Innovative Food Science and Emerging Technologies 8 (2007) 269 – 278

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Comparative study of gelation and cross-link formation during

enzymatic texturisation of leguminous proteins
Christian Schäfer a,1 , Christian Zacherl a , Karl-Heinz Engel b , Sybille Neidhart c,⁎, Reinhold Carle c
a
Fraunhofer Institute for Process Engineering and Packaging,Giggenhauser Strasse 35, 85354 Freising, Germany
b
Technical University Munich, Chair of General Food Technology, Am Forum 2, 85350 Freising-Weihenstephan, Germany
c
Hohenheim University, Institute of Food Technology, Plant Foodstuff Technology, August-von-Hartmann Strasse 3, 70599 Stuttgart, Germany
Received 4 September 2006; accepted 27 January 2007

Abstract

Isopeptide bonds that resulted from protein cross-linking, catalysed by a microbial transglutaminase (MTG), substantially contributed to the
physicochemical modification of leguminous proteins. For the development of texturised vegetable protein (TVP)-based foodstuffs using MTG,
valid methods for an efficient control of the gelation process are a prerequisite. Formation of ε-(γ-glutamyl)lysine cross-links in a simple food
model system, containing proteins from soy [Glycine max (L.) Merr.] or pea (Pisum sativum L.), was monitored by quantification of the ε-(γ-
glutamyl)lysine bond via HPLC-MS and by determining the depletion of free amino groups during cross-linking spectrophotometrically after
derivatisation with o-phthaldialdehyde (OPA). Increasing gel strengths during incubation with MTG were measured via texture analysis. The OPA
method proved too unspecific for controlling the enzymatic gelation process of leguminous proteins. Specifically for each substrate, the levels of
isopeptide cross-links, detected via HPLC-MS analysis, correlated well with the gel strength of the texturised proteins (R2 = 0.961–0.994). Rapidly
measurable, gel strength was shown to be a reliable command variable for managing MTG-induced gelation. Its use also allowed indirect
estimation of the degree of feasible cross-linking.
© 2007 Elsevier Ltd. All rights reserved.

Keywords: Texturised vegetable protein; Transglutaminase; Cross-linking; HPLC-MS; o-Phthaldialdehyde derivatisation; Texture analysis

Industrial relevance: Leguminous proteins represent a valuable alternative to animal proteins for the manufacture of texturised foodstuffs. However, due to the poor
gelling properties of the native proteins, their potential is still unexploited. For the development of TVP using MTG, simple gel strength measurements were shown
to be a valid tool for the control of the gelation process. For this purpose, unlike OPA determination of free amino groups and LC-MS analysis of isopeptide bonds,
tedious sample preparation is not required.

1. Introduction of the enzymatically modified proteins (Dickinson, 1997), such

Microbial transglutaminase (MTG, protein-glutamine γ-
glutamyltransferase, EC 2.3.2.13) catalyses the formation of
stable isopeptide bonds between protein lysyl and glutamyl
residues (De Jong & Koppelman, 2002; Yokoyama, Nio,
& Kikuchi, 2004). Through intermolecular and intramolecular
ε-(γ-glutamyl)lysine cross-links, a protein-based network is
formed, thus altering fundamentally the functional properties
⁎ Corresponding author. Tel.: +49 711 459 22317; fax: +49 711 459 24110.
E-mail address: neidhasy@uni-hohenheim.de (S. Neidhart).
1
Current address: DSM Nutritional Products Ltd., 4002 Basel, Switzerland.
1466-8564/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ifset.2007.01.005
as texture (Motoki & Kumazawa, 2000), heat stability
(Kuraishi, Yamazaki, & Susa, 2001), water binding (Kuraishi,
Sakamoto, & Soeda, 1996), rheological behaviour (Kang et al.,
1994; Chanyongvorakul et al., 1994; Larré et al., 2000),
emulsification (Motoki & Seguro, 1998), and foaming prop-
erties (Gerrard et al., 2000).
Commercial MTG preparations for food applications are
available since several years. Recent reviews (De Jong &
Koppelman, 2002; Yokoyama et al., 2004; Motoki &
Kumazawa, 2000) and consideration of patent literature
concerning MTG cross-linking technology (Dube, Schäfer,
Neidhart, & Carle, 2006) reveal that the majority of applications
refer to meat, fish, seafood, and dairy products. In fact, MTG
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cross-linking is an established process for the texturisation of
various foods, primarily in the meat and fish industry.
Moreover, enzymatic cross-linking is regarded a powerful tool
for the development of new foodstuffs based on texturised
vegetable protein (TVP) (Yokoyama et al., 2004; Zhu, Bol,
Rinzema, Tramper, & Wijngaards, 1999). However, as
reviewed by Dube et al. (2006), enzymatically texturised
plant proteins are still unexploited, apart from a few prepara-
tions derived from defatted soy and wheat flours and
concentrates or isolates thereof.
As shown for four leguminous plant proteins (Schäfer,
Schott, Brandl, Neidhart, & Carle, 2005), significant differences
in their affinity to MTG and the relative contribution of their
lysine contents to ε-(γ-glutamyl)lysine, formed under stan-
dard conditions, may even be observed among rather similar
proteins. For the potential use of alternative raw material
sources, the cross-linking behaviour of such proteins has to be
evaluated specifically and process parameters may have to be
further adjusted. Monitoring of the enzymatic reaction by fast
detection of the generated cross-links, while mimicking relevant
process conditions, is therefore crucial in product development.
Since the reaction of lysine ε-amino groups with γ-carb-
oxylamide groups of glutamine results in a depletion of free amino
groups, the formation of ε-(γ-glutamyl)lysine cross-links can be
quantified spectrophotometrically after derivatisation with o-
phthaldialdehyde (OPA) (Nielsen, Petersen, & Dambmann,
2001; Frister, Meisel, & Schlimme, 1988). This method has
been applied to determine MTG activity during cross-linking of
proteins (Dinnella, Gargaro, Rossano, & Monteleone, 2002;
Flanagan & FitzGerald, 2003). Quantification of newly formed ε-
(γ-glutamyl)lysine bonds provides an alternative way to trace
enzymatic texturisation. Accordingly, enzymatic protein digestion
followed by HPLC separation and mass-spectrometric (MS)
detection has been proposed for the quantitative determination of
ε-(γ-glutamyl)lysine isopeptide bonds in cross-linked plant
proteins (Schäfer et al., 2005). From the molar conversion rates,
both initial and average MTG activity under the given conditions
could be deduced. However, both quantitative methods require
tedious sample preparation. Enzymatic digestion is necessary prior
to HPLC separation and MS detection of isopeptide bonds,
whereas derivatisation of the analyte is indispensable before
photometric determination of derivatised amino groups. In
addition, time-consuming freeze-drying of the samples is required
in both cases.
As reported by Sakamoto, Kumazawa, and Motoki (1994),
the increase in gel strength during enzymatic cross-linking may
present a more feasible analytical tool to monitor the
transglutaminase reaction, because the gel strength can easily
be measured via consecutive texture analyses, as described for
gelatine (Association of Official Analytical Chemists, 1990).
The contributions of the generated cross-links to the textural
properties vary among protein and peptide molecules of different
size and depend on the location of the new bonds within the
molecules and networks. Thus, such a techno-functional
parameter may be of higher practical importance due to its
direct relationship with attributes of sensory relevance and the
purpose of the cross-linking process, even though simple gel
strength measurements are far from reflecting the full spectrum
of associated textural changes.
In view of the demand for both fast and reliable tools for the
monitoring of the enzymatic cross-linking reaction in the
development of TVP products, the present work aimed at the
appraisal of both techno-functional and chemical approaches
regarding the obtainable information on the response of protein
sources and attainable extents of cross-linking. For the
treatments of plant proteins with MTG, exemplarily selected
protein isolates from soy [Glycine max (L.) Merr.] and pea
(Pisum sativum L.) were used. Their gelation properties were
assessed for the comparison of the increases in gel strength with
the growing number of bonds that was deduced from chemical
approaches. For the latter purpose, the concomitant changes
in free amino groups, determined with the OPA method, and the
ε-(γ-glutamyl)lysine isopeptide bonds, quantified by HPLC-
MS after enzymatic digestion of the textured proteins, were
monitored.
2. Materials and methods
2.1. Materials
2.1.1. Chemicals
Disodium tetraborate decahydrate (borax), o-phthaldialde-
hyde (OPA) and L-serine were from Fluka, Buchs, Switzerland.
Sodium dodecyl sulphate (SDS) was obtained from Sigma-
Aldrich, Seelze, Germany. 2-Dimethylaminoethanethiol hydro-
chloride was purchased from Aldrich, Milwaukee, WI, USA.
Ethanol (absolute), boric acid, thymol (2-isopropyl-5-methyl-
phenol), trichloroacetic acid (TCA), tris(hydroxymethyl)ami-
nomethane (Tris), ferric chloride hexahydrate (FeCl3·6H2O),
hydrochloric acid (HCl), and trifluoroacetic acid (TFA) were
from VWR International, Darmstadt, Germany. Water (HPLC
grade) was obtained from Fluka, Buchs, Switzerland. Peptide
standards, such as ε-(γ-glutamyl)lysine and N-carbobenzoxy-L-
glutamyl-glycinine (Z-Gln-Gly), glutathione (reduced form),
hydroxylamine, and L-glutamic acid γ-monohydroxamate were
from Bachem, Weil am Rhein, Germany.
2.1.2. Enzymes
Microbial transglutaminase (“Activa® WM”) from Strepto-
myces mobaraensis (syn. Streptoverticillium mobaraense) was
provided by Ajinomoto, Hamburg, Germany, for enzymatic
cross-linking of the plant proteins. The powdery enzyme
preparation consisted of 99% maltodextrin and 1% active
enzyme. Following the instructions of the enzyme supplier, the
activity of the enzyme used in this study was 94.8 ± 0.7 units/g of
enzyme preparation on the basis of the colorimetric hydroxamate
assay, where one unit is defined as the amount of enzyme which
catalyses the conversion of 1 μmol of substrate into product
(hydroxamate) per min at 37 °C. MTG activity was determined
in triplicate with a UV/VIS spectrophotometer Lambda 25
(Perkin Elmer Life and Analytical Sciences, Boston, MA,
USA). In brief, MTG catalysed the formation of a glutamic acid
γ-hydroxamate from a glutaminyl residue of the substrate peptide,
which was N-carbobenzoxy-L-glutamyl-glycinine (Z-Gln-Gly,
 C. Schäfer et al. / Innovative Food Science and Emerging Technologies 8 (2007) 269–278
9.2 g/L), with hydroxylamine (6.32 g/L) as second substrate in
0.2 M Tris–HCl buffer (pH 6.0) in the presence of glutathione in
its reduced form (2.79 g/L). The reaction was stopped after 10 min
of incubation by adding 2 mL of a reagent, consisting of ferric
chloride hexahydrate (16.7 g/L) in TCA (40 g/L) and 1 N
HCl, to 2.2 mL of incubated solution. The amount of glutamic
acid γ-hydroxamate was detected as a red complex with
ferric ions at a wavelength of 525 nm, using the MTG-free
peptide sample as blank. For the five-point calibration, solutions
of L-glutamic acid γ-monohydroxamate, subjected to equivalent
complexation, were analysed by analogy.
The following enzyme preparations were used for proteolytic
digestion prior to HPLC-MS analysis according to Schäfer et al.
(2005): Pronase (7.0 units/g) from Streptomyces griseus, in the
form of a lyophilised powder, was acquired from Roche
Diagnostics, Mannheim, Germany. An ammonium sulphate
suspension of leucine aminopeptidase (77–86 units/g) and a
salt-free lyophilised powder of prolidase (194 units/g), both
from porcine kidney, as well as an aqueous suspension of
carboxypeptidase A (77–86 units/g) from bovine pancreas were
purchased from Sigma-Aldrich, St. Louis, MO, USA.
2.1.3. Protein samples
Two commercial leguminous protein isolates, derived from
soy [G. max (L.) Merr.] and pea (P. sativum L.), were used as
substrates for enzymatic cross-linking. The soy protein isolate
(SPI; “SUPRO® Ex 33”) was purchased from DuPont Protein
Technologies, Ieper, Belgium. The pea protein isolate (PPI;
“Pisane® HD”) was obtained from Cosucra, Fontenoy,
Belgium. According to the product specification provided
by the producers, protein contents of these protein isolates were
90 ± 2%, while the contents of those amino acids relevant for the
cross-linking reaction were 19.1 and 20.6 g/100 g of protein for
glutamine, calculated as glutamic acid, as well as 6.3 and 8.7 g/
100 g of protein for lysine in SPI and PPI, respectively.
2.2. Enzymatic cross-linking of proteins
Composition of the model systems was deduced from a
preliminary feasibility study, screening test conditions for
technical aspects, such as the solubility of the proteins at
concentrations up to ∼ 20% w/w and sample viscosities allowing
homogeneous distribution of the ingredients by stirring. For the
production of enzymatically texturised plant protein samples at a
protein content of 18% (w/w), a total of 360 g of PPI was
suspended in portions in 1640 mL of demineralised water, which
contained 2% (w/w) sodium chloride. SPI samples with protein
contents of 14% (w/w) were produced by analogy from 280 g of
SPI and 1720 mL of salt solution. Homogenous suspensions of
2 kg were obtained by stirring with an IKA Eurostar anchor
stirrer (Staufen, Germany) at 200–400 rpm for 30 min at 20 °C in
a transparent 5 L polypropylene beaker (Plastibrand®, VWR
International, Darmstadt, Germany). An aqueous suspension
(10% w/w) of the MTG preparation was subsequently added,
adjusting the ratio of nominal MTG activity to the protein
content of the samples to 948 units/kg, consistent with doses of
“Activa WM” and MTG of 10 and 0.1 g/kg of protein,
271
respectively. After mixing for 30 s at 20 °C by the use of the
anchor stirrer at 400 rpm, aliquots of 100 mL were immediately
transferred into 12 standard Bloom jars of 150 mL (Schott,
Mainz, Germany) and incubated in a water bath at 40 °C for 0,
10, 30, 60, 120, and 240 min to monitor the progress of cross-
linking. The characteristic dimensions of the flat-bottom jar were
85 mm of total height and 65 mm of shoulder height at diameters
of 66 mm outside, 59 mm inside, but only 41 mm inside at the
neck. After the desired incubation, enzymatic cross-linking was
stopped each time prior to further chemical and physical
analyses by subjecting the Bloom jars to heating in a water
bath at 95 °C for 30 min, a procedure that had preliminarily been
proven equivalent to a core temperature of 90 °C for 5 min. Per
incubation time, 2 Bloom jars were used for gel strength
measurements in duplicate and the following pooling of their
contents for freeze-drying and subsequent chemical analyses.
Concomitantly, further Bloom jars with aliquots of 100 mL
were investigated by analogy, but immediately after each period
of incubation at 40 °C, without prior thermal enzyme
deactivation. The comparison of the data sets obtained from
corresponding samples before and after thermal MTG deacti-
vation aimed at distinguishing potential thermal texturising
effects as a consequence of heat-induced isopeptide formation
(Sakamoto, Kumazawa, Kawajiri, & Motoki, 1995) from those
of MTG-catalysed cross-linking.
2.3. Monitoring of cross-linking by determination of free amino
groups
The samples collected at each incubation time were frozen
and stored at − 50 °C for 10–16 h to lyophilise them for 48 h in
a Beta 1–8 Christ freeze dryer (Osterode/Harz, Germany),
with supplementary drying below 25 °C and 1.03 mbar. Quan-
titative analyses of free ε- and α-amino groups were carried
out according to Nielsen et al. (2001), but with the substitu-
tion of N,N-dimethyl-2-mercaptoethylammonium chloride (2-
dimethylaminoethanethiol hydrochloride) for dithiothreitol as
thiol component as recommended by Frister et al. (1988). The
reaction products were determined at a wavelength of 340 nm
with a UV/VIS spectrophotometer Lambda 25 (Perkin Elmer
Life and Analytical Sciences, Boston, MA, USA) against
demineralised water. Based on calibration using serine
standards of 0, 25, 50, and 100 mg/L, the contents of free
amino groups were expressed as milli equivalents (meqv) of
serine-NH2 as proposed by Adler-Nissen (1986). The serine
standard of 100 mg/L corresponded to a NH2 amount of
0.9516 meqv/L. All measurements were performed in triplicate.
2.4. Enzymatic digestion of cross-linked proteins
Aliquots of the lyophilised samples, described in Section 2.3,
were additionally subjected to enzymatic digestion for quanti-
fication of ε-(γ-glutamyl)lysine in the proteolytic digests by
HPLC separation with subsequent ESI-MS detection according
to Schäfer et al. (2005). A 20 mg aliquot of the lyophilised cross-
linked protein was dissolved in 3 mL of 0.1 M borate
buffer (pH 8.0) containing a small thymol crystal. Exhaustive
272 C. Schäfer et al. / Innovative Food Science and Emerging Technologies 8 (2007) 269–278
proteolytic digestion was performed by sequential addition of
proteolytic enzymes, following the procedure of Sakamoto et al.
(1995). For each proteolytic enzyme, the ratio of enzyme activity
to leguminous protein was adjusted as detailed previously
(Schäfer et al., 2005). Deactivation of the enzymes was
performed by heating the mixtures at 100 °C for 10 min after
each incubation step. Finally, the proteolytic digests were frozen
and lyophilised as described in Section 2.3 for the cross-linked
protein samples.
2.5. Monitoring of cross-linking by HPLC-MS determination of
ε-(γ-glutamyl)lysine
HPLC-MS analysis was performed as previously described
(Schäfer et al., 2005). In brief, the total amount of each
lyophilised digest was dissolved in 7.0 mL of demineralised
water. After filtration of the solution through a PTFE syringe
filter with a pore size of 0.45 μm into an HPLC vial, an aliquot
of 20 μL was injected into a Surveyor MS HPLC system that
was connected with a LCQ Deca ion trap mass spectrometer
(Thermo Finnigan, San José, CA, USA). On a Hypersil ODS
C18 column (250 × 4.6 mm i.d., 5 μm; CS Chromatographie
Service, Langerwehe, Germany), HPLC separation of the
isopeptides from proteolytic digests was achieved by isocratic
elution at 15 °C and a flow rate of 0.6 mL/min with water,
containing 0.1% TFA. MS detection of the ε-(γ-glutamyl)lysine
isopeptide was based on electrospray ionisation (ESI). Quan-
titative determination of the isopeptide was in the MS/MS
mode by monitoring two fragmentations with m/z = 276 to 147
and m/z = 276 to 130. The first may be assigned to the
protonated lysine fragment, the latter to the deaminated lysine
fragment (Schäfer et al., 2005).
The detection limit was previously found at an isopeptide
level of approximately 50 μmol/100 g of protein (Schäfer et al.,
2005). Consistent with calibration on the basis of aqueous ε-(γ-
glutamyl)lysine standards in the range of 0.5-10 μg/mL,
isopeptide concentrations of the cross-linked samples were
expected to range from approximately 50 to 1000 μmol/100 g of
protein (Schäfer et al., 2005). Hence, the detection limit of the
HPLC-MS method was considered sufficient. ε-(γ-Glutamyl)
lysine contents were determined in duplicate for each sample.
The highly specific detection of the isopeptide via its MS/MS
fragmentation pattern enhanced sample throughput, since its
fluorimetric detection (Sakamoto et al., 1995) would require
tedious chromatographic fractionation and precolumn derivati-
sation of the proteolytic digests with OPA. Similarly, phenyl
isothiocyanate (PITC) derivatisation required for UV detection
(Sato et al., 1992) was avoided by the method used.
2.6. Texture analysis of cross-linked proteins
By penetration tests performed with a TA XTplus/5 texture
analyser [Stable Micro Systems (SMS), Surrey, UK], gel
strength of the samples was determined in duplicate after each
incubation time, both prior and past thermal MTG deactivation.
After enzymatic cross-linking of each protein isolate at 40 °C
and subsequent optional heating for MTG deactivation, 2
Bloom jars per sample were cooled in ice-water for 30 min
before texture analysis. At a gel core temperature of 20 °C,
analyses were carried out with the SMS P/0-5 probe unit,
according to a modified AOAC method for determining the gel
strength of gelatine (Association of Official Analytical
Chemists, 1990). Unlike the cited method, penetration was
extended from 4 to 20 mm, since the samples tended to produce
a superficial skin following heat deactivation of MTG. At a pre-
test speed of 10 mm/s, the plunger was moved until it reached
the sample surface, as indicated by a resistance force of 3 g
(trigger force) resulting from initial compression. Subsequent
penetration of the gel sample up to the depth of 20 mm was
performed at a constant test velocity of 0.5 mm/s. From the
penetration forces occurring during this phase, gel strength was
calculated as the average penetration load (in N/cm2), using the
SMS texture analysis software Exponent.
2.7. Statistical analyses
Statistical dispersion of arithmetic means was indicated
by their standard deviations for measurements in triplicate and
by their average absolute deviations from the means for those
in duplicate. For correlation analysis between selected pairs
of parameters, linear regression based on the method of
least squares was calculated, using the RGP and F-statistic
functions of Microsoft® Office Excel 2003. Significance of the
coefficients of determination (R2) were indicated based on
P = 0.05 (R2 ⁎), 0.01 (R2 ⁎⁎), and 0.001 (R2 ⁎⁎⁎).
3. Results and discussion
3.1. Gel strength of cross-linked leguminous protein systems
As shown by the comparison of the respective samples after
0 and 240 min of incubation with MTG and subsequent thermal
enzyme deactivation (Fig. 1), cross-linking of SPI caused a total
increase in gel strength of 155% to a final level of 1.6 N/cm2,
whilst the firmness of analogously treated PPI gels even rose by
300% to 2.9 N/cm2. Achievable gel strengths depended on both
the protein source and the concentration of the protein isolates
used for texturisation. Both higher initial gel firmness and faster
progress in texture development of PPI systems may partly be
ascribed to their elevated protein content.
To control the gel strength of texturised foods, precise
termination of gelation by MTG deactivation after an adequate
reaction time is a prerequisite. However, heat-induced formation
of protein cross-links, such as those based on ε-(γ-glutamyl)
lysine (Otterburn, 1983; Sakamoto et al., 1995), lysinoalanine
(Friedman, 1999; Schwass & Finley, 1984), histidinoalanine
(Lauber, Klostermeyer, & Henle, 2001), and further cross-linked
amino acids (Maga, 1984), may occur under the given thermal
conditions (90 °C, 5 min). Furthermore, gelling of proteins
through the formation of disulphide bridges has been described
(Ma & Harwalker, 1992; Boye, Ma, & Harwalker, 1997;
Oakenfull, Pearce, & Burley, 1997; Acton & Dick, 1989).
To distinguish the texturising effects of thermal mechanisms
from those of MTG-catalysed cross-linking, non-thermally
 C. Schäfer et al. / Innovative Food Science and Emerging Technologies 8 (2007) 269–278 273

(Fig. 2). For both leguminous protein isolates, MTG-catalysed

Fig. 1. Gel strengths (GS) of cross-linked protein isolates measured before (GSE;
open symbols) and after (GSE/H; filled symbols) thermal MTG deactivation,
respectively. PPI: 18% w/w (rhombs); SPI: 14% w/w (circles); MTG: 948 units/
kg of protein, NaCl 2%. Error bars: average absolute deviation from the mean.
cross-linking included initial linear accumulation of the
isopeptide bonds up to 120 min of incubation, irrespective of
thermal enzyme deactivation or its omission prior to analysis.
The initial reaction rates, i.e. the molar rates of isopeptide
formation under the test conditions, were thus calculated from
the average slopes of the graphs within this incubation period.
In systems analysed after thermal MTG deactivation, they
amounted to ε-(γ-glutamyl)lysine accumulation rates of
approximately 16 and 11 μmol/min per kg of protein for PPI
and SPI, respectively. Based on an enzyme dose of 10 g of the
enzyme preparation “Activa WM” (i.e., 0.1 g of MTG) per kg of
protein, corresponding to 948 units/kg, the respective substrate-
specific activities of the microbial transglutaminase used were
1.6 and 1.1 μmol min− 1 g− 1 of “Activa WM” under the applied
conditions (40 °C). After this initial linear phase, plateaus at
maximum ε-(γ-glutamyl)lysine contents were mostly reached
until 240 min of incubation, as particularly shown by systems
without thermal MTG deactivation prior to analysis.
When ε-(γ-glutamyl)lysine formation (Fig. 2) was compared
with the textural development of heated PPI and SPI samples
(Fig. 1) during the first 120 min, the 3.5-fold growth rate of the
gel strength in the SPI system resulted for that of PPI, whereas
the rates of concurrent ε-(γ-glutamyl)lysine formation were in a
similar range for both proteins. Indeed, MTG-induced ε-(γ-
glutamyl)lysine formation was slightly faster, when PPI was

treated samples were compared with heated ones. Although

MTG was still active during texture analysis, when the
enzymatically cross-linked PPI and SPI samples were analysed
without prior thermal deactivation step, considerably lower gel
strengths (GSE) were recorded than for the cross-linked systems
after heating (GSE/H) (Fig. 1). At the beginning of incubation
with MTG, gel strength measured after the deactivation step
(GS E/H ) was approximately 10 times higher than the
corresponding one determined without the thermal treatment
(GSE), irrespective of the protein isolate. The two graphs, which
corresponded to the monitoring of gelation after thermal MTG
deactivation or with its omission, almost paralleled per protein
isolate in Fig. 1. From 10 to 240 min of incubation with MTG,
the GSE/H/GSE ratio hardly decreased from 6.5 and 7.5 to 1.3
and 2.8 for PPI and SPI, respectively. Therefore, it could be
concluded that an additive contribution of heat-induced firming
to enzymatically generated gel strength resulted from thermal
MTG deactivation. This effect was virtually comparable for
both protein sources. It may largely be ascribed to additional
cross-links generated non-enzymatically during heating, even
though some minor increase in dry matter and a superficial skin
as a consequence of possible water loss on the sample surface
during the deactivation step cannot be excluded under the given
experimental conditions. Fig. 2. Contents of ε-(γ-glutamyl)lysine isopeptide (IPC) in cross-linked
protein isolates quantified before (IPCE; open symbols) and after (IPCE/H;
3.2. Isopeptide contents of cross-linked leguminous protein filled symbols) thermal MTG deactivation. PPI: 18% w/w (rhombs); SPI: 14%
systems w/w (circles); MTG: 948 units/kg of protein, NaCl 2%. Error bars: average
absolute deviation from the mean. Slopes, as ε-(γ-glutamyl)lysine isopeptide
contents per time unit between 0 and 120 min, were (⋄) 21 μmol kg− 1 min− 1
The ε-(γ-glutamyl)lysine contents of the cross-linked and (♦) 16 μmol kg− 1 min− 1 for PPI as well as (○) 14 μmol kg− 1 min− 1 and (●)
proteins were quantified for each incubation time studied 11 μmol kg− 1 min− 1 for SPI.
274 C. Schäfer et al. / Innovative Food Science and Emerging Technologies 8 (2007) 269–278
Fig. 3. Concentrations of free amino groups in cross-linked protein isolates
analysed before (open symbols) and after (filled symbols) thermal MTG
deactivation. PPI: 18% w/w (rhombs); SPI: 14% w/w (circles); MTG: 948 units/
kg of protein, NaCl 2%. Error bars: standard deviation.
used as substrate rather than SPI (Fig. 2), but resulted in
disproportionately faster setting of samples from the former
protein isolate (Fig. 1). The amounts of newly formed ε-(γ-
glutamyl)lysine cross-links were comparable after identical
reaction times. For example, after 240 min of incubation and
subsequent MTG deactivation, ε-(γ-glutamyl)lysine levels of
225 and 192 μmol/100 g of protein were found for the PPI and
SPI systems, respectively, whereas corresponding gel firmness
ranged at 2.9 N/cm2 for the PPI sample but only at 1.6 N/cm2
for SPI (Fig. 1). Since the total ε-(γ-glutamyl)lysine content
increased likewise by ∼200 μmol/100 g of protein during
240 min of incubation, irrespective of the protein isolate
(Fig. 2), bonding sites for MTG were assumed to be more easily
accessible in pea protein, especially during the linear phase of
the cross-linking reaction (0–120 min). Superior gel firmness of
PPI samples may not exclusively be attributed to the higher
concentration of pea protein (18%), which exceeded that of
the soy protein by 4%. It is more likely that different positions
of ε-(γ-glutamyl)lysine cross-links, generated in PPI, have a
greater impact on gelation than in the case of SPI. Increased
average molecular mass of the protein substrate would
additionally favour initial gel strength. However, similar initial
gel strength (Fig. 1) may indicate rather resembling molecular
size properties of the substrates used.
Easily accessible reaction sites for transglutaminase (bovine
plasma factor XIII) were localised in the non-repetitive domain
of γ-gliadin, whereas chemical deamidation affected amino
residues of both the repetitive and the non-repetitive domain of
the protein (Chobert et al., 1996). Using bovine caseins,
Christensen, Sørensen, Højrup, Petersen, and Rasmussen
(1996) reported limitation in the number of potential transglu-
taminase-reactive glutamine residues, despite relatively high
glutamine contents. For PPI and SPI, respectively, 0.22 and
0.21% of the glutamyl residues as well as 0.51 and 0.62% of the
lysyl residues were involved in the ε-(γ-glutamyl)lysine cross-
links detected after 240 min of incubation with subsequent MTG
deactivation.
By modifying the protein structure, accessibility of trans-
glutaminase, derived from guinea pig liver, to the reaction sites
of pea legumin could be enhanced (Larré, Chiarello, Dudek,
Chenu, & Gueguen, 1993). Modified legumin substrates were
obtained by chromatographic separation of subunits or by
complete dissociation of the protein through partial acylation of
its amino groups. Furthermore, the peculiarities of oligomeric
legume 7S and 11S storage proteins are of importance among
the complex of structural features of the proteins influencing
functionality, in particular protein surface and conformation
(Schwenke, 2001). Conformation and amino acid sequences of
globular proteins from pea (Subirade, Pezolet, & Gueguen,
1993; Boulter, 1983; Gatehouse, Croy, & Boulter, 1984) and
soy (Mizuno, Mitsuiki, Motoki, Ebisawa, & Suzuki, 2000;
Renkema, 2004) showed a great variation, even within the same
species. Moreover, secondary structures of seed storage
proteins, such as α-helix or β-structure (Pernollet & Mossé,
1983), may be affected by protein denaturation (Sikorski,
1997), e.g. heat treatments during production processes. Major
modifications of the protein structures, such as dissociation of
the oligomers (quaternary structure) into subunits, unfolding of
the tertiary structures through ruptured disulphide bridges and
ionic bonds, and uncoiling of secondary structures (α-helix or
β-structure), may be caused by different factors like shift in pH,
change in temperature and ionic strength, and addition of
various detergents or solvents. Extreme heat conditions
(Sikorski, 1997) and enzymatic or chemical hydrolysis
(Adler-Nissen, 1986) may even affect the primary structure
and break down the amino acid sequence. In a recent study of
preheating conditions influencing MTG-catalysed cross-linking
reactions of β-caseins, αs-caseins, κ-casein, and whey proteins
(β-lactoglobulin and α-lactalbumin), Rodriguez-Nogales (2006)
could identify optimum parameters for preheating of cows' milk
at 84.5 °C for 60 min.
In the light of the manifold factors influencing protein
conformation, broad variation in the accessibility of reaction
sites is quite evident. As a consequence, the highly specific
affinity of a protein towards MTG has to be verified for each
individual substrate experimentally. However, diverse conclu-
sions are likely from the available methods, as shown by the
different rates of gel strength development and isopeptide
formation that were observed for each substrate. The degree, at
which the information, obtainable from those methods regarding
the response to MTG, correlates for given protein substrates, is
of practical importance. Similarly, an improved understanding of
occurring side-effects, influencing the results of each method, is
needed.
Unlike gel strength (Fig. 1), ε-(γ-glutamyl)lysine isopeptide
contents of the gels were higher in thermally untreated samples
 C. Schäfer et al. / Innovative Food Science and Emerging Technologies 8 (2007) 269–278
(Fig. 2), involving 0.31 and 0.28% of the glutamyl residues as
well as 0.73 and 0.85% of the lysyl residues available in PPI and
SPI, respectively, after 240 min of incubation. Average IPCE/H/
IPCE ratios of isopeptide contents of heated (IPCE/H) and
unheated (IPCE) samples were 0.61 ± 0.10 and 0.50 ± 0.18 for
PPI and SPI, respectively. Without the previous heating step,
minor MTG activity, persisting at very low temperatures until
complete sample freezing, seemed to cause considerable
isopeptide bonds during sample preparation for chemical
analyses, whereas active MTG during the rather short firmness
measurements performed immediately after incubation hardly
contributed to overall isopeptide formation. Furthermore,
residual MTG activity, after lyophilisation of the unheated
gels, may result in a progressing cross-linking reaction after
rehydration of the samples for proteolytic digestion prior
to HPLC-MS analysis. At least, the heating step used for
MTG deactivation obviously contributed far less to the for-
mation of ε-(γ-glutamyl)lysine than previously expected
(Sakamoto et al., 1995) and its improvement of gel firmness,
observed under the given conditions, was due to other reasons
discussed in Section 3.1.
3.3. Concentrations of free amino groups
When PPI and SPI were compared in terms of the concen-
trations of free amino groups as determined by the OPA method,
clear differences were observed between the protein sources.
The depletion of free amino groups was greater in cross-linked
SPI than in PPI (Fig. 3). Since both properties changed with
radically different rates during individual phases of incubation
(Figs. 2 and 3), interrelation between decreasing free amino
groups (Fig. 3) and rising ε-(γ-glutamyl)lysine isopeptide bonds
(Fig. 2) was poor, especially for SPI. Unlike the significant
influence of the thermal deactivation step on the detectable
isopeptide contents (Fig. 2), the time-dependent depletion of free
amino groups was merely characteristic of the individual protein
substrate, as indicated by almost congruent graphs for the
respective heated and unheated samples (Fig. 3). Obviously,
neither the heat treatment on the one hand nor ongoing MTG
catalysis during sample preparation on the other hand did
significantly contribute to the depletion of free amino groups.
Different rates were observed for SPI and PPI, both at the
beginning of the reaction and throughout incubation. In the
course of cross-linking via MTG, the overall depletion of
free amino groups was significantly higher for SPI than for PPI
(Fig. 3). The enzymatic conversion of PPI ended in a plateau, as
previously detected via gel strength and isopeptide levels
(Figs. 1 and 2). However, the content of free amino groups
from SPI samples dropped disproportionately with proceeding
gelation, in view of the plateaus shown after 120–240 min of
incubation by analyses of gel strength and ε-(γ-glutamyl)lysine.
Correlation of cross-linking lysine residues with decreasing
free amino groups was expected, but could be superposed by
reactions of lysine ε-amino groups that were different from
MTG-induced ε-(γ-glutamyl)lysine formation. The excessive
decrease of free amino groups in polymerising SPI was also
attributed to masking of amino groups within the gel network,
275
making them inaccessible to the OPA reagent, consistent with
the findings of Flanagan and FitzGerald (2003) for MTG-
induced cross-linking of sodium caseinate.
On the whole, the less specific OPA method did not allow the
precise determination of MTG activity, unlike the specific
determination of isopeptide bonds. In particular, the formation of
lysinoalanine (Friedman, 1999; Schwass & Finley, 1984), the
reaction with proteinaceous asparagine to form the ε-(β-
aspartyl)lysine-isopeptide (Asquith, Otterburn, & Sinclair,
1974), and reactions with reducing sugars to form Maillard
compounds (Hurrell, Carpenter, Sinclair, Otterburn, & Asquith,
1976) cannot be distinguished from the MTG reaction, when
applying the OPA method. As carbohydrate contents amounted
to 1 and 4.5 g/100 g of SPI and PPI, respectively, according to
suppliers' information, occurrence of reducing sugars is likely.
Hence, the OPA method was unsuitable for monitoring of
MTG-induced texturisation of PPI and SPI, in contrast to the
report of Dinnella et al. (2002), where the OPA method was used
for the determination of MTG activity to study the cross-linking
kinetics for casein. Dinnella et al. (2002) performed MTG cross-
linking, additionally using the small-size acyl donor N-carbo-
benzoxy-glutamyl-glycinine (Z-Gln-Gly) as easily accessible
dipeptide to minimize steric hindrance. When complex legumi-
nous proteins are to be applied as ingredients of food-like systems,
as in this study, steric hindrance of MTG and numerous com-
petitive reactions of free amino groups are much more probable.
3.4. Monitoring of the cross-linking process
The present work confirmed the substantial contribution of
MTG-catalysed formation of ε-(γ-glutamyl)lysine-isopeptide
bonds to the generation of gel structures for leguminous
proteins. Isopeptide contents and gel strength were significantly
correlated, as shown by coefficients of determination (R2) in the
range of 0.961-0.994 that characterised the protein-specific
linear regression equations between both parameters (Fig. 4A).
Irrespective of the different contributions of heat during thermal
MTG deactivation prior to analysis and of residual enzyme
activities in the analysis of unheated samples, the same
characteristic relationships between isopeptide bonds and gel
strength were found for heated and unheated samples of the
respective proteins, as expressed by slopes of the graphs in
Fig. 4A of 0.073–0.093 N kg μmol− 1 cm− 2 for PPI and 0.03–
0.043 N kg μmol− 1 cm− 2 for SPI.
For a more precise evaluation of the degree of cross-linking,
ε-(γ-glutamyl)lysine contents and gel strengths of the samples,
studied after thermal MTG deactivation, were normalised
relative to the respective maximum values that were set at
100% (Fig. 4B). With xo and xs referring to the respective
values at the beginning of cross-linking and to their maxima,
normalised values y were calculated from the recordings of gel
strength and isopeptide contents according to Eq. (1).
y ¼ ðx−x0 Þ=ðxs −x0 Þd 100% ð1Þ
During cross-linking, a substrate-specific saturation stage
may finally be reached when all the reaction sites, which are
276 C. Schäfer et al. / Innovative Food Science and Emerging Technologies 8 (2007) 269–278
Fig. 4. Correlation of gel strength (GS) and ε-γ-(glutamyl)lysine isopeptide
contents (IPC). A. Linear regression of the type GS = s · IPC +c between both
parameters for cross-linked PPI (18% w/w; rhombs) and SPI (14% w/w; circles)
analysed before (open symbols) and after (filled symbols) thermal MTG
deactivation, with slopes s in N kg μmol− 1 cm− 2, constants c in N cm− 2, and
coefficient of determination R2: (⋄) 0.073, −1.2331, 0.961⁎; (♦) 0.093, 0.0778,
0.970 ⁎; (○) 0.030, − 0.5468, 0.986 ⁎⁎ ; (●) 0.043, 0.4326, 0.994 ⁎⁎ .
B. Development of normalised values of IPC (triangles) and corresponding GS
(squares) during cross-linking of PPI (black symbols) and SPI (grey symbols)
analysed after thermal MTG deactivation. MTG: 948 units/kg of protein, NaCl 2%.
accessible before the onset of syneresis, would have been cross-
linked. Assuming that monitoring is performed until this stage,
the characteristic percentage of feasible cross-linking could be
deduced for each proteinaceous substrate from its amino acid
composition and the reached isopeptide level. By contrast, a
merely theoretical prediction of the maximum degree of cross-
linking from the glutamine and lysine contents of the protein
was deemed to be unrealistic.
Comparison of the normalised values for PPI and SPI
(Fig. 4B) revealed good substrate-specific correlations between
the concentration of ε-(γ-glutamyl)lysine isopeptide links and
the resulting gel strengths. Both parameters approached to a
maximum during incubation, even though sampling intervals
did not allow the identification of a true saturation stage.
However, proportional increase in normalised gel strength (y2)
with rising normalised isopeptide contents (y1) was documented
by linear regression between y1 and y2, with R2 of 0.983⁎⁎⁎
(PPI) and 0.995⁎⁎⁎ (SPI) as well as slopes m of 1.0039 and
1.0098 for PPI and SPI, respectively (lines through origin of the
type y1 = m·y2 not shown). Therefore, gel firmness can be
considered a suitable indirect command variable in managing
MTG-catalysed cross-linking of protein isolates in practice.
Apart from being rapid and convenient, the suggested texture
measurements only require simple laboratory equipment.
In product development, the use of gel strength as techno-
functional command variable, instead of chemical attributes of
the MTG-catalysed reaction, for the control of incubation time
facilitates the adjustment of the product properties to the desired
profile during the identification of suitable substrates and
operating conditions among alternative protein sources. High
amounts of ε-(γ-glutamyl)lysine cross-links do not necessarily
result in high values for gel strength, as previously shown for a
lupin protein in comparison with other substrates (Schäfer et al.,
2005). Particularly, in very heterogeneous substrates, smaller
peptides accessible for MTG may considerably contribute to the
total isopeptide content, but with poor firming effect. For the
type of correlation shown in Fig. 4, only relationships that are
highly specific for each individual substrate can be expected.
However, in routine application of a well-specified protein
substrate in food processing, the instantaneous level of feasible
enzymatic cross-linking could be estimated for any incubation
time, according to Eq. (1) as percentage of the gel strength at the
saturation stage, from the characteristic firmness development
under the operating conditions used.
4. Conclusions
MTG-induced formation of ε-(γ-glutamyl)lysine cross-links
significantly enhanced the gelation of pea and soy protein
isolates in the food model used. However, depletion of free
amino groups, as determined by the OPA method, proved
unspecific and inaccurate in evaluating the levels of MTG-
induced ε-(γ-glutamyl)lysine isopeptides of cross-linked pro-
tein isolates studied. This was ascribed to interfering reactions
of the lysine ε-amino groups, e.g. with reducing sugars or amino
acids, and inaccessibility of free amino groups to the OPA
reagent due to polymerisation.
 C. Schäfer et al. / Innovative Food Science and Emerging Technologies 8 (2007) 269–278
Precise monitoring of cross-linking was enabled by quanti-
fication of the isopeptide cross-links by HPLC-MS detection
after proteolytic digestion of the samples. The contents of ε-(γ-
glutamyl)lysine cross-links correlated well, but specifically for
each substrate, with the gel strength development traced by
texture analysis. A rapid and convenient method for the
measurement of gel strengths was shown to be suitable for
indirect estimation of instantaneous levels of feasible cross-
linking in enzymatically texturised leguminous proteins.
Acknowledgements
This research project was supported by the FEI (Forschungs-
kreis der Ernährungsindustrie e. V., Bonn, Germany), the AiF
and the German Ministry of Economics and Labour. AiF-
Project No.: 13177N.
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