CyTA - Journal of Food

ISSN: 1947-6337 (Print) 1947-6345 (Online) Journal homepage: http://www.tandfonline.com/loi/tcyt20

Evaluation of proteolytic and physicochemical

changes during storage of fresh Panela cheese
from Queretaro, Mexico and its impact in texture

J.A. Guerra-Martnez , J.G. Montejano & S.T. Martn-del-Campo

To cite this article: J.A. Guerra-Martnez , J.G. Montejano & S.T. Martn-del-Campo (2012)
Evaluation of proteolytic and physicochemical changes during storage of fresh Panela cheese
from Queretaro, Mexico and its impact in texture, CyTA - Journal of Food, 10:4, 296-305, DOI:
10.1080/19476337.2011.653791

To link to this article: http://dx.doi.org/10.1080/19476337.2011.653791

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 CyTA Journal of Food
Vol. 10, No. 4, November 2012, 296305

Evaluation of proteolytic and physicochemical changes during storage of fresh Panela cheese from
Queretaro, Mexico and its impact in texture
Evaluacion de cambios proteolticos y fisicoqumicos durante almacenamiento de queso panela de
Queretaro, Mexico, y su impacto en la textura
J.A. Guerra-Martnez, J.G. Montejano and S.T. Martn-del-Campo*
Escuela de Ingeniera en Alimentos, Biotecnologa y Agronoma, Tecnologico de Monterrey, Campus Queretaro. Epigmenio
Gonzalez 500, Fracc. San Pablo. CP 76130, Santiago de Queretaro, Qro. Mexico
(Received 22 August 2011; nal version received 22 December 2011)

Mexican Panela cheeses manufactured with pasteurized cows milk at a pilot plant scale were analyzed in order to determine the
relationship between physicochemical and textural changes during 15-day storage. Changes in hardness, cohesiveness, springiness
and chewiness were measured by texture prole analysis (TPA). Moisture, pH (superior and inferior rind and center) and total
protein were evaluated with ocial methods. Proteolytic indexes (acid soluble nitrogen (ASN), non-protein nitrogen and 70%
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ethanol soluble nitrogen (EtOH-SN)) were obtained by selective precipitation and quantied by Kjeldahl method. Organic acids
(lactic (LA), propionic (PA) and butyric acid (BA)) quantitation was done by high performance liquid chromatography. Analysis of
variance (ANOVA) showed signicant dierences for all the physicochemical parameters evaluated, but only cohesiveness and
springiness showed signicant dierences in texture parameters. By principal component analysis (PCA) two groups were separated,
one from day 1 to 7 and one from day 9 to 15.
Keywords: Panela cheese; storage; textural changes; physicochemical changes; PCA

Quesos panela mexicanos elaborados a escala piloto con leche pasteurizada de vaca fueron analizados para determinar la relacion
entre los cambios sicoqumicos y de textura durante 15 das de almacenamiento. Los cambios en dureza, cohesividad, resortividad
y masticabilidad fueron medidos a partir de pruebas de perl de textura. Los cambios sicoqumicos en humedad, pH (corteza
superior, inferior y centro) y protena total fueron evaluados con metodos ociales. Los ndices proteolticos (nitrogeno soluble en
acido y en etanol 70% y nitrogeno no proteico) fueron precipitados selectivamente y cuanticados con metodo Kjeldahl. Los acidos
lactico, propionico y butrico se cuanticaron por cromatografa de lquidos. El analisis de varianza arrojo diferencias signicativas
en todos los parametros sicoqumicos, pero solo cohesividad y resortividad mostraron diferencias signicativas en los parametros
de textura. El analisis de componentes principales diferencio dos grupos, uno del da 1 al da 7 y otro del da 9 al da 15.
Palabras claves: queso panela; almacenamiento; cambios en textura; cambios sicoqumicos; PCA

Introduction has a good acceptance among consumers due to its crumbling

Panela cheese is a popular handcrafted Mexican soft fresh
cheese manufactured from pasteurized (NOM-243-SSA1-
2010, 2010) cows skim or partially skimmed milk (Cervantes
Escoto, Villegas de Gante, Cesn Vargas, & Espinosa Ortega,
2008), with little or no starter culture acidication (Cervantes
Escoto et al., 2008; Farkye & Vedamuthu, 2002) and with
usual average yields of 1314 kg of cheese per 100 l of
pasteurized milk. It is usually found in local markets as a 0.5
2 kg white cheese and characterized by its high moisture
content and susceptibility to spoilage, therefore, better if
consumed within the rst days after being manufactured
(Villegas de Gante, 2003). Panela is described as a unique
basket shaped (usually molded in palm or plastic baskets)
and a mild pleasant avor cheese obtained by rennet
coagulation (Farkye & Vedamuthu, 2002) with a slightly
salty avor. Its chemical composition consists of 5358%
moisture, 1929% fat, 1620% protein and 1.52.2% salt
(Cervantes Escoto et al., 2008; Farkye & Vedamuthu, 2002;
PROFECO, 2007; Villegas de Gante, 2003). Panela cheese
and good slicing properties; thus, it is manufactured by large-
scale dairy Mexican industry in great volumes due to its high
demand throughout the country. The production of Panela
cheese in Mexico was 23,059,000 kg in 2007 and 27,010,
000 kg by 2008 (http://www.inegi.org.mx). According to a
new regulation in the Mexican law, it is mandatory to
pasteurize the milk used in cheese making (NOM-243-SSA1-
2010, 2010) in order to assure food safety. The pasteurized
milk Panela cheese sales in 2008 accounted for $1,201,525
Mexican pesos; this represents 12% total sales of pasteurized
milk cheese manufactured by large-scale Mexican dairy
industry (http://www.inegi.org.mx). More recently in the
United States, manufacture of Hispanic-type cheeses such as
Chihuahua type cheese (Van Hekken, Drake, Corral, Prieto, &
Gardea, 2006), increased from 86,446 kg in 2007 to 87,650 kg
in 2008; globally, a growth of 8.6% from 2004 to 2005 and
1.4% from 2007 to 2008 was registered by the U.S national
agricultural statistics service (Barriere et al., 2008). Statistics
provided by USDA in April 2011 (http://www.nass.usda.gov/)

*Corresponding author. Email: smartinde@itesm.mx

ISSN 1947-6337 print/ISSN 1947-6345 online

2012 Taylor & Francis
http://dx.doi.org/10.1080/19476337.2011.653791
http://www.tandfonline.com

showed that production amounts of Hispanic cheeses in the
United States are comparable, yet lower, than production
amounts of some Italian cheese varieties such as Ricotta and
Parmesan (93,845 kg against 111,481 kg and 101,841 kg,
respectively); this is a proof of increasing interest and
acceptance of these types of cheeses among consumers, mainly
attributed to the growth of Hispanic population in the United
States (Moore, Richter, & Dill, 1986; Van Hekken et al., 2006)
and the interest of US dairy industry to satisfy the current
demand of not only Hispanic, but also non-Hispanic
consumers who are interested in trying Latin American foods
(Wall & Calderon de la Barca, 2006). As many as 20 out of 30
dierent types of Mexican cheeses are manufactured in small-
scale and family type operations (Moore et al., 1986; Villegas
de Gante, 2003) where very limited technology is available and
poor processing standardization results in very low yields
(Cervantes Escoto et al., 2008). The heterogeneity in milk and
nal product composition makes the study of sensory, textural
and physicochemical properties of handcrafted Mexican
cheeses, an extremely dicult task.
In the US, only a few kinds of raw milk cheeses are
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allowed (ripened at least 60 days at not more than 1.678C or
35 8F) (FDA, 2011), so fresh cheeses are not included and
hence companies there use pasteurized milk, as do many
plants in Latin America. In the US, a type of fresh cheese
similar in composition to Panela cheese is known as Queso
Fresco (QF) and is one of the most popular Hispanic fresh
cheeses found in the US market. Its manufacture and
composition are very similar to that of Panela cheese: 46
57% moisture, 1829% fat, 1721% protein and 13% salt
(Guo, Van Hekken, Tomasula, Shieh, & Tunick, 2011). QF is
exclusively manufactured with pasteurized milk; several
studies have been conducted to characterize properties of
QF (Renye, Somkuti, Vallejo-Cordoba, Van Hekken, &
Gonzalez-Cordova, 2008; Tunick & Van Hekken, 2010). A
major dierence between QF and Panela cheese is that the
latter is manufactured with or without addition of starter
culture, while QF has no starter culture addition (Genigeor-
gis, Toledo, & Garayzabal, 1991). In large-scale dairy
industry, sanitary regulations are complied and standardiza-
tion of manufacturing processes of Hispanic cheese was
assured. Nevertheless, information regarding textural and
chemical properties of these cheeses is scarcely found
(Villegas de Gante, 2003). Due to the increase in domestic
and international demand of Mexican type cheeses, further
research into sensory attributes (avor, aroma and texture)
and biochemical phenomena such as proteolysis, glycolysis
and lipolysis assessment is needed as a mean to characterize
extensively, the quality traits that dene this particular type
of cheese.
The current study aimed to measure physicochemical and
textural changes of Panela cheese with the same character-
istics of those produced and commercialized by large-scale
dairy industry. Statistical correlations of cheese properties
were carried out in 1-day-old to 15-day-old pasteurized milk
Panela cheeses stored at 2.5 + 0.68C temperature.
Materials and methods
Cheese manufacture
Whole milk (3.19% fat, 3.11% protein, initial pH 6.69) was
cooled and stored at 48C for 18 h before cheese making. Milk
was pasteurized in a plate heat exchanger (758C for 15 s), and
CyTA Journal of Food 297
cheeses manufactured on a pilot plant scale in the food
engineering facilities of Tecnologico de Monterrey, Quere-
taro, Mexico.
After pasteurization, the milk was heated gradually to
358C and 4 UC/L of commercial type O starter was added.
This starter was mixed with mesophyllic Lactococcus lactis
subsp. lactis and L. lactis subsp. cremoris MO 032 culture
(Lyofast1, Sacco, Via Manzoni, Italy) was used as a
starter. CaCl2 was added to milk by diluting 51.2 mL
CaCl2 50% (Cal-Sol501, Industrias Cuamex SA, Mexico)
in 500 mL water. The milk was allowed to reach 2021
Dornic degrees (8D) and kept at 358C before adding liquid
calf rennet (Qualact1, Altecsa SA, Mexico) at a sucient
level to coagulate milk in 45 min at 358C. Coagulation of
milk was completed in 4550 min; then, curd was cut into
approximately 1 cm3 cubes and allowed to rest in whey for
5 min before stirring without heating for 10 min. Cooking
of curd consisted on gradually increasing temperature from
35 to 408C within 20 min (increasing *28C every 5 min).
After cooking, approximately 2/3 of whey was drained at
pH 6.4. Salt was added to curd at 0.7% and mixed
manually for 10 min, afterward, cheeses were molded in
100 g plastic molds and slightly pressed by piling them up
on one another for 20 min (by each side), allowing natural
whey drainage at room temperature. Cheeses were packed
in plastic bags and stored in refrigeration at 93.8 + 1% of
relative humidity (RH) and 2.5 + 0.68C temperature for
15 d. With this procedure, 25 cheeses of 100 g each were
obtained.
Sampling
Two whole 100 g cheeses were removed after 24 h (noted day
1), 4, 7, 9, 11, 13 and 15 d of storage. One complete cheese
was used to perform pH measurements at superior rind,
inferior rind and cheese center. Measurements were per-
formed by triplicate on the same cheese, but at dierent and
random regions located at superior, inferior and center of
cheese. pH values at center of cheese were measured at
approximately 1 cm from surface, thereafter, same cheese
used for pH measurements was grated. Second cheese was
used to obtain six 1718 mm diameter 6 1.5 cm height
cylinders for instrumental texture prole analysis (TPA)
after discarding 1 mm rind of cheese. Cheese cylinders were
discarded after TPA test concluded. The remaining cheese
sample was grated and mixed with rst cheese used for pH
measurements. An aliquot was used immediately for moisture
determination; the rest was weighted in the necessary
proportions for protein analysis, proteolysis assessment and
organic acid determinations. Grated weighted samples were
stored at 7808C until analysis.
Physicochemical analysis of cheeses
Moisture was determined by oven drying at 100 + 28C
(NOM-116-SSA1-1994, 1995) by triplicate. Protein was
measured by determining total nitrogen (TN) content by
Kjeldahl method (AOAC, 2001) by triplicate, and multi-
plying by the conversion factor 6.38. The pH values of
cheeses were determined directly by triplicate with an Orion
3-Star1 pH meter (Thermo Fisher Scientic, MA, USA)
equipped with an Orion 9135APWP1 at-surface pH
electrode (Thermo Fisher Scientic).
 298 J.A. Guerra Martnez et al.
Proteolysis assessment
Crude fractionation of nitrogenous fractions was used to
quantify the extent of proteolysis in these types of cheeses. Acid
soluble nitrogen (ASN) at pH 4.6, non-protein nitrogen (NPN)
and 70% ethanol soluble nitrogen (EtOH-SN) were used as
proteolytic indexes. ASN and NPN were prepared according to
the method described by (Leclercq-Perlat et al., 2000) with
slight modications. A cheese suspension with 10 g of ground
cheese and 100 mL of NaCl solution (9 g/L) was homogenized
(10 min, 258C) using a Stomacher BagMixer 400W1 (Inter-
science, St. Nom La Breteche, France). For ASN, the pH of the
suspension was adjusted to 4.6 by adding 2N HCl. It was then
incubated 20 min at 258C, centrifuged (30 min, 6000 rpm), and
ltered through Whatman No. 42 paper. For NPN 25 mL of
cheese suspension was mixed with 15 mL water and 10 mL of
60% (w/v) Trichloroacetic acid (TCA) to achieve a 12% TCA
nal concentration. After homogenization, it was incubated for
20 min at 258C and ltered through Whatman No. 42 paper.
EtOH-SN was prepared according to the method described by
Rohm et al. (1996) with slight modications: absolute ethanol
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was added to an aliquot of 10 mL of the ASN in an appropriate
volume to achieve a 70% (v/v) nal concentration; the
suspension was held at 48C overnight and subsequently ltered
through Whatman No. 1 paper. All nitrogenous fractions were
held at 7808C until analysis. All nitrogenous fractions were
analyzed by duplicate with the Kjeldahl method as described by
Collomb, Spahni, and Steiger (1990).
Instrumental texture prole analysis (TPA)
Texture properties of Panela Cheese were evaluated on six
replicated samples with a MTS-QTEST11 (Universal Testing
Machine, Eden Prairie, MN, USA), using a two-bite compres-
sion test of cylindrical samples of 1.8 cm of diameter and 1.5 cm
height by using two parallel plates (10 cm diameter) and a 50 N
load cell. The compression ratio used was 50% deformation
from the initial height at a rate of 200 mm/min to simulate a
deformation rate similar to that between ngers during
squeezing (Voisey & Crete, 1973). After removing a complete
sample from refrigeration, cheeses were left at room temperature
for 40 min before obtaining cheese cylinders and proceeding
with the TPA tests. Parameters measured were hardness, co-
hesiveness, springiness and chewiness, and were obtained from
the force-time plots of Tension Zero ver. 1.0 software developed
by Tecnologico de Monterrey (Hernandez & Sarinana, 2007).
Hardness was dened as the maximum force required to
compress the cheese sample 50% from its original height during
the rst compression. Cohesiveness was dened as the strength
of the internal bonds making up the body of the product and it
was calculated as the area under the second-bite curve divided by
the area under the rst-bite curve. Springiness was dened as the
distance regained by the sample during the time between the end
of rst compression and the beginning of second compression.
Chewiness was dened as the product of hardness, cohesiveness
and springiness as describe by Bourne (1982).
Organic acids determinations by HPLC
Sample preparation
Organic acid production was quantied by high perfor-
mance liquid chromatography according to the method
described by Leclercq-Perlat, Oumer, Bergere, Spinnler and
Corrieu (1999). Cheese suspensions from day 1 to day 15
were prepared by mixing 10 g of nely ground cheese and
10 mL distilled water. Samples were incubated at 508C for
1 h; afterward, they were homogenized using an Ultra-
Turrax homogenizer (CAT 6 1201, Staufen, Germany) at
30,000 revolutions/min, during 2 min. After they had been
cooled to 258C, 10 mL trichloroacetic acid (240 g/L) and
10 mL water were added to the samples. After homo-
genization and incubation for 1 h at 258C, the mixture was
ltered twice; one through Whatman No. 42 lter paper
and second ltration trough 0.45 mm PTFE lter (Agilent
RC-Econolter1, MN, USA). Samples were stored in vials
at 48C until analysis. Analysis was carried out no more
than 15 d after extraction. Results were calculated by
performing two injections of same vial for each day
monitored.
Chromatography conditions
Lactic (LA), propionic (PA) and butyric acids (BA) were
detected and quantied by high-performance liquid chro-
matography (HPLC). Analysis were carried out in an
HPLC system Agilent Technologies 1200 series1 (Palo
Alto, CA, USA) coupled with a diode array detector.
Compounds were separated in a Zorbax1 column (5 mm,
4.6 mm i.d. 6 150 mm, Agilent). Separations were con-
ducted at 508C using a 5 mM sulfuric acid solution as
mobile phase with an isocratic elution and a ow rate of
0.6 mL/min. Data acquisition was performed with a
Chemstation1 software (Agilent). The detection of LA,
PA and BA was set at 210 nm. The volume injected was
10 mL for both, samples and standards. Solutions with
known concentrations of lactic (Sigma-Aldrich, MO, USA),
butyric (Sigma-Aldrich) and PA (Fluka Analytical, Staufen,
Germany) were used to quantify those compounds in
samples. The quantication of the acids concentrations of
samples was done by multiplying the given concentration
obtained from a lineal regression curve of standards by the
corresponding dilution factor of sample.
Statistical analysis
The statistical analyses were carried out using the Statistica1
software v 8.0 (Statsoft, Inc., Tulsa, OK, USA). One-way
analysis of variance (ANOVA) was used to determine
signicant dierences (p 5 0.05) between the sampling days
for ASN, NPN, EtOH-SN, moisture, pH, organic acid
production and TPA analysis of cheeses. For each signicant
variable, dierences between means were detected using the
Fishers least square dierence (LSD) test with a 0.05.
Correlation analysis was also carried out to evaluate the
relationship between physicochemical and textural
parameters.
Additionally, a principal component analysis (PCA) was
applied using all the response variables and was carried out in
order to evaluate the evolution of those parameters through-
out cheese storage. PCA makes it possible to obtain an
overview of the data set information by replacing the original
variables by new ones called principal components (PCs).
The PCs contain almost all the information and are
orthogonal among them.
 CyTA Journal of Food 299

Results and discussion temperatures used during storage of Panela Cheese

Physicochemical parameters of cheeses
Mean values of pH at dierent cheese regions: center (pH-c),
superior rind (pH-s) and inferior rind (pH-i), as well as
moisture content during 15 d storage are given in Table 1.
Overall, pH values at the three cheese regions evaluated
in this study remained above 6.0 during the rst 7 d of
storage. pH values developed at cheese center (pH-c) showed
highly signicant dierences in a 15 d period (p 5 0.01). pH-c
values ranged from pH 6.4 in d 1 to pH 5.94 in d 15, pH value
in d 1 is similar to that reported by other authors in high
moisture QF with 0.05% salt added (Jimenez-Guzman,
Flores-Najera, Cruz-Guerrero, & Garca-Garibay, 2009). In
this type of Panela cheese, the starter culture made it possible
to enhance acidication, and the rate at which pH decreased
to approximately 5.9 in d 15. pH at cheese center decreased
notably between d 1 and d 7 at an average rate of 0.06 units/
day, lower pH values obtained at cheese center in comparison
to those obtained at rinds, can be explained with the suitable
metabolism of Lactococci under microaerophyllic conditions
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(2.5 + 0.68C), may explain the lower acidication pattern
observed in both, superior and inferior rinds of Panela
cheese. Other organic acids, besides LA, such as PA and BA
were monitored in Panela cheese during storage, amounts of
these organic acids ranged from 10.12 to 23 mg g71 of cheese
and 18.76 to 20.4 mg g71 of cheese, respectively. LA was in
greatest concentration among all other organic acids
monitored in this study. Organic acids are the major product
of carbohydrate metabolism; however, important avor
compounds are also produced as a result of hydrolysis of
milk fat during lipolysis. In Panela cheese, PA and BA were
used as an activity indicator of non-starter lactic acid bacteria
(NSLAB), indigenous milk lipase (mainly lipoprotein lipase)
and microbes developed during storage and ageing (Collins,
McSweeney, & Wilkinson, 2003). Amounts of organic acids
produced were, as expected, negatively correlated with pH
changes, but only PA showed signicant correlation
(p 5 0.05). It is important to mention that despite signicant
pH changes were obtained at dierent cheese regions, the
average pH range of Panela cheese remained relatively close

(Farkye & Vedamuthu, 2002). The rapid use of residual
lactose by Lactococci metabolism during the rst 24 h after
manufacture provides 95% of the LA production in cheeses
that use this type of SLAB (Hassan & Frank, 2001). LA
production in Panela cheese was highly signicant (p 5 0.01)
throughout 15 d storage, values obtained ranged from 25.8 to
36.7 mg g71 of cheese. Panela cheese has the optimal pH for
growth of Lactococci because it is found between a range of
5.56.5 (Mayra-Makinen & Bigret, 2004); therefore, suitable
conditions are encountered by starter lactic acid bacteria
(SLAB) used in this type of cheese. pH values at superior
(pH-s) and inferior (pH-i) rinds also showed highly sig-
nicant dierences between storage days (p 5 0.01), but were
lower than those values obtained at the center. Growth of
mesophillic Lactococci takes place within a wide temperature
range of about 10408C (Hutkins, 2006), however, below the
extreme end of this range, low growth is expected. Lower
to that pH reported as typical by other authors in Panela
cheeses (Tunick, 2007; Villegas de Gante, 2003). High pH in
these cheeses (pH 4 6.8) gives Panela its crumbling char-
acteristics and prevents melting due to presence of calcium
phosphate in casein micelles as structural component, and
calcium phosphate solubilization takes place after pH drops
below pH 5.8, but this conditions are unlikely in this type of
fresh cheese due to its high susceptibility to spoilage.
Signicant dierences (p 5 0.05) were detected between
storage days for moisture values. Overall moisture content
remained quite constant throughout 15 d storage (approxi-
mately at 52%); nevertheless, it increased slightly during the
rst 7 d, stabilized from d 9 to d 13 and decreased slightly at
d 15. Increase in moisture content within rst 7 d may be
attributed to freshly curd taking up moisture when stored at
low temperatures and high RH (%). This is conrmed as
moisture was positively correlated with RH at storage

Table 1. Mean values of physicochemical parameters of Panela cheese added with O type lactic acid culture and studied during 15 d storage
under refrigeration at 2.5 + 0.68C.
Tabla 1. Valores promedio de parametros sicoqumicos de queso panela adicionado con cultivo lactico tipo O y analizados durante su
almacenamiento en refrigeracion por 15 das a 2.5 + 0.68C.
Storage dayc
Parametera pb Day 1 Day 4 Day 7 Day 9 Day 11 Day 13 Day15
f e d c a bc
pH-c 0.0000** 6.40 6.26 6.03 5.99 5.91 5.96 5.94ab
pH-s 0.0001** 6.48e 6.39d 6.30c 6.20b 6.17ab 6.11a 6.14ab
pH-i 0.0001** 6.47e 6.35d 6.21b 6.17ab 6.11ac 6.15ab 6.03c
Moisture (%) 0.0143* 51.76a 52.16ab 52.61b 51.71a 51.95ab 51.69a 50.84a
NPN (%/TN%) 0.0013** 0.55a 0.61a 1.62b 1.51b 1.55b 1.69b 1.77b
ASN (%/TN%) 0.0000** 4.17a 6.05b 6.28bc 7.00bc 7.30c 7.76d 9.17e
EtOHSN (%/TN%) 0.0000** 2.16a 2.27ab 3.09b 4.80c 4.73c 7.04d 7.15d
TN (%) 0.0025* 3.04a 3.15ab 3.45c 3.09ab 3.14ab 3.08ab 3.17b
Protein (%) 0.0025* 19.39a 20.11ab 22.00c 19.69ab 20.04ab 19.65ab 20.24b
LA (mg g71) 0.0000** 25.80b 31.34c 19.97a 36.49f 33.33d 36.71f 35.82e
PA (mg g71) 0.0000** 10.12a 14.67c 12.27b 21.04e 19.68d 23.01f 22.86f
BA (mg g71) 0.0000** 18.76a 19.77b 18.95a 19.02a 20.31c 20.40c 20.39c
Notes: apH-c, pH values at cheese center; pH-s, pH values at superior rind; pH-I, pH values at inferior rind; NPN, non-protein nitrogen; ASN, acid soluble nitrogen;
EtOH-SN, ethanol soluble nitrogen; TN, total nitrogen; LA, lactic acid; PA, propionic acid; BA, butyric acid; bSignicant at *p 5 0.05, **p 5 0.01; cMeans with
dierent letters within the same row are signicantly dierent (p 5 0.05).
Notas: apH-c (valores de pH en el centro), pH-s (valores de pH en corteza superior), pH-i (valores de pH en corteza inferior), NPN (nitrogeno no proteico), ASN
(nitrogeno soluble en acido), EtOH-SN (nitrogeno soluble en etanol), TN (nitrogeno total), LA (acido lactico), PA (acido propionico), BA (acido butrico);
b
Signicancia a *p 5 0.05, **p 5 0.01; cValores promedio con letras distintas en la misma lnea presentan diferencias signicativas (p 5 0.05).
 300 J.A. Guerra Martnez et al.

Table 2. Texture prole analysis (TPA) of Panela cheese added with O type lactic acid culture studied during storage under refrigeration at
2.5 + 0.68C.
Tabla 2. Analisis de perl de textura (TPA) de quesos panela adicionados con cultivo lactico tipo O y analizados durante su almacenamiento en
refrigeracion por 15 das a 2.5 + 0.68C.
Texture parametera
Hardness (N)b* Springiness* Cohesiveness* Chewiness (N)b*
Time (days) n X (SD)
1 6 38.58a (0.86) 0.89a (0.007) 0.84a (0.006) 25.83ab (2.1)
4 6 31.91a (4.31) 0.95c (0.031) 0.80ad (0.05) 29.82a (3.09)
7 6 31.73b (1.58) 0.86ab (0.02) 0.74acd (0.03) 21.37b (0.83)
9 6 39.75a (2.68) 0.89a (0.01) 0.62b (0.07) 25.19ab (5.29)
11 6 39.91a (0.67) 0.86ab (0.01) 0.71bcd (0.03) 24.54ab (1.68)
13 6 43.15a (0.26) 0.81b (0.01) 0.71bcd (0.06) 24.54ab (2.31)
15 6 40.25a (2.65) 0.95c (0.03) 0.84a (0.07) 31.81a (3.72)
Notes: n, number of samples; X, mean values; SD, standard deviation; aValues within a column not sharing a common superscript signicantly diered (p 5 0.05);
b
Newton (N); *Mean values (with standard deviation in parenthesis).
Notas: n, numero de muestras; X, valores promedio; SD, desviacion estandar; aLos valores que no comparten un mismo superndice en la misma columna presentan
diferencias signicativas (p 5 0.05); bNewton (N); *Valores promedio (con desviacion estandar entre parentesis).
Downloaded by [187.208.192.54] at 21:27 18 April 2016

chamber (R 0.82, data not shown). Moisture content
exhibited good correlation with pH developed at rind
(R 0.52) which is in accordance to that reported by Fallico
et al. (2004) and Kristensen and Skibsted (1999). However,
more importantly was the high inuence moisture has on
dening Panela cheese Hardness and Chewiness, as they were
negatively and highly correlated to moisture content
(R 70.76 and R 70.88, respectively). Moisture and pH
were negatively and highly correlated with all proteolytic
indexes (R 4 7 0.82), while moisture itself was positively
associated with total protein content (R 0.53).
Textural properties
Evolution of texture properties monitored by TPA is given in
Table 2. In this study, Panela cheeses showed considerable
uctuating changes in texture properties during 15 days
storage. Cohesiveness and springiness showed more variation
within days with signicant changes (p 5 0.01) throughout
storage, while hardness and chewiness remained stable
without signicant changes (p 4 0.05), especially after d 7.
The correlations observed between texture parameters and
physicochemical parameters measured in this study at a level
of p 5 0.05 are shown in Table 3. Hardness showed high
correlation with moisture levels (R 70.76) as expected,
thus increase in moisture levels during rst 7 days of storage,
results in hardness decrease; nevertheless, hardness also
showed high correlations with chewiness (R 0.59),
EtOHSN (R 0.70), protein content (R 70.83), RH%
(70.53, data not shown) and organic acid productions,
especially with that of LA (R 0.89). This is under-
standable, considering that cheese texture is pH-dependent,
thus colloidal phosphate gradually dissociates from sub-
micelles and weakness protein network due to early a s1-
casein proteolysis by residual chymosin and plasmin. Also,
cheese moisture itself showed to be an important factor
dening cheese attributes, especially those regarding hardness
and chewiness; it seems that higher hydration during rst
days of storage may have contributed for the decrease in
hardness observed between d 1 and d 7. From d 9 to d 11
hardness increased and remained somehow constant without
signicant changes (p 4 0.05). These variations are in
accordance to that reported by Lawrence, Creamer and
Gilles (1987), who reported considerable changes in casein
network structure during the rst 7 d to 14 d after
manufacture. Also, de Jong (1978) who studied the inuence
of rennet content on the rate of softening of high moisture
cheeses.
Springiness did not show high correlation with any
parameter measured in this study that may help us to
explain its behavior. Nevertheless, cohesiveness presented
signicant dierences (p 5 0.05) throughout storage days.
An overall decrease of cohesiveness took place within the
rst 13 days of storage, which is understandable due to
lack of cohesion at this point, inuenced by pH values
close to 6.1 at superior and inferior rind, nevertheless, in
day 15 cohesion increases considerably as pH-c gets closer
to pH 5.8. According to Lawrence et al. (1987), it is closer
to this pH value when fusion of curd particles occurs;
therefore, it is not surprising that pH values at rind and
center showed good correlation with cohesiveness. Also,
chewiness showed good correlation with this parameter
(R 0.51), this seems reasonable considering that a
corresponding increase in curd particle fusion leads to a
rmer and closer structure, thus more bites are needed to
disrupt the cheese structure before swallowing. More
important is the high negative correlation between cohe-
siveness and the proteolysis index NPN (R 70.63), this
tells us that cheese matrix will eventually lose cohesiveness
as proteolysis progresses over time and pH continues to
decrease to approximately pH 4.8, when cohesiveness is
completely lost. Nevertheless, all proteolysis indexes were
highly inuenced by pH, as very important correlations
were obtained for these parameters. Chewiness presented a
good correlation with cohesiveness, but it was more
importantly correlated with moisture content (R 70.88)
and organic acid production, particularly with LA and PA
levels (R 0.60 and R 0.43, respectively). Additionally,
chewiness was well correlated with protein content of
cheese (R 70.51); this is understandable considering that
hydrolysis and hydration of casein contribute to disinte-
gration of casein matrix, thus hardness and chewiness are
reduced.
 Downloaded by [187.208.192.54] at 21:27 18 April 2016

Table 3. Correlation coecients for the physicochemical, textural and proteolysis parameters analyzed in Panela cheese added with O type lactic acid culture and studied during storage under refrigeration
at 2.5 + 0.68C.
Tabla 3. Coecientes de correlacion de parametros sicoqumicos, textura y proteolticos analizados en queso panela adicionado con cultivo lactico tipo O y analizados durante su almacenamiento en
refrigeracion por 15 das a 2.5 + 0.68C.
Variablesa,b Hardness Springiness Cohesiveness Chewiness pH-s pHc pH-i Moisture NPN ASN EtOHSN Protein Butyric acid Propionic acid Lactic acid TN
s
Hardness 1.00
Springiness 70.32 1.00
Cohesiveness 70.06 0.48 1.00
Chewinesss 0.59 0.49 0.51 1.00
pHs 70.48 0.32 0.60 70.21 1.00
pHc 70.32 0.33 0.69 70.05 0.98* 1.00
pHi 70.35 0.16 0.62 70.20 0.98* 0.98* 1.00
Moisturex 70.76* 70.07 70.30 70.88* 0.52 0.36 0.45 1.00
NPNy 0.31 70.42 70.63 0.03 70.96* 70.98* 70.94* 70.38 1.00
ASNy 0.46 70.03 70.39 0.44 70.94* 70.88* 70.94* 70.66 0.85* 1.00
EtOHSNy 0.70 70.37 70.35 0.42 70.91* 70.82* 70.84* 70.75 0.85* 0.90* 1.00
Proteinx 70.83* 0.19 70.16 70.51 0.04 70.07 70.05 0.53 0.06 0.01 70.28 1.00
BAz 0.64 70.07 70.12 0.52 70.71 70.59 70.62 70.72 0.54 0.77* 0.72 70.19 1.00
PAz 0.74 70.23 70.50 0.43 70.91* 70.83* 70.88* 70.67 0.79* 0.89* 0.92* 70.36 0.74 1.00
LAz 0.89* 70.07 70.29 0.60 70.63 70.51 70.59 70.68 0.44 0.65 0.72 70.66 0.66 0.89* 1.00
TNx 70.83* 0.19 70.16 70.51 0.04 70.07 70.05 0.53 0.06 0.01 70.28 1.00* 70.19 70.36 70.66 1.00
Notes: *Correlations are signicant at p 5 0.05; apH-c, pH values at cheese center; pH-s, pH values at superior rind; pH-i, pH values at inferior rind; NPN, non-protein nitrogen; ASN, acid soluble nitrogen; EtOHSN, ethanol soluble
nitrogen; TN, total nitrogen; LA, lactic acid; PA, propionic acid; BA, butyric acid; RH%, relative-humidity; bExpressed as: x%; y%/TN%; zmg g71 of cheese; s(N).
Notas: *Correlaciones signicativas a p 5 0.05; apH-c, valores de pH en centro; pH-s, valores de pH en corteza superior; pH-I, valores de pH en corteza inferior; NPN, nitrogeno no proteico; ASN, nitrogeno soluble en acido; EtOHSN,
nitrogeno soluble en etanol; TN, nitrogeno total; LA, acido lactico; PA, acido propionico; BA, acido butrico; RH%, humedad relative; bExpresado como: x%; y%/TN%; zmg g71 de queso; s (N).
CyTA Journal of Food
301
 302 J.A. Guerra Martnez et al.

(2006), major changes in ASN are observed during the initial

Proteolysis assessment during storage
Crude fractionation of nitrogenous compounds of cheese was
used to determine the primary proteolysis in Panela cheese
during ageing in refrigeration conditions. Changes in
nitrogenous fractions (ASN, NPN, EtOH-SN) during storage
are presented in Figure 1.
ASN index (ASN/TN%) remained somehow constant
during the rst 7 days of storage at 46. From d 1 to d 4, it
increased at a rate of approximately 0.63 per day. From d 11
to d 13 it increased at a rate of approximately 0.23 per day
and exhibited an important increasing rate of 0.70 per day
from d 13 to d 15, reaching a maximum of 9.17 ASN/TN%
by day 15. ASN levels in this type of cheese were similar to
those reported by other authors in fresh and 21 d ripened
cheddar cheeses (Lucey, Johnson, & Horne, 2003; OMah-
ony, Lucey, & McSweeney, 2005).
ASN index showed highly signicant dierences within
15 d storage (p 5 0.01). Correlations between ASN and
physicochemical parameters provided valuable information
(Table 3). ASN presented very high negative correlations
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stages of primary proteolysis.
Changes in NPN indexes (NPN/TN%) presented highly
signicant changes during storage (p 5 0.01) and remained
below 2 by d 15. During the rst 3 days of storage NPN
increased at a rate of approximately 0.02 per day. From d 4
to d 7, it increased slightly to a rate of approximately 0.33 per
day. Afterwards and until d 15, the increase was small
reaching 1.77 NPN/TN%. As expected, NPN was highly
dependent on ASN and EtOH-SN levels due to very high
positive correlations obtained for these parameters (Table 2).
Signicant changes in EtOH-SN index (EtOH-SN/TN%)
(p 5 0.05) were observed, it showed to be highly inuenced
by pH changes but also by moisture levels. EtOH-SN barely
increased during the rst 4 d of storage at a rate of 0.036 per
day. From d 4 to d 7 it increased to 0.27 per day, afterwards
and until d 9 it increased considerably to a rate of 0.85 per
day. From d 11 to d 13 it increased even more at a rate of
1.15, remaining steady until d 15 to achieve a total 7.15
EtOH-SN/TN% by the end of the storage time. EtOH-SN
was inversely and negatively correlated as well (R 70.28)

with pH values at the three dierent cheese regions evaluated
(superior rind, center, inferior rind: R 70.94, R 70.88
and R 70.94, respectively), also high positive correlation
with NPN (R 0.85) and EtOH-SN (R 0.85). Strong
positive correlations within proteolysis indexes were reported
before (Fallico et al., 2004; Martn del Campo, Coso-
Ramrez, Picque, & Corrieu, 2007). Greater changing rates
for ASN were observed in comparison to NPN and
EtOHSN; we believe this can be attributed to early casein
hydrolysis by residual chymosin and plasmin and also to
SLAB proteolytic system, but in more extent to residual
chymosin activity. According to Upreti, Metzger and Hayes
with protein content, as expected.
All the nitrogen fractions seemed to be related to organic
acid production, especially to that of LA and PA (Table 2).
Although weak proteolysis levels were detected in high
moisture Panela cheese, it appears that in more extent
moisture is the variable that plays a major role on dening
some texture properties of these cheese and in a lesser extent,
proteolysis. Although this biochemical phenomenon is weak
in this variety, the resulting activity can be attributed to
enzymatic reactions of residual chymosin and SLAB intra-
cellular peptidases, as happens in most young cheeses. In
conclusion, ASN was the most abundant proteolytic fraction

Figure 1. Changes in acid-soluble nitrogen (ASN) (}), ethanol-soluble nitrogen (EtOHSN) (4) and non-protein nitrogen (NPN)
() indexes during 15 d of storage of Panela cheese manufactured with addition of O type starter culture.
Figura 1. Cambios en ndices de nitrogeno soluble en acido (ASN) (}), nitrogeno soluble en etanol (EtOHSN) (4) y nitrogeno no
proteico (NPN) () en queso panela adicionado con cultivo iniciador tipo O y analizado durante 15 das de almacenamiento.
 CyTA Journal of Food 303

extracted from Panela cheese; this fraction mainly contains a

mixture of high and low molecular weight peptides. ETOH-SN
and NPN contain low molecular weight peptides and free
amino acids (McSweeney & Fox, 1997). All indexes studied
showed signicant changes (p 5 0.01) throughout 15 d storage
but compared to the extent of proteolysis undergone by ripened
varieties, proteolysis in Panela cheese is limited.
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Principal component analysis (PCA)
The factorial map and the factor loading plot obtained from
PCA of the physicochemical, textural and proteolytic indexes
are shown in Figure 2((A), (B)). PC1 and PC2 explained
respectively 55.50% and 23.38% of the total variation
between all measured parameters.

Figure 2. Biplot of two principal components: PC1 and PC2: (A) Factorial map of scores of variables obtained by principal components analysis
(PCA) of samples with dierent storage days at 2.5 + 0.68C. Dx samples codes where x denotes storage day. (B) Factor loadings of
physicochemical, texture and proteolysis parameters obtained during 15 d of storage of Panela cheese manufactured with addition of O type
starter culture.
Figura 2. Graco de dos componentes principales: PC1 y PC2: (A) mapa factorial de valores obtenidos mediante analisis de componentes
principales (PCA) de muestras con diferentes das de almacenamiento a 2.5 + 0.68C. Dx codigo de muestra donde x denota el numero de das de
almacenamiento. (B) carga de factores de parametros sicoqumicos, texturales y proteolticos de queso panela obtenidos durante 15 das de
almacenamiento y con adicion de cultivo iniciador tipo O.
 304 J.A. Guerra Martnez et al.
In the factorial map (Figure 2(A)), changes in physico-
chemical, textural and proteolytic properties are dened two
groups described by PC1, one group from d 1 to d 7 and a
second group from d 9 to d 15. On the second axis, the value
of the factor coordinates which represents days of storage
decreases continuously from d 1 to d 7, then, increased from
d 9 to d 15. This behavior makes us believe that Panela
cheeses exhibit similar structural and textural changes within
each other during rst 7 d after manufacture (when most
changes in texture and chemical parameters take place).
Nevertheless, from d 9 to d 15, it seems that textural and
physicochemical changes starting at d 7 end up dening
texturally and chemically dierent cheeses by d 15, even if
they are still considered fresh. These results are similar to
those observed by Martn del Campo et al. (2007) who
measured similar physicochemical parameters and proteoly-
tic indexes in soft-mold cheeses during 27 d ripening and
observed an evolution of physicochemical parameter in two
phases, from d 1 to d 7 and from d 8 to d 27.
Factor loading plots (Figure 2(B)), showed that
most measured variables presented a strong correlation
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with PC1. Hardness (R 70.74), ASN (R 70.91), NPN
(R 70.91), EtOH-SN (R 70.96), LA (R 70.84), PA
(R 70.98) and BA (R 70.79) acids, presented strong
negative correlations with PC1, whereas pH at center (pH-c),
superior rind (pH-s), inferior rind (pH-i) and moisture
showed strong positive correlations with PC1 (R 4 0.73).
Cohesiveness, chewiness, protein content and TN were
more suitably described by PC2. Cohesiveness and chewiness
presented positive correlations (R 0.68) and (R 0.72)
respectively, while Protein and TN showed negative correla-
tions with PC2 (R 70.79).
Conclusions
The results obtained in this study show that during 15-day
storage, Panela cheeses present signicant changes in
textural and physicochemical parameters that could separate
the cheeses according to their storage time. Textural changes
were correlated with physicochemical changes even if those
changes were slight as expected for a fresh cheese. PCA
made it possible to separate the two dierent groups that
could be explained by the textural and physicochemical
changes during storage. A further study is necessary to
obtain detailed information regarding free fatty acids
prole, aroma compounds, peptides proles and sensory
attributes. It would be interesting to evaluate how
consumers perceive changes evaluated in this study in a
sensory panel evaluation.
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