd for

expcriments with the N a m e may have provided additional stresses in the developing network

[sce also Lucey et d., 1998e). (Cettainly this underlines the dificulty of comparing appatcntly

related characteristics estimatd through differcnt types of mcasurcments.) The argument is not

easy to spell out but thcm are dues for suggcsting that phcnomena homologous to imvcrsible

thixotropy (rheomalaxis), Le., non-Newtonian flow khaviour, may have k c n impl icatsd.

Thixotropic systems arc chatacterizcd by a continuous-reversibledtcrease in apparent viscosity

with time when subjected to shearing [van Vlict, 19991. fhe (tempocaiy) reâuction in viscosity
may be due to (partial) brerking down of internai structure under &eu. Various gels, including

(pre-heatcd) milk gels of the yoghurt type N i c & Kurmann, 1978; Benezech & Maingonnat,

1994 for a nvicw], exhibit (imvmible) thixotmpic flow behaviour or 'shesr-thinning' as it is

commonly refemd to. The publications by Labropoulos et al. [1984], Dannenberg & Kessler

[1998b], and Attia et al. [1993] provide interesting pieces of information related to the subject.

(Note that most of these studies used empirical test methods and that flow behaviour was often

detemincd on disturbed 'gels' after mixing and transfer to the rheometer for testing.)

Labropoulos and colleagues [1984] showed that the optimum firmness (estimated as curd

tension by pcnetrometry at 4°C) and apparent viscosity (Bmokfield LVT rotational viscometry at

4OC) of yoghurt gels prepared h m milk pre-heated at 82% for 30 min went along with the

greatest time-dependent duction in viscosity on shearing. (For purposes of discussion. and in

light of the data of Danncnberg & Kessler [1988u,b] about the extent of denaturation of PLg in

standard fiesh milk, the heat load at 82T-30min c m bc deemed equivalent to that et 90°C-1

min.) Gels fiom low-heat (pasteurized) milk had properties intermediate between those from milk

pn-heated at 82OC-30 min and UHT milk (149"C-3.3 s). The authors suggested that the higher

apparent viscosity and more pronounced sheu-thinning for the gels h m milk pre-heated at 82T-

30 min were due to the increased water-holding capacity of the (denatured) milk proteins

following this pre-heating and the subsequent l o s (reduction) of same (so-called 'lyophoresis' or

migration of the solvent) on shcaring of gel at incmsing rates. (PamelCClunies [1986]

emphasized a samingly rclatcd issue, vu., the necessawy d e - o f f betwecn gel firmness (or

apparent viscosity) and hydmphilic propnies (as deteminant of gel overall stability) for

achieving optimum physical characteristics of yoghurt chrough prc-heating milk by different

m&ods.) Similuly, Dannenbwg & Kcsskr [1988b] showed that yoghurt gels made fmm high-

heat milk (i.e., milk with a dcgm of denatuntioa of 84 2 90%) only had distinctly higher
finnncss and apparent viscosity (Rhcomat rotational viscomcûy at 1W)cornparcd to the gels
h m kss intensely pre-heated rnilks (10 and 603C denaturation of PLg) at low shear rates (Iess

than 20 TI), ie., pre-heated gel systems werc more d i l y destroyed by shuring.
Attia and collaborators [1993] documented the ultrafiltration and rkological pmpecties of

coagula obtained h m differently acidified low-heat RSM (with or without renneting at 25%).

They showed that the appatent viscosity of the coagula fonncd h m biologically acidified and

renneted milk decteased sherply with time of shearing at S R , in contrast with the nearly

constant viscosity for gels fomed by biologica! or chernical acidification. n i e authoa

commented that the nlatively 'unstable' behaviour of mrnet-acid gels was suggestive of

thixotropic-like behaviour, but point4 out that the hgility of such gels (especially at the

relatively high temperature of their experiments) would make it dif'licult, if not impossible, to

check the extent to which the original (undisturkd) gel structure ncovers when the sheating

process is discontinued. (Overall higher apparent viscosity for rennet-acid gels compared to

strictly acid analogues was also evident in the work of Attia et al., as well as higher finnness and

susceptibiiity to syneresis, which the authors attributed to stmget and mon numerous binding

forces in renneted systems.)

It is uncertain whether (how) like effeçts related to sheat (md/or sample weight and time)

manifested in the prernt study. It is possible that the distinctly lower consistency (i.e., apparent

viscosityxdensity) thughout the coagulation by rennet and acid of RSM pre-heated at 90°C-1

m i b a s compared ta their low-heat analoguei-rrflected more pronounced shear-thinning nature

of pre-hated gel syptems, Le., their p a t e r susccptibility to the e f k t s of (rclatively important)

shcar as expetienced in the Nameter visameter. partial disruption or destabilization of the

assembling gel structure on continwusly measuring would account for part of the decreased

rcproducibility of Nametrc coagulation experiments encountd with pm-heated milks (Section

a). Appmntly relatcd difficulties in obtaining reproducible results of shear stress and

deformation at yielding for strictly acid gels formed h m milk pre-heatcd at 8S°C for 30 min
comparai to gels f h n low-hcat milk have been reportai by Lucey et al. [1997u, 1998e1.

Actually, important variability is not unusual for hcture or large defortnation tests. A Bohlin

VOR controlled m i n rheometer w u used in the study of Lucey et al. since this is a more
suitable instrument for condwting large deformation tests than a controlkd stms instrument such

as the Cmi-Med. The authon reportcd that an important plut of experimental variability was
coneibuted by the important brittleness of pic-heated systems, Le., their susceptibility to hcture

locally.] This characteristic of gel would not be as apparent under the conditions of shear applitd

in the Carri-Med rheometer. It is intemting that conditions of ~fiicientpie-heating and

renneting of milk semicd to be required for the reduction in instrumental gel consistency. to be

appreciabk in our work, as if combination of the cffects of hcat and mzyrnatic modification

amplified the putative shear-thinning khaviour of the devel-opingacidic gels.

This suggestion raises difficult-teanswer questions about, e.g., the fundamental properties

that would underlie differcntial khaviour of such gels under extemal shear, and how

susceptibility to shear may vary with degree of gel development. Differences in dynamic

properties (e.g., tan 6) and/or fhgilizstion of the demineralizing para-caseidwhey protein

structure may corne into play, along with alteration of the capacity of the protein matrix to

effectively retain the serum, m l i n g the latter more available for flow. It is noteworthy that the

phenomena r c f d to as lyophorcsis [i.e., migration (mlwe) of the solvent (setum) phase;

Labropoulos et al., 19841 and (micro) synetcsis [Le., separation of the serum and protein phases]

both have a bearing on changes in the efféctive hydrophilic propcrties of the gel, and so, likely

encompass related physicoshemical d i t i e s . To be sure, the mechanisms by which high pre-

heating modifies the propehes of acid milk gels, with or without remet, still have to be clarified.

Z2.4. Effceb of ~ C O M N M WMUk

R ~by Ulh-o~

Gel formation in concentratal milk was invcstigated using the retentates obtaincd h m th2

ultrafiltration of standd and p r c - h d (90°C-1 min) RSM. (Few sunplcs were prcpared by
hcating Mer concenûating milk.) The direct UF procedure was continued until volumetric

concentration factors in the range 2 to 4x were achieved, with concentration factor 1x referring to

non-concentratcd (standard or pn-heated) RSM. Total solids and protein contents of the
retentites rangcd h m CO. 10 ?O 14% w/w and 3.2 to 8.5% w/w, mpectively (sec Section 6.2.3,

Tabk 6.2). which in the contcxt of chese and yoghurt-making would comspond to the
composition of low or medium-concentisted UF retentates. (Fewsampks were also prepamd by

diluting with üF pemeste milk that had been conccnbated Cfold.) Rheological coagulation

profiles for the different retentate series wem obtained at 40°C, at concentrations of bacterial

cultures and rennet C/4-Rx4 (occasionally CI8-Rx64), afier adjusting renneting pH to 6.4.

Control cxpriments were carried out with 3x concentrates.

(4 As illustmted in Figures 7.2.10 and 7.2.1 1 (A7.2.35-37 and A7.2.39-41, Narnetre and

Carri-Med data for non-pre-heated concenttates; Figures A7.2.4345 and A7.2.47-49 for pte-

heated concentrates), the generic features of rennet-acid gel development in both unheated and

pre-heated milks were little affected by increasing volumetnc concentration factor up to about 4x.

For cultured controls fiom UF conccnttated milk a h , the characteristic variations in the rate

of acid gel development (as dC/dt and dG W ) were much in evidence (Figures A7.2.386 and

A7.2.42c&d for non-pre-heated concentratcd RSM,and Figures A7.2.466 and A7.2.506 for pre-

heated and concentrated RSM). Additionally, it seemed that the modulus of cultured UF-milk did

not mach a stcaây value within experimental t i r n e - h e as was also reported by Ozer et al.

[1998]. (Renneted controls h m standard UF milk also exhibitcd conspicuous variations in rate of

development as dG '/di;Figure A7.2.42b.)

The time-sale of ovemll gel devclopmmt was comparabk at al1 levels of concentration

testcd, except it ocemcd at the highcr concentrations for low-heat retentates in which case a slight

delay was noted compued to standard milk. This last effcct may have kni contributcd by a

retoidation of effective lowering of the pH on lactic fermentation due to the elevated buffering
 Incubation tirne a 40°C (h)

Figure 7.2.10. Overview of consistency development curves for differrntlv

=C
-O- cultured and renneted at Q4-w at 40°C. Pmfiles
of consistency C and pH vs. time for each concentration of milk are show for
single representative experiments carried out with the Namette rheometer. Anows
point to Le regions of local maximum and minimum in consistency (or its deemed
quivalent) for the milks concentrated 2x and 4x. and to the comsponding
(approximate) values of. pH. (Profiles for replicated and corresponding
measurements of milk consistency and pH are displayed individually in Figures
A7.2.36adb .)
 + œ

- Elastic modulw G'

RSM pre-conccntrated &

Figure 7.2.11. Ovewiew of elastic modulus development curves for

bik&) ~IWOEGU$TJW cultureci and renncted at Cf4-wat 40°C. Profiles
of elastic modulus 0' and loss angk 6 (tan6= GW/G')vs .time for each concenmtion
of milk are shown for single reprcrntative experiments carricd out with the
-. (Profiles for rcplicated and comsponding mwurements of milk
viscous and elastic moduli and loss mgle are displaycd individually in Figures
A7.2.40adb .) Compare with the counterpart timc-profiles of consistency (Nametre
rhcometer) and pH show in Figure 7.2.10.
capacity and ionic strength of the more concentratcd retentates, as outlincd in Section 6.3.46.

Apparently, the values of pH at characteristic stages of gel formation were comparable for UF

concentrated milks and standard milk.

It should be noted that more diftïculties were encountered in measuring the pH of

concenûated systems, malhinctioning of the pH ekctrode usually translating in clearly over-

estimated readings. Certainly, no efTect akin to that reportcd by Gastaldi et a!. [1997] (apparent

decrease of the pH by 0.2 unit at the onset of GDL-Uiduced gdation of fortifid skim milks with

increased total solids in the range 10 to 20%; Section 2.2.66) stood out in our investigations.

The general resemblance between qualitative coagulation behaviour of diffhently

concentrated milks and standard milk likely originated also h m similar succession of renneting

and acidification, at lest under the conditions of moderate concentration and combined

coagulation considered. lncreasing the concentration of micellar casein (and assoçiated Ca

phosphate) undoubtedly modulateci the mechanisms by which the gels developed as was reviewed

under Sections 2.2.5 and 2.2.66, but without appreciably modifying global coagulation behaviour.

Most likely, gradua1 acidification of the renneted concentrates favoured the development of gel

structure by allowing more efféctive incorporation of the partly renneted casein particles into the

gel. Mon distinct coagulation khaviour may be expecteû for more extensively conceneatcd

rnilks (e.g., above 4 or 5-fold) given the considerable changes in overall composition (including

proportions of whey proteins and minerais) that are known to result h m important concentration

of milk by UF.

(io Certainly, the magnitude of gel consistency and elastic modulus (and absolute rate of

change thereof) during coagulation increased substantially with increasing milk concentration

(especially at concentration factors greatcr than 2x), as cxpectcd. Apparently, the effects of

the mapitude of gel apparent rofiening (demindiution) betwecn

important acidificatio~~e.,

P m and P,,,merc cornmensurate to the extent of casein (i.e., m i n a l ) concentration. This

would k consistent with the amplification of viscous-likc khaviour i n f e d h m the evolution

of loss tangent for concentratcd systerns (to be discussed).

It is noteworthy bat thm tended to be an attenuation of the maximum-minimum in the time-

profiles of consistency for the 4x retentates h m low-heat milk. [A similar feature was apparent

for retentates fiom pre-heated milk at concentrations 4x and (albeit not systematically) below,

although in these cases it is difficult to distinpish between the specific contributions of

concentration vs. heating. (Recall that pre-heated milk uf standard concentration also exhibitcd

somewhat shallower maximum-minimum at C14-Rx4 compared to non-pre-heated standard milk,

as commented under Section 723a.)] No such an aîtenuation was apparent in the profiles of

elastic modulus (on the contrary; con- Figure 7.2.1 1 to 7.2.10 for unheated concentrates and

Figure A7.2.47 to A7.2.43 for pre-heated kncentrates). This last observation suggests that

perhaps the Nametre viscometer became less sensitive to changes in the physical characteristics

of the coagulated sarnples when excessively firm/cornpact structures developed, i.e., when

apparent consistencies higher by 1 to 1.5 order(s) of magnitude compared to those for standard

milk gels were measuted. It is noteworthy elso that at concentrations 4x lower values of l o s

angle G were measured.

Globally, the positive efTect of milk concentration on the development of consistency and

especially modulus was enhanced for the rctentates obtained from pn-heated milk (Figures

A7.2.43-44 and A7.2.47-48), much in kccping with the effect of pre-heating on the firming

(development of dynamic modulus that is) of m t - a c i d gels h m milk of standard

concentration (Section 7.2.3~).It is known that UF processing can help restorc the rc~etabiiity

of (ultra) high heated milk (in tenns of gclation time et amund neutral pH that is) but low rates of

gel finning and wedc gels typically iesult bwrcnce, 1989; Shanna, 1992; Guinee et al., 19%].

Prcsumably, the close proximity of ( p u ) csscin particla in pn-heated concentratcd milk

increascs the pmbability of collision and aggrcgation, but the prcscncc of dcnatured whcy
proteins at their s d r e hinders their effective integaion into the gel matrix. T k measurements

in this work clcarly show that it is possible to obtain finn gels h m prc-heated concentratcd milk

when gradua1 acidification occm during renneting. (This is consistent with the results of Savello

& Dargan [1995] about the positive effect of combining üF and subsequent heat treatment

ktwecn 100-I2O"Cin ternis of the rtnngth and stimd viscosity of yoghun gels.) As alluded to

previously, attenuation of the maximum-minimum behaviour (i.e., the less negative values of

dC(t)ldr and dG '(r)/dt minima than for non-pre-heateâ systems) chûncteristic for remet-acid

coagulation of pre-heated milk of standard concmtration was also apparent for pre-heated

concentrated milk, especially at concentration factor 4x in the profiles of modulus.

fui) Temporal evolution of ton 6 (= G "IG3 for concentrated milks was characteristic of the
setting of nnnet-acid gels and also exhibited the qualitative and quantitative modulations typical

of unhcated us. pre-hcated systems of standard concentration (Figures 72.11, A7.2.39-40,

A7.2.4748, and 7.2.12 to 7.2.14). In cultured and renneted concentrates, the relative initial

stabilization of fun 6 supported interpretation of coagulation behaviour as relative decoupling

(succession) baween mainly enzymatic and acidic stages of gel development.

Most notable differences brought about by pre-concentrating milk concemed the increase in

the magnitude of tm 6over the initial stages of remet-acid coagulation, especially d u h g the

transition toward incrcasingly acidic (demineralized) gel. The incmse in the peak values of 6

cornparcd to non-conccntrated milks was by about 5-11" (from 3 1-32O to 35-40') for concentrates

h m low-heat milk, and about 2" (h to 27-28') for concentrates fiwn pre-heaîed milk.
25026~

Theoc observations concumd with the higher (peak) values of 6 measured for mneted and

cultuml controls h m concentratcd milk. A similar trend was rcported by Gastaldi et al. [1997]

on strictly acid coagulation of IOW-hcatRSM fortified to khucm lO-2Wh total solids. The

amplification of gel viscous-like character upon important daninenlization may k related to the

inhercntly high viscosity of conmtnted systems. Apparcntiy incrcasing milk concentration had
 1 C/4-Rr4 and naaet & lactic acid

C/4-RxO (lacr
acid gel. 1x) 1
\

(*For the 3x lactic acid g

at C/4-RxO, 6 essentially follows the hsce of 6
for 3x RSM at C14-Rx4 afltr the "viscouspeak".)
-
Incubation time at 40°C (h)

Figure 7.2.12. Typical evolution of loss angle 6 (tan6 = G"/û')upon the

coagulation of culhind and renneted (C/4-Rx4) diffcrrntlv g r c - c m
vs. mneted (CO-Rx4 at pH 6.4) and biologically acidified (C/4-RxO) ppe,
at 40°C. Profiles of 6 vs. time for cach set of coagulation
conditions M show for single representative experiments carried out with the Carri-
Med rheometer. (Profiles for replicated and comsponding measurements of milk
loss angle, viscous and elastic moduli G" and G',and approximate pH are show
elsewhere in the dissertation.) Contrast to the counterpart time-profiles of S for pre-
heated and/or concentrated RSM shown in Figures 7.2.13 and 14.
 Incubation time at 40°C (h)

Figure 7.2.13. Typical evolution of loss angle S (tans = G"/Gf)upon the

coagulation of cultund and renncted (Cf4Rx4) & diffmntlv
vs. renneted (CO-Rx4 at pH 6.4) and biologically acidified
a at 40°C. Profiks of 6 vs. time for each set of
coagulation conditions are show for single repmentative experiments canied out
with the Carri-Med theorneter. (Rotiles for replicated and corresponding
measurements of milk loss angle, viscous and clastic moduli G" and G', a d
approximate pH arc show elsewherc in the dissertation.) Conttast to the
counterpart time-profiles of S for non pre-heated concentratcd RSM shown in Figure
7.2.12.
 Incubation time at 40 or 2S°C (h)

Figure 73.14. Overview of typical evolution of loss angle 6 (tan6 = G"/G1)upon

the coagulation of diffrrmtlv p-m & cultured and renneted
at Cl4-Rx4 (or CR-Rx8) a 40°C. Profiles of 6 vs. time for cach set of coagulation
conditions are shown for single representative experiments carried out with the Cani.
Med rheometer. (Profiles for nplicated and comsponding rneasurements of milk
loss angle, viscous and elastic moduli G" and G',and approximate pH are shown
elsewhere in the dissertation.)
littk effect on the relative elasticity of the mature gels since loss angle invariably stabilizcd nsu

13-14' ultimately. [Gastsldi and CO-workersreportai a limited d e c m e (1 O; questionable) in the

final value of 6 for acid milk gels enriched with total solids, which they attributed to the

enhancement of interactions between gel particles on increuing casein concentration.]

7.3. General Discussion

Different levels of perspective for the preceding analyses are brought together in the

following subsections, starting with a recapitulation of the parameters most notabk for their

effects in the evolution of milk gel viscoelastic properties on combined acidification and

nnneting, and a discussion of some experimental nsults in relation to prcviously published work.

Physico-chernical arguments for what processes are likely to underlie the different coagulation

paths evidenced are presented next, followed by considentions about the possible technological

and nutritional significsnce of the effects obsewed, general conclusions and implications for
funher research on the subject.

7.3.1. Kcy Parametcm In the Progrtss of Gel Developnw~ on Combined Biolugical

Addification und Reuneting of Mil&

A central theme in this chapter is that variations in the scquence of renneting and continuous

(biological) acidification of milk are the foundation for the gndations in coagulation behaviour

evidenced. Compositional and treabnent parruneten most notabk for their effects on (qualitative)

gel development as resarched herein thus op- to modify the succession of renneting and

acidification through effeçts on the coagulating efficiency of either or both processes relative to

one another (see schematics of Figures 7.3.2u&band 73.3 under Section 7.3.3).

(0 For the combinations of concentrations of remet and lactic bacteria considercd, the
predominant influence of =Ma concentration on ovenll coagulation behaviour obviously

concurs with like interpretations. The distinct effbcts of lowering incuôation and coagulation
temperature (40-20°C) and of changing milk composition through adding NaCl (0.6% w/w),

indirectly cycling the pH (6.7 + 5.8 + 6.7), or ultra-high heating of milk prior to settng at given
concentrations of acidifying starter bacteria and rennet enzymes could also be reasonably well

explained, in part, by considering the unfavourable effects of these manipulations on the relative

efficiency of mainly renneting (enymatic proteolysis andlor aggregation).

In cornparison, parameters such as milk fat content and homogenization, addition of CaCb

(0.01% wiw), direct cycling of the pH, and to some extent, moderately high pre-heating ~nd/or

UF concentration of milk seemed to have moderate effects on qualitative coagulation behaviour,

presumably because o f minimum (or less pronounced) repercussions on effective progression of

overall renneting under the conditions of simultaneous acidification investigated. [Rc-heating

and concentration certainly had important effccts, qualitatively, on gel (apparent) fming.] Also,

the sequence of renneting relative to bacteriological acidification appeared rnoderately modified

by the addition of 5% (vlv) ethanol to standard RSM (results not shown), i.e., signature profiles of

coagulation by rennet and acidification (e.g., Cil-Rxl6) were clearly evidenced at 40 and 20°C

for such milks.

(ii) It is interesting that the conditions of coagulation that resulted in appreciable attenwtion

of the maximum-minimum pattern of gel consistency/modulus development (in particular, the

conditions of low nnnet concentration, low coagulation temperature, added NaCl, or to some

extent pre-heating and important concentration, te., conditions that amounted to apparently

limited efkiency of mincting) am also known to gcnemlly =duce the occurrence of gel

syneresis. Some comspondencc ktween coagulation and syneresis behaviour is to be expected

given the prominent influence of rcnneting-morc precirly, degree of renneting-on both

coagulation end syneresis pracesses (especially in non-pre-heated nor concentnted mik). It may

k rationalized that the less the net contribution of muieting to gel devclopmcnt (i.e., the l e s

developcd the remet character of gel), the kss the susceptibility to (Iater) synmsis phenomena.
 It is not clear whctha the relatively low values of loss tangent (- G"/G3 measured in the

early stages of the sctting of prc-heated milk and of milk incubeted klow 300C (e.g., F i p

7.2.14) may have to do with the maikedly reduced susceptibility of such systems to syneresis.

This allusion derives from suggestions that the magnihidc of tm 6(an indication of the relaxation

behaviour of the interactions within the gel, Le., of the dynamic character of gel structure) goes

along with the tendency of milk gels to exhibit synensis [van Vliet et al., 19910; van Vliet &

Walstn, 19941.

Modifications of milk ionic (Ca phosphate) equilibria certainly are a major determinant of

coagulation/syneresis behaviour. The possible relation between propoflion of micellar Ca

phosphate at nnneting and maximum-minimum khaviour of coagulation may be understood by

considering the state of MCP as an indication of the acquisition of more or less acid-like character

(perhaps through modulating the ef'fïciency of renneting). In the cases of milk modified by adding

NaCI, by i n d i d y cycling the pH, or by cooling, for example, conespondence between

(expected) reduction in MCP content initially (which may k thought of as a promotion of overall

acid-like character) and attenuation of maximum-minimum behaviour would be in keeping with

the fact that this coagulation pattern is most typical of systems with relatively limited

development of nnnet chsractet.

In the following subsection we con- some of the experimental mults to (seemingly)

related specific observations in the iitemture.

7.3.2. Relation of Ejcpcrhntd Rcsvla ta k i u u s W o ~ k

(a) Studies of Gelation o f A c i d i h k and Rennetintz Milk. Essentially, the results describeû in this

chapter substantiate and expand on the multifactorial rheological study of combined coagulation

carrieâ out by Noël et al. [1989,1991) (0.1 Hz; low-heat RSM;apparent concentrations of culture

and rennct = Cl4 and Rx0.5-Rx23, mpcetively; pH a rennct addition 6.6-6.0; CaCI, addition at

0-0.04% wlw ;30-34OC; main conclusions are outlined under Section 2.2.7).
 There also is a broad convergence between the results obtained in the present study and the

observations made by Dalgleish & Home [1991a,b] in a phcnomenological investigation of gel

asscmbling in fermentcd and mnetcd milk by diffusing wave spectroscopy ( k h pastcurized

skim milk; apparent concentrations of culture and rennet a C18 and s Rxl-Rx4; a p p n t l y
unadjusted start pH; 25-33OC). An important characteristic of DWS (or fibre optic DLS, as

described in Chapter 3) as compareci to conventional dynamic r h e o m e ~is that the measurements

are perfomed under conditions of zero-shear (no moving parts), Le., without perturbation of the

gel at any stage of formation. Individual profiles of gelation can be established in terms of cuwes

of intensity of the multiply-scatîend light and of apparent particle size over time.

It is not stmightfonvard how the information about the optical and mechanical (viscoelastic)

properties of gelling milk compare exactly, partly because of difticulties inherent to the

interpretation of DWS data at the cumnt stage of development of the technique, and partly

because of differences behiveen the experimentaf conditions in our work and in that of Dalgleish

Br Home. The two sets of observations showed similar trends, however, regarding (0 the
gradations in the coagulation behaviout of standard milk as the relative contributions of renneting

and bacteriological acidification were varied, 0 the predominant influence of rennet

concentration vs. starter concentration on the overall shape of gelation profiles, and (UI) the

relative resemblance in rems of apparent gel stmigth between rennet-lactic acid milk gels and

strictly rcnnet gels.

The general comspondence betwtcn rheological and Iight scattering analyses providcd

additional confidence in the overall validity of the rheological approach adopted in this study ( s e

discussion under Section 1.13). The scattend intensity measund in DWS appean to be inversely

related to the elastic piopenies (rigidity) of the casein gels (analogue to the viscoelastic parameter

tun 8)[Dalgleish & Home, 199la,b]. It may be signifiant that in DWS analyses of combineâ

coagulation with rclativcly high amounts of mnet enzymes at 33OC, the primary profiles of
scattercd intensity md apparent radius vs. time show an apparent tuming point (small but distinct

duction in slopc) about halCwry thmugh the docming portion of the curves (i.e., about 1 h

after the detection of plation which reportedly occumd amund pH 5-45). Pelhrps such changes
in the slope of DWS curves signal transitions in the behaviour of the gel, which may coincidc

with characteristic points in the consistency (dynamic moduli) curves obtained in this study.

A tentative contrasting of typical intensity and viscalastic coagulation prbfiles for milk

culhind in the presence of relatively high remet is show in Figure 7.3.1 (designed to illush.ate

trends only). Note the possible comspndence khwen tuming points changes in the dope of the

curve of scattered intensity by DWS, and the points Pi (inflection), PH. (local maximum), or PI*

(local minimum) in the curve of elastic modulus G' as estirnated at 40°C using the Carri-Med

rheometer.

It is intercsting also that at the lowest concentration of rennet and 33OC. Dalgleish & Home

[1991a.b]noted an effect of changing the concentration of acidifying starter on the character of

gelation profiles. For the combination of low rcnnet and high starter at 33OC. the gels seemed to

develop lower apparent (DWS) clastkitylrigidity, and in a more monotonous way than for the

combination at low starter. These observations arc not without reminding us about the effeets of

high concentration of starter on the development of lower gel consistency at 40°C in milk

containing no or minimal amount of rcnnet (sec Sections 7.1.4~and 7.1 Sa). The monotonie-like

chmcter of DWS gelation profiles obtained at high concentration of starter may correspond to

the seemingly decrcased reodution of the secondaiy changes in rate of gel consistency

developmcnt evidenced in Our work. We M e r note that the non-monotonous patterns of

combincd coagulation reportai by Dalgleish & Home appcar distinctly attenwted at 2S°C

comparcâ to 33OC. which would k in keeping with the effcets of gelation temperature

documentcd in Section 7.2.2.

 G' (Pa) and loss angk 6 (degrees) Elastic modulus G'(Pa) and loss angle S (degrees)
by dynamic rheometry by dynamic rhwmetry (Carri-Med heumeter)

O -
0
C 3
0
t ) P
0
V
0
I O
0
\ 4
0 o z l s z 8 g 8 8
Scattered intensity by DWS (arbitrary units) Scattered intensity by DWS (orbitnry units)
(b) Studies of Gelation of Acidifiinn Milk. We also see a possible parallel ktween the particulm

of gel development evidenced upon bacteriological acidification of control milk (Sections

7.1.4d&e) and the observations nported by Kim & Kinsella [1989b] about the rheological
changes in pasteurized skim milk during acid-induced gelation by glucono-Glactone (GDL),as

defined using an Instron Universal Testing Machine (applied fnquency = 0.2 Hz, fRsh

pasteurized skim milk; 40-50°C).

( ' Kim & Kinseila observed a distinctly-resolved, transient de~reasein gel rigidity (shear

modulus) about halfbay through gel formation as rnonitored over about an hour. (Rather

intriguingly, the overall coagulation profiles presented are in fact nminiscent of the profiles

obtained in this study on addition of the lowest amount of rennet.) For reasons that were not

explained, the maximum-minimum behaviour, with the drop in gel modulus occumng around

values of modulus of 2-3 Pa, was only detected when fairly high concentrations of GDL were

used (2 1.5 g GDUlûû mL milk). We note that subtle changes in the rate of modulus

development appear to k present also in the coagulation profiles showing a monotonous increase

in modulus with subsequent leveling off around 1.5 Pa (1 .O g GDUl O0 mL milk) [see aiso the

rheological profiles of acid gelation of high heat-treated milk reponed by Lucey et al., 19984.

No pH data were given by Kim & Kinsella (and their values of shear modulus seem

unrealistically low), but it is probable that the difference in rheological coagulation behaviour in

their work had to do with effects of the rate of gluconic acid production on gel characteristics,

including its tendency to rearrange and synerese. Perhaps, the combination of different regime of

acidification, Le., relatively hi@ rates of acidification end high coagulation temperature in Kim &

Kinsella's study amplified the magnitude of the effects exemplified in our control experiments

with RSM cultured at 40°C, although it is not clear whether the effects evidenced were of an

allied nature. It is possible in fact that the hoological and physical propcrties of gels msulting

h m the hydrolysis of GDL differ h m the properties of gels derived h m lactic fermentation,
particularly at high gclation tempentwes, as was pointcd out alrio by Lucey & Singh [1997] and

Luccy et al. [199ûdJ.

(The results of an expriment we conductcd with standard RSM acidified with 1% (wh)

GDL and renneted at level Rx4 at 40°C are shown in Figure A7.1.41~under Section 7.1.4. The
coagulation curve suggests a possible deviation h m strictly sigmoidal evolution of consistency

after inflection amund pH 5.4-5.3, but this was limiteci. That largely sigrnoid-like kinetics of gel

development resulted (much like for contiol remet gels) is likely relatecl to the fact that the pH of

renneted milk did not decrease klow the critical level of about pH 5.2 throughout its sctting, Le.,

similar sequence of renneting and acidificaîion as for milk renneted at constant pH around 6.0.)

(U) Famelart & Maubois [1988] actually reported distinct profiles for the coagulation of

medium heat RSM by bacteriological acidification, depending on starter culture activity in

producing lactic acid. On relatively npid acidification at 42OC, a marked decline in gel apparent

viscosity (as monitored using a Contraves viscorneter) was found around pH 5.0 within about an

hour of gelation (increasing viscosity up to a peak of a 2.3 mPa.s), which the authors attributed to

destruction of the gel network on shearing. This khaviour contrasted with the largely continuous

increase in apparent viscosity obsnved on gelation by more slowly acidifying bactcria at 37OC

(the value of viscosity ahr IO h was 1.8 mPa.s and the pH n: 5.3). Note that the lower

temperature may have played a de in the evolution of viscosity.

[The studies by Roefs [1986] and Roefs et al. [1990b] are of interest also because of a

treatment of the dynamic (mechanical) propertics of casein gels prcparcd by combined

acidification and nnnct action, albcit in a diffennt way (Le., der acidification to pH 4.6 in the

cold and subxquent warming). It was showed in pu<icular that rrnnet-acid casein gels haâ a

maximum in tan S around pH 5.2, and this was nlated to a transition h m gels with a

predominant rcnnct character to gels with a predominant acid chamter. The interesteci mader is

invited to refer to the actual publications for details.]

 An important point hem also is that direct cornparison of (rheological) coagulation profiles

established using different instruments may k difEcult. Certainly, as brought up a numkr of

times in previous sections, the possibility that eff- ssociated with instrument penommce

may be reflected in the evolution of the gel characteristic(s)monitored ought to be borne in mind,
in particular when it is dificult to be sure that measurements do not bias (damage) fiagile gel
structure.

A synthetic view about the physico-chernical processes that may be a the rooi of the basic

pathways fur gel development in acidifying and renneting milk systems is given next. The

proposed interpretation combines experimental evidence derived fmm the present study with

elements and fonnalism of existing views about the mechanisms for casein coagulation in milk

(Chapter 2).

X3.3. Ptoposed Interptetatimt of the Processes O/ Gd Devefopment in Acidfiing und

Rennehg MUk

Available evidence converges in showing that interplay between the acidifioation and

s central to the progrcss and outcome of combined coagulation of milk. The

renneting ~ c t i o n is

influence of milk pre-tmtmcnt and other puuneters investigated on gel-fonning properties

cannot be ignored, but these factors appear to affect more the magnitude and time-rate-of-change

of the state of the systems (Le., in the present woik, the precise shape of the rheological

coagulation curves) than the generic fmtutts of gel temporal dcvelopment, that is, the basic

reaction SChemes.

Deviation h m sigrnoid-like kinetics of gel development in bct&iologically acidified milk,

with or without rennet added, seems to k a common thread that nins through the rhcological

patterns of coagulation exemplificd in îhe prcceding sections. It may k suggestcd that the

physico-chernical picturc for the differcnccs in coagulation behaviour rcvolves around the

differcntial effects of acidification depending on the stage of gel development (aging) at which

the shifi in ionic status occurs.

 Important (concomitant) changes during acidifiution of milk at temperatures above 20°C

include: gradua1 dissolution of micellar Ca phosphate (Le., steady demineralization of the cosein

structure), increase of soluble Ca, little dissociation of micellar caseins, and neutralization of

charges. Essentially, the coagulation conditions examined may bc reduced to situations in which

the trpnsfer, on continuously decreasing milk pH, of micellar Ca phosphate to the serum phase

either: (4 precedes and/or is largely concurrent with initial formation of gel structure, with no or

little coagula<ingenzymes [scenarios (a) and (b) below]; or (U) !a@ M i n d pl formation [rnostly

by enymatic action that is; sceniuio (c) J.

In other words, the deme of intcnration koordinationl of the effects of acid ~roductionand
rcnnet action is essential in definine how the gel fonns.

Interpretation of gelation processes in the limiting case of strictly acidifying standard milk is

developed first as a grounding for the interpretation of the coagulation behaviour of differently

renneted acidifying milk. In fact it is useful to think of the modes of coagulation of acidifying

milk as king on a continuum. The diagrams in Figures 7.3.2u&b (and 7.3.3) highlight the

possible conespondence between the different stages of gel development, as infemd by dynamic

rheometry, in the diffemnt cases envisaged. Average values of expcrimental coagulation

parameters am summarized in Figures A7.1.58 to 63. Note that since the focus hae is on the

differcnces in the succession of acidification vs. ienneting, it does not matter in furt analysis

whether acidification of milk is by using bacterial starters or GDL.Essentially similar patterns of

gel development are expected in both cases (given that similar acidification profiles arc obtaincd,

that is), albeit, pehaps, with some modulation of gel properties.

 I I

I
m
b La-tly concurrent t I Stages 3 & 4
I I
acidification and formation of 1
1
1
I
acid-minet gel [minimal R] I
i
I
"* ......".".*

c. Largely sequential formation it /r---

1
Stages 4 & 5
of remet (acid) gel and ,r 1 # # d
I
@

acidilication [iiibstrntial RI j 1
I
/
I
I
tI 1 Stage 3
I ,*.* C1...*.-...-
Stage 1 I
I
[ Stage2 1 *..'
**.*'*".

I I
w I
I
p H (incubation timc) > 5.5 ! j ~5.0
!
< 4.5

Figure 7 . 3 1 ~ . Schematic representation of the basic patterns of

succession of continuous (bacteriological) acidification and renneting as
influenced pndominantly by the effective concentration of rennet
enzymes in standard milk. Acidification and renneting curves are only
meant to approximate the relative extent of the acidification and
renneting pmcesses and their contributions to gel development.
Choracteristic comsponding rheological profiles of gel development are
contrasted in the facing figure. In scenarios (a) and fi),Stage 1 refers
to acidification (and renneting in b ) with gel formation and firming,
Stage 2 = secondary gel firming, and Stages 3 & 4 = gel consolidation
and 'stabilization' (& synetesis). In scenario (c), Stage I = renneting
and moderate acidification with gel formation and finning, Stage 2 =
acidification with gel softming, Stage 3 = secondary gel firming, and
Stages 4 & 5 = gel consolidation and 'stabilization' (& syneresis).
 Irubatioo time (b)
PH > 5.5 < 5.0

Figure 73.26. Basic patterns of coagulation of (standard) acidifjhg milk as

defined by the (approximative) contrastcd evolution of elastic modulus G',
tint time-derivative thereof dG'/dt (i.e., instantancous rate of change in G'
with time), and lors tangent tan5 (= G"/G') over time of incubation (pH)
under the conditions of continuous (bactcriological) acidification relative to
rcmeting illustntcd in Fig. 7.3.2~.Scenarios (a), 0, (c), and the diffmnt
stages of milk gel development are as detined uid discussed in Figure 7.3.2~
& Section 7.3.3. Pb & ,P refer to the points of local maximum and
minimum in gel dynamic modulus, icspcctively. Generic numerical values of
G', dG'ldt, tans, and incubation time are given to illustrate the magnitude of
the changes in the viscoelastic propcrties of gelling systems hcrein considercd
as standard at about 40°C. 322
 I i
b. Lirgely concurrent j @
I
Stages 3 & 4
/\ aeMUlcrtion rad formrtkn O

Figure 7.3.3. Schematic representation of the effects of certain

treatment and compositional panmeters on the succession of continuous
(bacteriological) acidification and renneting in milk. Treatments such
as lowering the incubation & coagulation temperature (40-20°c), adding
NaCI (0.6% w/w), indirectly cycling the pH of milk (6.7->5.8 ovemight
-%.7), or ultra-high heating pnor to setting at given concentrations of
starter bacteria and rennct enzymes importantly reduce the efficiency of
predominantly-renncting, thereby reducing the successiveness1decou-
pling ktween îhe renneting and acidification processes. Conditions of
coagulation that amount to scenario (c) (or 6 ) in untreated standard
milk thus bccome quivalent to conditions more typical of scenuio (bl
(or a) in so-treated milk (upwad arrows; Sections 7.2 & 7.3.1 for
details). Scenuios (a), (a), (c), und the diffemnt stages of gel
development have the same meaning as in Fig. 7.3.2 & Section 73.3.
323
 * . .
and Gel Fonnatioq. In standard acidifjring mil4 in the absence of
(a) ccmurrent A~1dlfi~@!!l~

rcnnet, the shiR toward incrcasingly soluble Ca phosphate and decrcasing electmtatic charge

(surface potential) is integral to bringing on dcstabilization of the casein particies and, thmugh

not well-known processes of dissolution-rearrangement-pncipitation of the caseins, subsequent

aggregation and gel formation (saSections 2.1.3 and 22.6 of Chapter 2). Extensive modification

(disniption) of the casein system on changing ionic conditions occurs at the micellar level-in the

pre-gel -te during the induction pend (hgstage)-and during the early stages of gel assembiy.

Stage I. Acidijkation with gelformution andflrming. The overall effect is to increase intra-

and inter-particle interactions-ie., predominantly electrostatic and hydrophobic forces of

attraction-, which is rcflected experimentally in the net continuous increase in consistency and

dynamic moduli (netwodc rigidity or density) on gelation.

It is noteworthy that initial development of gel modulus during the acceleraiion phase seems

to be accompanied by an increasing relaxation (partial loosening) of the assernbling gel structure,

as suggested by the distinct transient increw in tun 6(= Gt'/G')following the transition fiorn

fluid (sol) to viscoelastic (acid gel) phase. (For standard RSM fermented at 40°C, this occumd

within the region of mort rapid and important acidity development, between pH 5.7 and 5.2.1

Ongoing removal of the Ca phosphate that contributes to the structure of the casein particles

originally likely is a major determinant of this relaxation behaviour, the assembling gel

temporarily behaving in a relatively more viscous and less elastic way. Loosening ('swelling' or

increasing voluminosity) of the aggrcgating peiticlr on deminenlization may be expected to

hindet (delay) dynamic dcvelopment of gel modulus and consistcncy-the more so, pcrhaps, the

f a e s t the acidification. By cornparison and anticipation of the interpretation for the coagulation

khaviour in situations (b) and (c), it may k reasoned that loosening of aggregated (coagulated)

uni& through altering the cohesiveness of a pm-foimcd pl base, would have a more prominent

effect on lowering gel modulus/consistcncy.

 The pnvailing paradigrn holds that fundamentally diremnt types of c w i n foms are prcsent

above and below a transition pH amund 52-5.1. That most pronounced changes in the physico-

chemical statu of milk micelles happen in the region between pH 6.0 and 5.0 has indeed bcen

extensively documented, but we still have mostly best guesses at the stnictural and functional

evolution of the casein particles between these two states. There are views that the particles p a s

through a kind of mesophase state between pH 5.5 and 5.0 to compensate for the gradual loss of
interactions involving colloidal Ca phosphate by alternative cesein-cesein interactions [ G a d d i et

al., 19961. It seems that rather short-lived, intemediate structures must form but their relative

populations and the (possibly cooperative) mechanism of their diffenntiation remain poorly

undetstood. It is not clear either whether a similar procesr of structural rearrangement wodd

apply to interacting (gelled) particles. A cornplicating factor in aggregated systems is that the

recasting may initiate micro-phase cparation (microsyneresis), the caseins clustering togethcr,

thereby cmting micro-environments of fm whey within the gel network.

As the particles proceed toward the tuming point around pH 5.2, it may be assumed that the

shift in the type of interactions spark major re-conformation of the casein rnolecules (both intra-

and inter-particle), which may set the stage for diffetent arrays of attractive forces subsequently,

Le., more stable configuration of gel components. Such a transformation may be deteminant in

particular for the establishment of hinctional hydrophobic interactions rince the influence of these

interactions probably is expected to be via eff- on protein conformation (hence particle

structure), rather than by acting directly among the particies.

Stage 2. Secondmy g e l m i n g . As the pH appmaches the iso-electric mgion of the different

classes of caseindwhey protcins (pH 5.3-4.6), the effects of decharging predominate, favouring

intra and inter-partick attraction, thercby coneibuting to the devclopment of consistency and

moduius (mainly elastic pmpaties as it tums out since tun 6 decre~ws)by reinforcing gel

structure. It is possible that the shouldcr detccted in some time-derivative (fiming) curvcs during

the decelention phase of devalopment bc a maniftstation of this proccss of secondary

'agpgation' (strengthening) during which oolubilizcd or loosely aggrcgated pmtein material (P
casein?) rnay be (re-)incoiponted into the casein gel matrix with pome concomitant tightening of

network elements.

Despite the essential differences between the physico-chernical m t i o n s involved, this

process may be viewed as the acid counterpart of the apparently binuy or two-stage development

of gel strength documented by Storry dé Ford [1982u,b] for fksh whole milk renneted at 30°C

and constant pH (details in Section 2.2.36). That die effect rnay be more pronouncal in pre-

heated acidifying milk cornparrd to standard milk rnay have to do with the co-aggregation of

casein and whey proteins andlor the apparently distinct associative properties of the caseins-whey

proteins in pre-heated milk towards interchange reactions with the senim phase upon acidification

MW,
1996; Singh et al., 1996; Lucey et al., l998c,dJ.

Stage 3. Gel consolidation und 'stabifuation'. The so-envisaged secondary strengthening

would initiate the tinal stages of gel development with further consolidation of the network and

attainment of metastable structural unifomity upon completion of fermentation (Le., stabilization

of the pH around 4.5-4.0). This stage is characterized by the attainrnent of an apparently more

stable rheological behaviour, Le., lower, asymptotic valws of rate of firming and of tan 6 as

paudo-equilibrium is appmched. This khaviour rnay be interpretcd as moderate or unresolved

evolution of the nature of the interactions that contribute to gel stmigth (sinichue). Some

decrease in pl consistency and/or modulus a very low pH rnay nsult fmm incrrase in the net

positive charge of casein molecules, Le., incrcasc in clectrostlaic npulsion. [An additional, partly

overlapping stage of gel syneresis rnay be distinguishd.]

(b) Larnelv Concurrent Acidification and Gel Fonnatioq. Addition of low arnounts of rennet

relative to the concentration of rcidifying agent crcateo conditions such that the efkts of

continuous acidification and spccih enzymatic action overlap and pioduce coagulation within or

in the vicinity of the region of important ridity development, below pH 6.0. Renneting does not
go near to completion and coagulation is triggcd by the cumulative destabilizing effects of

limited acid and enzymatic modification o f the casein particles, as well as changes in semm iunic

(Ca) composition. For sta~dardsxpcriments in this wodc, this conespondcd to concentration of

rennet Rxl, and average pH and degtce of K-casein hydrolysis at the onset of measurable

coagulation between 5.8-5.7 and 30035% at 4 û T , respectively.] Perhaps it is those conditions

Dalgleish & Home [1991a,b] described as 'approximately balanced', implying relative

adequation (intcgration) between the coincident (direct) contributions of acidification and

renneting in the course of milk gel development.

There is a broad similarity ôetween the dynamic conditions for gel setting in acidifying milk

with no and little coagulating enzymes, and approximately equivalent stages of gel evolution may

k distinguished (Figure 7.3.2a, panels a&@. An important distinction is that the partial nmoval

of pseudo-micellar surface it-casein in renneted milk modifies the reactivity of the casein

particles and allows for destabilization and gel fornation at somewhat higher pH-and at higher

rate-than for milk coagulated by acid exclusively.

Stage 1. Acid@?cationand renneting with gelformation andjirnting. The distinct influence of

acidification during the early phases of gelation (pH 5.6-5.0 at 40°C) is still (much) in evidence in

'minimally' renneted milk, gel modulus and consistency developing with a simultaneous increase

of tm 6 initially. Renneting might contribute-albeit thmugh different reactions-to the latter

response since a shallow local maximum in tan 6 is sometimes found immcdiately afler gelation

of non-acidifying renneted milk (only in this case, the response may point more to an

intemediate state during which a viscous fluid coexists witltpossibly re-amngingjxna-

cascin particles of gel). Since gel asscmbly sitar& slightly in advancc of important acidification, it

is expected that some colloiâal Ca phosphate is retained in the gel matrix at md shortly d e r

formation. Supposcdly, fùrther lowering of the pH (i.e., near completion of casein

demineraiization) alters the intcgrity of the gel just fonned, temporarily interfixing with (slowing)
structure development. The conspicuous slowing down of fiming centered amund pH 5.2 may bt

the hallmark for such somcwhat delayed dernineralization relative to gel formation.

Stage 2. Secondq gelfirming. With initial gel formation taking place mlatively late in the

acidification proccss, secondary sûcngthening and fiirther gel re-structuration take over soon aAer

dernineralization so thet gel rigidity is only modcrately affectcd (lugely retained) during

evolution toward extensively demineralized gel state on critical acidifcation. This particular

sequence, or sort of a 'rclay' mode of development, pmbably accounts for the characteristic step

wise evolution and rapid recovery of experimental gel modulus and consistency ôetween about

pH 5.0 and 4.6 (secondary increase in firming rate near pH %O), while elasticity develops (Le.,

tan Gdecreases).

The increasing divergence with time ktween the course of consistency (modulus)

development in strictly acidifying milk and in minimally renneted acidifying milk is worth

pointing out, and in particuliu the substantial enhancement in gel rigidity on lirnited renneting-

an efiect that was especially evident in our experiments past the transition stage around.pH 5.2-

5.0. The presence of supplementary (distinct) -ive sites on the renneted casein particles must

be determinant for the smicturing of what may be viewed as r icnnet-reinforcd acid gel. The
distinct and rapid strengthening may be interprcted to indicate a synergistic effect betwcen

renneting and acidification acidification potentiating the aggregating tendency of rennet-

converted (posdbly re-anangcd) particles, e.g., by diminishing electrostatic-and steric-

tepulsion and increasing frre cal'. The effect may k more effective at long times (increasingly

acidic conditions)-md possibly accentuateci by sporadic synemis pmcesses of contraction and

coarsening of UK gel.

Stage 3. Gel consolidation und 'slobiluution'. 'Ultimate' gel structure is established

subsequcntly with fiming mte and ton Grcaching stcady-like limiting values. The relatively high

rigidity confend by limited rmneting is still much in evidence at this stage, yet the low
magnitude of tan Gnlative to that for rcnnet coagula at constant (acidic) pH and long time (and

same measunment fiequency) seems to indicate that patterns of dominant interactions (and

structure) characteristicof acid systcms are in place. It is possible, however, that tm Gmay not be

a discriminatory enough parameter with respect to the type of interactions that govem pl

structure and dynamic rhcological behaviour. Perhaps the casein-cwin bonds introduced by

rennet action eventually get smeared out or their relaxation khaviour substantially modifieci on

embedding in an acidic gel matrix.

(c) Larnelv Seawntial Gel Formation and Acidification. Excess rennet relative to acidifying

agent mates conditions such that cnzymatic action-potentiated by slightly acidic miik

pKinduces coagulation (fu) in advance of the development of critical acidity. For typical

analyses in the present work, this comsponded to concentrations of remet r Rx4, and average

values of pH and u-casein hydrolysis at the onset of coagulation between 6.3-6.0 and 55-60% at

40T, respectively.] Actually, the terni 'combine# may not k the most appropriate way of

defining the coagulation process under such conditions: confusion may arise if the maximum-

minimum rheological khaviour that results experimentally is to k undersiood as a manifestation

of mainly rennet coagulation and consecutive-as distinct fiom simuItuneoirc, as envisaged in

/a)-acidificrtion. The partial overlap, initially, ktween renneting and acidification (i.e., the

positive effcetr of moderate acidity on the eficiency of the renneting pioccps) cannot be ignored,

but the situation seems better viewed, ovcmll, as a decoupling+ather than a

combination-betwctn the two modes of dcstabilizationlcoagulation of milk casein.

Stuge 1. Rennethtg anà moderate acidification with gel /srmation and jhting. The tint

stages of gel formation, Iag stage inchdeci, correspond to the sctting of a largely rennet gel under

conditions of roughly static-inildly acidi~-pH(i.e., with moderate deminenliution of casein).

When acidification is by microbial fermentation, this more or less coincides with the initial
stationary phase of bacterial gtowth [ktwcm about pH 6.4 and 6.0 at 4û"C in this study]. Gel
formation and timing are evident h m the rapid rise of consistency and modulus, and fiom the

sudden deaease of tm 6to practically constant readings.

Hem the evolution of fun G points to the development of an important elastic component to

gel modulus and the acquisition, very e d y in gel development, of a rheological behaviour &in to

that of strictly rennet gels. Constant (relatively high) values of tan 6 suggest thnt elastic and

viscous components contribute in the same proportions to increasing gel fimness. This (short)

p e n d of initiai stabilization of tm 6 rnay be seen as an indication for the demarcation between

the effects of renneting [stage 11 and acidification [stage 2 and beyond, which may be interpreted

in analogy with stages 1 to 3 (4) of gel development in (a) and fi)].

Stage 2. Acidificafion with gel soflening. Important acidification in the advanced stages of gel

development changes an increasing proportion of the Ca phosphate in the pmo-cwin matrix to

soluble fom. Presumably, late demineralization decreases the interactions h e e n the different

caseins in and among the aggregated particks, which ultimately alters the cohesive properties of

the precursor rennet gel (i.e., apparent relaxation or softaiinvelative 'amorphization'?+f the

gel). The duration of this process would k of the order of magnitude of relaxation processes such

as syneresis for rennet milk gels, Le., about 10'-10' s [van Dijk & Walstra, 19861. Perhaps
acidiflcation triggers a configurational drift (disjointing) of gel components and major, slow de-

a d o r re-stnicturing (differentiation) of the network, possibly involving flow or diffusion of the

solid p h w . This transformation of the gel phase may account for the sustained decline of

experimental modulus and consistency and for the parallel increase of t a 6 in the region of pH
betwecn CU. 5.5 plus and 5.0 plus. Note that it is panicularly elusive whether the expectcd

collapse of the (remaining) K-caseinat the surface of the casein particles betw#n pH 6.4 and 6.0

(Le., initial stages of gel formation) may favour disjointing of gel elements at this stage.

That part of the softening may occur through pmcases of syneresis on a micro~copicscale

cannot bt rulcd out, but available experimental data permit no more than highly hypothetical
appmaches to this point plm. Microsynetesis may set the stage for macmsyneresis of liquid later

in gel development. Thcn is no unquivocal evidence in the litenture regarding the effect of

colloidal Ca phosphate on syncmis khaviour, although it would seem that lowering the mount

of Ca phosphate in rennctcd casein particks gives rise to cnhanceâ scparation of whey [Walstra,

19931. It has k e n suggested that high values of fun 6(an indication of relatively fast relaxing-

m-arranging-ôonds, i.e., relatively dynamic gel structure) comlate with an increased tendency

of milk gels to exhibit (macro) synercsis (van Vliet et of., 1 9 9 1 ~van Vliet & Walstra, 19941. It

might also be that shearing contributes to the development of relatively more viscous properties

and loss of consistency of the pseuda-rennet gel rneasureâ in this region of pH. This effcct would

be akin to (mostly irreversible) thixotropy or rheo-destruction, i.e., temporary soflening (thinning)

attributable to disruption of intemal structure on shearing.

Stage 3. Secondmy gelfirming. New patterns of interactions of the type of those in strictly

acid gels probably fonn near the end of the demineralization period, allowing for restoring and

fiming, ie., further differentiation of the gel analogous to secondary sûengthening. It is possible

that secondary increase of the rate of biological acidification around pH 5.5-5.0 (Chapter 6,

Section 6.3.4) also contributed to the nfirming of gel evidenced in the presmt work. Syneresis

processes (gel contraction) may overlap and possibly mask part of the efT'ects of demineralization.

This would explain the secondary increase in modulus and consistency and concomitant decrease

in fan Gmeasured below pH 5.3-5.0.

Stage 4. Gel consolidation und 'stabilharion'. Furtha changes would leid to additional

densification (coarsening) and acquisition of long-tem propcrties. Le., levcling off of finning rate

and fun 6. Apporcntly, gel chmctcristics measureâ at this stage arc similtu to those for minimally

renncted systems, Le., relatively high values of modulus and consistency compared to snictly acid

gels, and mlatively low values of tun Gcompambk to those for acid gels.
 Admittedly, the above intcrprctation spadcs many questions about the details of the

rcidification-dtivcn difkentiation piocess that cannot be answercd easily. One cm only guess

for e m p k at whether (how) the pu-casein gel produced on renneting rnay get 'recycled' in

the process of reorganization (inter- andor in-micellar rcarrangements after the start of gel

formation? mle of particle fusion?), the specificity of the (hybrid?) structures that result, and the

interrelations ktween coagulation and syneresis (and latter) behaviours. The main argument here

is that interaction e&ts of acid and renntt on milk coagulation rnay bc determineci by stage of

gel development. In this view, the accent is on variations in the sequencdextent of renneting and

acidification as a key dimension to the gradations in coagulation behaviour explored in this

chapter.

The final section focuses attention on the i m p o ~ c eof achieving a pmper balance
(replation) ktween Ennet action and acid production in practice.

7.3.4. Possible Technological and Nuttiîiond ReIevance

(a) Processinn of Daiw Products. If it is important to foster rationalization of the different

structures and reactions that govem the formation and properties of milk gels/curds at the

research Ievel, the most important (and challenging) aspect remains how the understanding may

translate into profitable opportunities for the dairy industry. This may mean either improving

existing products or manufachiring processes, or developing new ones, possibly expanding the

use of milk beyond rraditional markets and applications.

(0 In standard manufacture of chceses such as Cheddar, for which gel formation depcnds
predominantly on nnncting with datively little &utCr culture, cutting and draining of the casein

coagulum certainly take place kforc the e f f of

~ acidification (solubilization of colloidal Ca
phosphate) cm manifest to the extent obscrvcd in this work at nlativcly high concentrations of

remet and long incubation t h e s (> 1 h) [sccnatio (c) undcr Section 73.31. Typically, thcrcforc,

about 6045% of thc Ca and 50-6û?! of the phosphorus in the statting milk are rctained in

Cheddar cheese [Ernstrom & Won& 1974; Hill, 1995a].

 Still, the dynarnic conditions that bring about coagulation and changes in gel physico-

chemical pmprties initidly arc expected to k influentialin detemining the evolution of the curû

during later stages of the chces-making ptocess as well. Basic underpinning investigation of the

mechanisms by which rennct-acid milk gels are produced may thercfore also provide fiirther

insights into the mechanisms of. e.g., synercsis. This in tum may lead to improvements in out

ability io objectively pdict-and thereby control-the potential of the curd to drain (an

important pmctica! consideration in relation to chcese yield and quality).

It may be noteworthy that the values of pH in the region between Pn*,(local maximum in gel

consistency and elastic modulus) and Pmh (local minimum in consistency and modulus)

comspond to the pH for optimum meltability/softening of cheese, i.e., pH around 5.2-5.4. The

amount of colloidal calcium phosphate certainly plays a key role in regulating melting properties

also. It is well known also that high pre-heating is detrimental to melting. This may be related to

the quantitative differences in the coagulation behaviour of pre-heated vs. unheated milk

evidenced herein.

(ii) The particular pattern of gel deveiopment evidenced under experimental conditions of

minimal m e t i n g and continuously decreasing pH [scenario (b) of Section 7-33]has a direct

bearing on the technology of cottage cheese-the starting point of the study as it was-as well as

other soft unripened cheexs, including quark types (ôaker's cheese) and cream cheese. In the

manufacture of cottage chcese products, a period of houa [5-16 h d e p d i n g on the level of

culture addition (O.ES%)and the set temperature (25-32T)]is gennrlly allowed kfore cutting

when the pH of gel miches rn 4.64.8 (a 5.1-52 for milk pre-heated beyond pasnuriation)

m i n o n s & Tuckcy, 1%7]. Gel formation for making cottage cheese thus accurs under more

acidic conditions than for the production of most oiher types of cheescs, so that about 2 W and

35% of the Ca and phosphorus, respectively, am typically found in the fins1 chcese [Emstrom &

Wong, 1974; Hill, 1995~1.

 For minimally renneteâ acidifed milk, the tuming point (secondary firming) in the
experimental evolution of gel viscaelktic properties around pH 5.2 within 3-5 hours or so of

setting at 40°C may be related back to the well-recognized beneticial influence of small amounts

of rennet in the commercial making of cottage cheew. Limited addition of coagulating enzymes

to the cuîtured milk is customary to give a more 'elastic' ('firm' or 'strong') coagulum with a

better ability to drain, reduce matting of the curd during cooking, and ultimately improve the

textural properties of the cheese. (Concentrations of rennet S Rxl, Le., 1.2.2 mU1000 kg of milk

are typical for standard pasteurized skim milk.) This also makes the coagulum less subject to

breakage ('shattering' or 'dusting') at cutting, which contributes to incmsing the yield of cheese

by reducing the arnount of curd losses or 'fines' in the whey.

The tirne (pH) for cutting cottage cheese gel is critical and is cornmonly determined based on

results of the A-C test [Emmons & Tuckey, 19671. This test-at least when peifonned at 32OC-

gives a cutting pH of approximately 4.8 when standard pasteurized milk is used with little rennet.

(For strictly acid gel, the pH a the A-C end point is about 4.5.) For minimally renneted standard

RSM in our work, pH 4.8 cleariy occurred past the turning point in the cuwes of gel consistency
and viscoelastic moduli obtained at 40°C (e.g., Figure 7.3.4). In terms of the interpretation

propsed in Section 7.3.3, pH around 4.8 corresponds to a stage of gel development near the

completion of secondary firming (toward the end of Stage 2) and the beginning of consolidation

and stabilization (beginning of Stage 3). It Kcms logical indeed, to take advantagc of the

interaction c f f a between renneting and acidification, to cut the gel pasr the transition stage of

important acidification (casein demineralization)dunng which the rate of firming and the relative

elasticity [as recipmcal of loss tangent, i.e., (GP'/G~']

of the gel temporarily decmase. An

important considcration in the selection and adjustment of cutting pH for cottage cheese is the

ability of the curd to synercse and strcngthen adequately on subsequent heating and dnining.

[Recall that the ability of curd to synetese tends to go along with not too low value of t a 6.1
Figure 7.3.4. Typical cunies of consistency development for b w u
cultured at level C/8 and renneted at kvel (upper panel) and (lower panel) at
40°C. Comsponding primary and derivative profiles of consistency C and pH at each
level of rennet enzymes are shown for single reprcscntative expcrirnents carried out with
the Nmctre rhcometcr. h w s point to the tuming pointe) in the profiles of gel
consistency [i.e., lacal minimum or seroes in the instantancous rate of consistency
developmcnt dC/dt] and to the comsponding (approximatc) values of pH.
The rate of whcy expulsion and of curd shrinkage is gencrally slower as the pH dccrascs, hence

the tecornmendation to increase the pH at cutting (e.g., 0.1 unit) if the (cooked) curd is

consistcntly too SOA(Le., too high in moisture or insufficiently synercsed), and to decreasc the pH

if the curd is too fim andor mats [Emmons & Tuckey, 19671.

Typicaily, the pH at the A-C end point incrases with increasing the intensity of pre-heat

treatment of milk. The main problem with using highly pre-heated milk for making cottage

cheese is that the curd tends to be shatted md bdbre on cutting, even if adquately fim

[Emrnons & Tuckey, 1967; Emmons et al., 19811. For skim milk pre-heated at 80°C for 30 min,

Emmons and CO-worken [1967, 19811 nported that the most satisfactory curd in terms of

minimum breakage and of finnness was obtained by using 44 mL of rennecl1000 kg of mik (=

Rx2O) and cutting at pH 5.2, or by using 11 mL of rennet (m Rx5) and cutting at pH 5.15.

(Cmking times and temperatum and moistun content of the curd were standard. The setting

time was decreased by about an hour.) With Rx 1 and Rx4 of rennet, and pH values of 5.05-5.1 5

at cutting, the coagulum firmed adequately but shattered excessively. It seems that higher levels

of rennet and higher pH at cutting are required to limit the development of important brittlcness

(deficient plasticity) and stability against syncresis which are characteristic of acid gels obtained

from high-heated milk. [Recall that both properties tend to go along with solid-like (permanent)

charecter of the gel, Le., relatively low values of loss tangent.]

Our woik comparing the coagulation of minimaliy renneted non-pre-heated and pre-heated

(9Q0C-1min) RSM at 40°C shows that pH 4.8 and 5.2 correspond to distinct stages of gel

development (e.g., Figures 7.3.4 and 7.3.5). For heat-treated RSM rcnncted at R x l and Rx4

(Figure 7.33, pH 5.2 comsponded to about the M i n g point in the profiles of gel consistency

and rnoduli development, that is, ta the transition stage ôetween the near completion of gel

demineralization (end of Stage I at Rxl, Stage 2 at Rx4) and the start of secondary h i n g

(kginning of Sicge 2 at Rx 1, Stage 3 at Rx4).

 Consistency C ( c ~ . ~ . c m * ~ ) Consistency C ( c ~ . ~ . c r n - ~ )
and dC/dt ( c ~ . ~ . c m - ~ / h ) and dCldt (c~.~.crn-~/h)
I
& C L
z
0
L?,
0 0
- - W
r r r o m o m
0 0 0
h
0
)
0 - oov o
ioùu
o o o S 8 voi o
- - ) 3 h ) W b J &
o uoi o
o uor o
o

LC&PPrrNNWW.*pY'Y'?'?'
,~,ouraurouiourou,o~our
pH arid dpH/dt (pH uniWh)
 Rccall that this ûansition strge was characteritcd by a local minimum of the rate of firming

(03, which, at concentration of rennet Rxl essentially coincided with a minimum of relative
elasticity (maximum in tm, d) of the gel, and at 2 Rx4 seemed to precede it shortly. The values of

elastic modulus and relative elasticity (miprocal tan 4 for gels h m pre-heated RSM at x pH
5.2 were lowcr (but fairly high still) than for gels âom low-heat RSM at pH 4.8. Cutting gel

h m high-heated milk befire (important) secondas, firming may k kneficial because the

interaction effects ktween pm-heoting and acidification that irnpart increased brittieness and

stability to syneresis (too low value of ton 6)in practice rnay not be too manifest at this stage. The

relatively low values of maximum tan 6(i.e., more pronounced solid-like character of gel) and

the unexpectedly low values of instrumental consistcncy measured for gels fiom remet4 pre-

heated milk compared to standarâ gels may have to do with the inhemit tendency of such gels to

break on cutting. (Measurements of large deformation attributes such as brittleness would be

required to establish this correlation.)

Such observations empha~izethe important function of rennet in the production of cottage

cheese and the importance of fine-tuning rennet action and acidity development, as discussed in

Section 73.3, to the requinments of chee,making. Scientific understanding of the reactions that

modulate the technological properties of the gel on combined acidification and renneting m a i n s

sketchy, however. Mon integrated and quantitative accounts of curd-setting mcchanisms may

Iead to means of contmlling more closcly (and automatically) process/pmduct performance and

quaiity. As alludcd to in the pmcnt work in particular, integrated use of parameters such ru

'consistcncy', elastic modulus, tm a Luge deformation attribute, and pH may lead to more
precisc control of critical points (e.g., cutting tirne for differcnt checses) than the use of only one

or two such paramctcr(s). Down the line, a h , advances in understanding the technological

behaviour of milk may a h permit identification of alternative p o c w s of gel formation and

mise interesthg possibiiitics of triIoring novel products with specific functional pmpcrties.
(b) Gasûic Digestion of Milk bv the Re-Ruminant Calf. Natunlly, the coagulation behaviour of

milk must k amined to the physiologicd events, particularly digestion and absorption of

essential nutrimts, which take place in the gastrointestinal tract of the new-bom mammal. It is

interesting to expuid our f a u s and note that the conditions of rennet-acid coagulation we

investigated in vitro resmble those in the abomasal cornpartment (fourth or, rather, true digestive

stomach) of the pre-ruminant calf in the first week or two of life, that is, when calf rennet is high

in chymosin [Hill et al., 1969; Roy, IWO].

In the suckling calf, the action of the oesophageal grgroove usually ensures that the ingested

milk passes straight to the abomasum. On feeding, the pH of the abomasal contents rises rapidly

fiom behveen 2.0 and 3.0 to approach that of the milk, as approximately illumted in Figure

7.3.6. The pH then declines steadily over a period of 5-8 h to pre-fecding levels as a result of
gastric secretion of hydrochloric acid [Roy, 19801. In the hcalthy young calf given non-aciditied

tkesh milk, coagulation of micellar casein typically oecurs within about 5 min owing tc+

predominantly-the potent specific activity of chymosin at nearly neutral pH. (In humans, only

pepsin is produced and coagulation is mainly by stomach acids.) As in cheese-making, the casein

curd subsequently contracts and the whcy is quickly released, assisted by the peristaltic motility

of the abomasum, into the upper srnaIl intestine (duodenum).

The biological value of milk coagulation may be seen as a means by which a differcntial flow

of nutrimts is pmvided to the absorptivc a r a of the intestine. In essence, the clotting process

pmvides a timcly supply of mdily available and npidly assimilatcd nutrients in the form of

minerals and simple s u w fmm lactose, against a continuous background absorption of

degradation products h m protein and fat [Hill et al., 19691. Eff'tive coagulation of casein

initially (i.e., rapid formation of a fim dot, and thus, long enough retention time in the stomach)

dclays the passage of some of the milk canstitucnts into the intestine and incrrws the extent of

hydmlysis of protein and entnpped milk fat by gastric proteinases and pre-gastric lipases.
 - --

pH 6.5 = optimum pH for spccific proteolysis

)tMaguls<ion) of miik casein by gastric chymwin

f c w i n coagulum
in calf abomasum at about 37°C

pH 3.5 = optimum pH for non-specific

. '.
proteolysis of miik cascin by chymosin
.(pH 2.1 for pcpsin) at about 37°C

pH of caif abornasu&
(afkcr Roy (19801)
. -
%*. I

t - m .

Time following the ingestion of liquid milk (h)

Figure 73.6. Approximate evolution of the pH of the abornasal contents of the pre-
niminant calf following the ingestion of non-acidified k s h milk (ifter Roy [1980])
and putative parallel evolution of the initial consistency of the casein coagulum
(before extensive disintegntion, that is). Conjectured regions of maximum and
minimum "consistency" (i.e., Pl(, and PmiJ are indicated, together with the
conesponding values of pH (anows), and the optimum values of pH for specific and
non-specific proteolysis of milk casein by calf rennet (Le., chymosin and pepsin) at
physiological temperature.
Concomitant, gndual acidification in the stomach favours enzymatic digestion and, in the course

of over 6 h, disintegration of the curd.

Variations in the renncting pmpcrties of milk such as soft-curdling lead to different patterns
of digestive finction and may predispose the animal to digestive upset. It is well-known for

example that when a milk substitute based on overheated milk powder is included in the diet,

there is an increased escape of largely undigested protein with other constituents into the

duodenum ôecause o f the impaired coagulation characteristics of the feed (i.e., formation of an

excessively soft dot) [Hill et al., 1989; Roy, 1980; Buchheim, 1984; K a u h m , 1984~~6;
Meisel

& Hagemeister, 1984; Pfeil, 19841.

The rather high proportion of casein in ordinary cow's milk is (partly) responsible for the

compact, 'rubbery' nature of the clot normally formed on coagulation by rennet in vivo [Hill et

al., 19691. If the observations we discussed herein reflect the initial evolution of coagulum

consistency in the stomach of the young calf (certainly the conditions of peristaltic motility would

have to be taken into account), it may be suggested that transient decrease of the rate of firming

and soflening (loosening) of the relatively dense (coarsening) casein matrix on extensive Ieaking

out of Ca phosphate facilitates enzymatic decomposition, and hence digestibility, of the milk

proteino. (In humans, an analogous kneficial effect exists when cultured milk products are

consumed, the slow release of lactic acid by starter bacteria giving rise to a relatively soft

coagulum which is readily accessible to digestive enzymes in the gastrointestinal tract, and is

quite different îrom the dot fonncd when k s h milk enters the stomach [RaSic & Kumann,

1978; Renner, 1983; Robinson & Tamimc, 19861.) This might help ensure that the nutritional

boost of hi@ casein content does not undemine the degm to which milk nutrients cm be

pmperly utilized. The apparent stimulatory eff& of calcium release on gaseic acid secretion and

pepsin activity ~ u f m a n n 198461

, would k in kceping with maintaining optimal functioning of

the digestive system of the prc-ruminant calf.

 Important aspects of the coagulation of milk by rennet and acid have been thrown into relief

in this dissertation. Basically, the measurrments of apparent hydrdynamic size and

hydrophobicity presented in Chapter 4 are in kaping with the scheme of 'electroîteric'

destabilization of the casein particles, with progressive collapse of the surface layer of (primarily)

r-casein rnacropeptide upon exponue to increasingly acidic conditions of pH in the ranges 6.7-

6.0 and 6.0-5.5. The surface laycr of particles isolated h m milk pre-heated at 90°C-1 min seems

to be similarly affected by partial acidification but appcan thinnec the later characteristic may

contribute to differential intrinsic stability of the casein particles in unheated and pre-heated milk.

Actual analyses of gel developmcnt fiom standard and pre-eeiited milk in subsequent

chapters shed light on the gradations in coaplation behaviour nsulting h m varying the relative

contributions of renneting and bactcriological acidification. From the main features of rheological

gelation profiles, we have taken the view chat interaction effects of rennet and acid on milk

coagulation may be detemined by stage of gel development. In other words, distinct patterns of

coagulation behaviour seem to aria (in part) h m diffennt patterns of succession of renneting

and acidification. A conceptual scheme has k e n devclopcd to try to capture the underlying

physico-chemical processes for the behaviours cvidenccd by dynamic rheomeby.

If the research liner emphasized in this work have received relatively little attention in the

pastdirectly at least-, most observations, including comments on methodology, ceitainly echo

the availabie phenomenology and general understanding of enzymatic and acid coagulation. This

hardly cornes as a surprise in retrospcct. As brought up in the litcraturc survey in Chaptet 2, the

stability and coagulability of milk have bcni rescuchd fiom about evcry accessible angle ovcr

the 1 s t thirty plus y w s with a more or las direct beuing on milk manufacturing quality. Of

course, as in al1 complex situations, the multiplication of professional publications has not k e n in
proportion to the achial prognss made in undetstanding the details of the destabilization and

aggrcgation phenomcna in milk-hence the munent hand-waving explanations in the

dissertation also. Undoubtedly, them m a i n many elusive topics (e.g., the molecular and kinetic

dimensions of the many different reactions involvcd in the formation of minet and acid casein

gels or curds [also review by Home, 19981) but then, these are bmches in out knowkdge that

one cannot realistically expect to be filled any time smn. Of course, the rcstrictedinstrumentation

suitable to the variable (rnotecular) condition of milk casein 'micelles' continues to t>t an

important bottleneck limiting ntionalization of the technological functionality of milk systems.

What Ways Fomard?

To be sure, it has become increasingly difficuult to delineate promising, viable perspectives for

investigation within the =ope of milk (casein) coagulation properties. Relative saturation of

reseamh on the subject has restricted the window of oppottunities for mily innovative and usehl

contributions in the immediate Wre. Perhaps it would not be unwise, to further knowledge in

the area while limiting unnccessaty dundancy. to emphasize exploitation and assimilation of

accumulated information rather than aquisition (and impentivc publication) of first-hand data

(i.e.. 'fact grinding' rather than 'fact finding'; sec also the reflections by Horne [1998], Walstra

[1998], and Noël & Tessier [2000], and the general view outlined by Kazic [1994]). Knowledge

management, or the organizing of vast mounts of data into efficient and useful representations

and operations, is smly kcoming critical is in other disciplines. This prrsupposes effective and

affordable access to comlative and supportive information. Clarification of the tenninology used

may be beneficial in some cases.

Kinetic analyses of remet-acid modes of gel developmmt, for instance, although ' p l

constitutive equations' would m e as a powefil tool for the conbol and optirniution of

industrial processes (and formulations), are often left out because o f the lack of fundamental

theories to account for the complex nmork of reactions and structures at play. Relations khuccn
gel characteristics in the initial and later stages of coagulation =main to be established in

particular, espccidly in highly pn-heated milk. A bmad expcrimental and analytical database is

available-if in a rather disparate and hgmented fonn. What is needed arc more quantitative,

integrated expressions of the phenornena leading to the formation and fiming of casein gels,

whereby the effects of specific process pariuneters could be factored in. This would go hand in

hand with attempts to refine theotetical and mechanistic interpretations of coagulation reactions.

lnvestigaton may kave molecular aspects aside for the time being and look for nonetheless
adequate accounts of the development of gel properties at higher levels. In the case of acidifying

and renneting milk, one obviously cornes up against the substantial difficulty of decomposing and

articulating the relative importance of charge neutralization, mineral (and protein) solubilization,

and nsidual steric stabilization-not to mention system dynamics-in the different States of

caseitdwhcy protein dispcnionlaggregation. The ultimate goal of such enterprises would be to

arrive at mathematical formulations of the global coagulation behaviour of milk under conditions

of processing with satisfactory explanatory (Le., descriptive or mechanism-based) and predictive

value. One may also seek to develop experimentally verified soAware tools implementing such

mathematical models. Growing scientific sophistication will likely be rcquired to take on

arnbitious tasks of this sort. For example, researchen may want to leam enough about the

concepts and potcntial of computer-assisted dynamic mdeling/numerical simulation to k able to

collaborate effcctively with skilled theorcticians.

Anothcr way to go would k to promote problcm-solving endeavours, with attempts toward

relating (applying) hindammtal knowledge to the changing realities industry and market present.

Such ventures may actually tike one back-and-forth along more typical invertigative paths, albeit

perhaps in different directions. This may mean, for example, compounding mik gels through

intentional mixing of diffcmit ptotcins andfor non-protein polymeric matenals such as modified

starch or patin or bacterial cxopolysiicchuides to hiitha diversi@ gel (textwal) propcrties

[rcview by S y r k et ai., 19981. One may also look for ways of promoting the physiological (both
nutritional and pharmacological) functionality of s~alllqd'probiotics' or cultutcd dairy products.

This approeeh would also knefit fiom sound crccition and utilization of knowledge assets

[overviews by Daly et ai., 1998; Ouwehand & Salminen, 1998; Sandea. 1998; Ziemer & Gibson,
IW8].
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