International Dairy Journal xxx (2013) 1e5

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International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj

Cross-linking with microbial transglutaminase: Relationship between

polymerisation degree and stiffness of acid casein gels
Doris Jaros a, *, Uwe Schwarzenbolz b, Norbert Raak a, Jürgen Löbner b, Thomas Henle b,
Harald Rohm a
a
Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, Bergstraße 120, D-01069 Dresden, Germany
b
Department of Chemistry and Food Chemistry, Technische Universität Dresden, Bergstraße 66, D-01069 Dresden, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Acid casein in phosphate buffer was used as a simplified substrate for cross-linking experiments with
Received 26 August 2013 microbial transglutaminase. The importance of the intensity of enzyme treatment on the rheology of acid
Received in revised form gels was evaluated by preparing mixtures of casein that were cross-linked to a different extent and a
15 October 2013
native reference to obtain samples with a similar polymerisation degree. The stiffness of acid gels from
Accepted 15 October 2013
caseins incubated longer then 3 h was always lower at identical polymerisation degree larger than
approximately 30%. The quantification of isopeptides formed by the enzyme reveals a substantial in-
crease of bonds at a stage of cross-linking where only 10% of monomers are available. This indicates that
cross-linking must occur between and/or within already existing aggregates, which become larger and
less flexible, and supports the hypothesis that, starting with a critical aggregate size the network for-
mation is somewhat impaired by the size and reduced flexibility of the proteins.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
Microbial transglutaminase (mTGase, EC 2.3.2.13) catalyses acyl
transfer reactions between the g-carboxyamide groups of peptide
or protein bound glutamine and primary amines, including the
3-amino group of lysine. In casein, an excellent substrate for
mTGase (Huppertz & de Kruif, 2008; Tang, Yang, Chen, Wu, & Peng,
2005), covalent intra- or intermolecular cross-links are formed
(Partanen, Forssell, Mackie, & Blomberg, 2013). The protein modi-
fication enhances firmness and improves other physical and texture
properties of acid gels made from mTGase treated milk (Buchert
et al., 2007; Jaros, Partschefeld, Henle, & Rohm, 2006a), but also
affects the kinetics of gel formation (Jacob, Nöbel, Jaros, & Rohm,
2011). Casein cross-linking is frequently quantified by using pro-
tein separation methods such as semi-quantitative electrophoresis
(Heber, Paasch, Partschefeld, Henle, & Brunner, 2012; Macedo,
Fazani Cavallieri, Lopes da Cunha, & Sato, 2010; Myllärinen,
Buchert, & Autio, 2007) or gel permeation chromatography (GPC).
Depending on the resolution of the latter technique, it is possible to
separate casein monomers and cross-linked dimers from fractions
comprising trimers and larger aggregates (Bönisch, Lauber, &
Kulozik, 2004; Lauber, Henle, & Klostermeyer, 2000).
* Corresponding author. Tel.: þ49 35146339283.
E-mail address: doris.jaros@tu-dresden.de (D. Jaros).
We have shown in a previous study (Jaros, Jacob, Otto, & Rohm,
2010) that the stiffness of gels that are made from milk or casein
solutions by chemical acidification with glucono-d-lactone (GDL)
increases with increasing reaction time of mTGase until a
maximum stiffness is reached. Upon exceeding this maximum,
which was approximately at 75% residual casein monomers for raw
skim milk and 20e30% residual monomers for casein (corre-
sponding to a polymerisation degree calculated from GPC of 25%
and 70e80%, respectively), gel stiffness started to decrease. Based
on these results, Jaros et al. (2010) proposed the hypothesis that
large casein aggregates in gelling systems sterically hinder proper
network formation by impairing the mobility of the smaller casein
molecules. Additional factors that are affected by the degree of
cross-linking are the pH at which the systems start to gel and the
velocity of gel stiffening as a function of time (gelation rate) (Jacob
et al., 2011). Recently we showed that the change of pH over time
(acidification rate), which may be influenced by acidification tem-
perature and GDL concentration, is also an important factor for the
stiffness of gels from cross-linked casein (Rohm, Ullrich, Schmidt,
Löbner, & Jaros, 2013).
Another important aspect that has to be considered in the
interpretation of mTGase induced effects on the physical properties
of acid milk gels is the number and the distribution of the 3-(g-
glutamyl)-lysine isopeptide bonds (GlueLys isopeptides). Only a
few studies addressed the reaction sites for intra- or intermolecular

0958-6946/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.idairyj.2013.10.011

Please cite this article in press as: Jaros, D., et al., Cross-linking with microbial transglutaminase: Relationship between polymerisation degree
and stiffness of acid casein gels, International Dairy Journal (2013), http://dx.doi.org/10.1016/j.idairyj.2013.10.011
2 D. Jaros et al. / International Dairy Journal xxx (2013) 1e5

cross-linking of casein (Christensen, Sørensen, Højrup, Petersen, & Germany) at room temperature. Details of the urea-containing
Rasmussen, 1996; Lilla et al., 2012; Menendez, Schwarzenbolz, buffer can be found elsewhere (Jaros et al., 2010). The fractions of
Partschefeld, & Henle, 2009), and isopeptide concentration (IC) monomers, dimers and polymers were calculated from the GPC
after mTGase incubation of milk was determined by Bönisch et al. peaks by relating the respective peak areas Ai to the sum of peak
(2004) and Lauber et al. (2000). Lauber et al. (2000) calculated areas. The degree of polymerisation is defined by:
the concentration of cross-linked amino acids from GPC results,
  1
which was in good agreement with measured IC. In these studies, PDð%Þ ¼ 100 Adimer þ Apolymer Amonomer þ Adimer þApolymer :
however, incubation was terminated after 120e150 min, and no
conclusions were drawn towards a dependency of gel properties on
that measure.
2.4. Determination of the N-3 -(g-L-Glutamyl)-L-Lysine isopeptides
The aim of the present study is to corroborate the hypothesis
that it is not the polymerisation degree alone but rather the size of
The samples were subjected to a three-step enzymatic hydro-
the cross-linked casein aggregates, as indicated by the quantity of
lysis as described previously (Henle, Walter, & Klostermeyer, 1991),
isopeptide bonds, that determines the rheology of acid gels. For this
with pepsin, pronase E and aminopeptidase M with prolidase to
purpose, mixtures were prepared from casein that had been
obtain free isopeptides, followed by amino acid analysis according
enzyme treated for different amounts of time with reference casein
to Lauber et al. (2000). The GlueLys concentration was quantified
(0 h) to obtain samples with a similar polymerisation degree (PD).
from the peak areas and is, by considering its molar mass, further
Acidification was induced by GDL, and the maximum storage
expressed as isopeptide concentration (IC) in g isopeptides 100 g1
modulus extracted from gelation curves of small deformation ex-
casein.
periments is discussed in relation with casein PD and GlueLys
isopeptide concentration.
3. Results and discussion
2. Materials and methods
3.1. Effects of polymerisation degree and isopeptide bonds on gel
stiffness
2.1. Substrate and enzyme treatment

Fig. 1 shows the stiffness of the casein acid gels as a function of

Commercial acid casein (SigmaeAldrich Chemie GmbH, Stein-
protein polymerisation, and also as a function of the isopeptide
heim, Germany) was dissolved in 0.1 mol L1 phosphate buffer (pH
content. The dependency of G0max on PD, which is, at constant
6.8) and mixed with a magnetic stirrer at room temperature over-
mTGase concentration and incubation temperature, determined
night. The target concentration of the casein solutions was
only by incubation time, follows the typical pattern that has been
27 g kg1.
reported in previous studies for both the casein solution and milk
Microbial transglutaminase Activa MP from Streptomyces
(Jaros et al., 2010; Jaros, Pätzold, Schwarzenbolz, & Rohm, 2006b):
mobaraensis (Ajinomoto Foods Deutschland GmbH, Hamburg,
gel stiffness increases until a PD of approximately 85% (3 h incu-
Germany) with an activity of 100 U g1 was used to cross-link
bation). If incubation time is prolonged, the stiffness of the acid
casein at 40  C (3 U g1 casein) and was inactivated after pre-
casein gels decreases. In this study incubation for 8, 20 and 24 h
defined periods of time by heating to 85  C and holding the tem-
resulted in PD values of 94, 97, and 98%, respectively. Jaros et al.
perature for 3 min. Incubation time was set to 1, 2, 3, 8, 20, and 24 h;
(2010) interpreted the decrease with the presence of large poly-
the reference sample without added enzyme (referred to as incu-
meric aggregates that partly impair gelation and prevent the for-
bation time of 0 h) was treated in the same way.
mation of a homogeneous network.
However, when considering gel stiffness versus isopeptide
2.2. Small strain oscillation experiments
content it can be seen that, for an IC w0.08 g 100 g1, G0max is
Gelation was monitored using a strain-controlled ARES RFS3
rheometer (TA Instruments, Eschborn, Germany). The instrument
was equipped with a cup-and-bob geometry (di ¼ 32 mm;
do ¼ 34 mm; h ¼ 33.5 mm). Measuring temperature was kept
constant at 30  0.1  C by a computer-controlled circulator. After
adding 40 g L1 glucono-d-lactone (GDL) and stirring for 20 s, the
sample was loaded into the geometry of the rheometer. Time-based
measurements were started immediately at constant strain of
0.003 and constant frequency of 1 rad s1, and the storage modulus,
G0 (Pa), was recorded. The maximum of the storage modulus, G0max
(Pa), was extracted from the curves and is further used as indicator
for gel stiffness. All measurements were done in duplicate.

2.3. Determination of protein polymerisation

Casein polymerisation was determined by gel permeation

chromatography after diluting samples with a dithiothreitol con-
taining elution buffer to a protein content of 2 g L1. Prior to in-
jection, the samples were filtered through membrane filters with a
pore size of 0.45 mm. Analyses were carried out at a flow rate of
Fig. 1. Gel stiffness ðG0max Þ extracted from gelation curves of 27 g kg1 casein in
0.5 mL min1 with a Superdex 200 HR column (Pharmacia, Frei- phosphate buffer (pH 6.8) as a function of casein polymerisation (open circles) and
burg, Germany) and a Smartline UV detector 2500 (Wissen- isopeptide content (closed circles). Enzyme treatment was with 3 U mTGase g1
schaftliche Gerätebau Dr. Ing. Herbert Knauer GmbH, Berlin, protein at 40  C for the specified time. Identical samples are connected by dotted lines.

Please cite this article in press as: Jaros, D., et al., Cross-linking with microbial transglutaminase: Relationship between polymerisation degree
and stiffness of acid casein gels, International Dairy Journal (2013), http://dx.doi.org/10.1016/j.idairyj.2013.10.011
 D. Jaros et al. / International Dairy Journal xxx (2013) 1e5
increased by approximately one order of magnitude, and that the
highest gel stiffness of 295 Pa is reached at IC ¼ 0.146 g 100 g1. On
further incubation, the isopeptide content still increases, and the
samples that were treated with mTGase for 8, 20 or 24 h can be
clearly distinguished by this measure.
How can the differences in the time-dependent progress be-
tween casein polymerisation and isopeptide content be inter-
preted? The amount of uncross-linked casein monomers is
described by (100PD)%, and the IC gives information on the total
number of covalent cross-links per unit protein. The time course of
casein polymerisation and isopeptide formation is depicted in Fig. 2.
A PD of 12% was calculated for the uncross-linked reference casein
from 8% dimers and 4% polymers that were present in the GPC.
However, in that sample, we did not detect any GlueLys isopeptides,
indicating that these bonds have to be caused by factors (e.g., heating
during production of the powder) other than mTGase (Bönisch,
Tolkach, & Kulozik, 2006; Henle, Schwarzenbolz, & Klostermeyer,
1996). There exists only rudimentary information on how mTGase
proceeds in cross-linking. After a rapid decrease of monomers
within the first hour of cross-linking, the process slowed down
considerably at a stage where approximately 35% monomers
remained. In the first 2 h of cross-linking there seems to be a linear
relationship between PD and IC up to a PD of w80% (see insert to
Fig. 2). However, it has to be mentioned that GlueLys isopeptides
were detected in a monomer fraction of casein treated for 1 h with
mTGase, which had been separated by preparative GPC. This in-
dicates that intramolecular cross-linking occurs even in the early
stage of enzyme action (data not shown). In a previous study we
reported amounts of dimers of 8, 22, 19 and 6% in the 0, 0.5, 1 and 3 h
sample, respectively (Rohm et al., 2013), proving that dimers also
serve as substrate for further cross-linking.
Together with a pronounced increase of isopeptides starting at a
stage where less than 10% monomers are present, this leads to the
conclusion that (a) the size of the polymeric casein aggregates further
increases, and/or (b) additional cross-links within already existing
aggregates are formed. Based on the molecular mass of GlueLys of
275 Da, and an average molecular mass of casein of 24,000 Da
(Walstra, Wouters, & Geurts, 2006), an IC of 0.4 g 100 g1 casein in the
sample cross-linked for 24 h is equivalent to approximately 0.35 mol
GlueLys per mol casein. This is lower than data from Mautner, Meisel,
Lorenzen, and Schlimme (1999) for casein, but in line with values
reported for cross-linked milk (Lauber et al., 2000).
Fig. 2. Casein polymerisation degree (PD, open circles) and isopeptide content (IC,
closed circles) of 27 g kg1 casein in phosphate buffer (pH 6.8) as a function of incu-
bation time with 3 U mTGase g1 protein at 40  C. Insert: IC versus PD.
Please cite this article in press as: Jaros, D., et al., Cross-linking with microbial transglutaminase: Relationship between polymerisation degree
and stiffness of acid casein gels, International Dairy Journal (2013), http://dx.doi.org/10.1016/j.idairyj.2013.10.011
3
3.2. Mixtures of casein with identical degree of polymerisation
Because it is hardly possible to control the amount and partic-
ularly the size of casein aggregates that are formed during incu-
bation with mTGase, we used the reference casein solution for
blending with casein samples that were cross-linked for different
times to obtain mixtures with almost identical degrees of poly-
merisation (Fig. 3). PD and IC were calculated on the basis of the
respective mixing partners. It is obvious that, for casein that was
incubated with mTGase for more than 3 h, the same monomer
content (or degree of polymerisation) of the respective binary
mixture does not result in the same isopeptide content. Taking 3 h
casein as an example, the 15% of monomers that were still present
in this sample as determined by GPC were also obtained by mixing
the reference casein with samples incubated for 8, 20 or 24 h; the
corresponding isopeptide contents were then 0.146, 0.234, 0.283, or
0.331, respectively. This means, on the other hand, that several
samples can be identified with identical IC at a different PD (e.g.,
IC ¼ 0.08 g 100 g1, corresponding PD ranges from 66 to 33%).
Generally, the longer the casein was cross-linked, the more refer-
ence sample that had to be added to achieve the same polymeri-
sation degree. For the above mentioned example (8, 20, 24 h), 10,
13, and 16% of reference solution was incorporated in the mixtures
to obtain a similar PD. Because all samples have the same casein
concentration, higher isopeptide content at an identical PD in-
dicates either the presence of larger and/or more extensively cross-
linked aggregates.
Fig. 4 shows the corresponding consequences on the stiffness of
acid gels from the casein solutions and their mixtures specified in
Fig. 3 as a function of the polymerisation degree, and as a function
of the isopeptide content. The last symbol in each curve refers again
to the pure casein solution that was incubated for the specified time
so that connecting these points would result in the plots shown in
Fig. 1. It is suggested that, as long as the formed aggregates do not
exceed a critical size or extent of cross-linking, gel stiffness is
determined by the concentration of the isopeptides. Under the
conditions applied in the present study, this is considered to be true
for casein cross-linked up to 3 h. For these samples it is also true
that a rather small IC is sufficient to increase gel stiffness consid-
erably, as was also found by others (Lauber et al., 2000; Mautner
Fig. 3. Monomeric casein and isopeptide content of 27 g kg1 casein in phosphate
buffer (pH 6.8) (closed circles). Enzyme treatment was with 3 U mTGase g1 protein at
40  C for the specified time. Open circles refer to mixtures that were made from the
reference (0 h) and any cross-linked casein solution. Dotted lines illustrate examples
with identical polymerisation degree, or isopeptide content.
4 D. Jaros et al. / International Dairy Journal xxx (2013) 1e5
Fig. 4. Gel stiffness ðG0max Þ extracted from gelation curves of 27 g kg1 casein in
phosphate buffer (pH 6.8) as a function of casein polymerisation (left) and isopeptide
content (right). Enzyme treatment was with 3 U mTGase g1 protein at 40  C for
different incubation time: 1 h, white circles; 2 h, grey circles; 3 h, black circles; 8 h,
white squares; 20 h, grey squares; 24 h, black squares. The last point in each curve
depicts the pure cross-linked casein, all others are samples mixed with the reference
sample of 0 h.
et al., 1999). The stiffness of gels that were made from mixtures
with casein that was incubated for 8 h was considerably lower at
comparable PD or IC. It is also evident that, for highly cross-linked
casein that was mixed with the reference, G0max remains constant or
even increases until a critical level of the uncross-linked sample
was added. For example, the 8 h casein gel had a maximum stiffness
of approximately 210 Pa. Mixing until a ratio of 63:37 (8 h:refer-
ence) reduced IC from 0.259 to 0.163 g 100 g1 and PD from 94 to
64%, but increased G0max to approximately 230 Pa. We suggest that,
initially, the uncross-linked casein molecules added to covalently
cross-linked aggregates support network formation and gel
strength because of their higher mobility during acidification that
allows a flexible incorporation into the gel. This, however, is less
pronounced for the 20 h sample and is no longer valid for the 24 h
sample wherein the largest or the most cross-linked and conse-
quently the least flexible casein aggregates are expected. When a
critical concentration is exceeded and the uncross-linked casein
molecules dominate the system, gel stiffness drops.
Fig. 5 shows gel permeation chromatograms, which give clear
evidence that there is indeed an increase of the size of covalently
Fig. 5. First part of the gel permeation chromatograms of 27 g kg1 casein in phos-
phate buffer (pH 6.8), treated with 3 U mTGase g1 protein at 40  C for the specified
time. 1 h, white circle; 2 h, grey circle; 3 h, black circle; 8 h, white square; 20 h, grey
square; 24 h, black square. Different line styles aid better visual discrimination of the
curves.
Please cite this article in press as: Jaros, D., et al., Cross-linking with microbial transglutaminase: Relationship between polymerisation degree
and stiffness of acid casein gels, International Dairy Journal (2013), http://dx.doi.org/10.1016/j.idairyj.2013.10.011
cross-linked casein aggregates with increased incubation time.
Although this method does not allow discriminating between
different casein polymers, a zoom into the part where the first
aggregates elute reveals a) the polymer peak clearly increases with
cross-linking time and b) the onset of the peaks is shifted to lower
elution time. As can be seen from the signal between 17 and 20 min,
smaller aggregates were mainly detected in the 1 h and the 2 h
sample. Several authors calculated a trimer fraction from this re-
gion (e.g., Bönisch et al., 2006; Lauber et al., 2000). Because we did
not observe a distinct peak, we decided to incorporate aggregates
larger then dimers into the polymeric fraction. Further experiments
with cross-linked fractions separated by preparative GPC are under
progress.
4. Conclusion
The gelation of casein solutions incubated with mTGase for
different times revealed a distinct dependency of gel stiffness on
the polymerisation degree. However, little is known on the modus
operandi of the enzyme and it is not possible to control the amount
and particularly the size of the casein aggregates during cross-
linking. Mixtures of caseins treated for different times with
mTGase with an uncross-linked reference made evident that the
same PD does not necessarily result in identical gel stiffness. The
quantification of isopeptides that resulted from mTGase action
leads to the assumption that, at the conditions applied in this study,
already formed aggregates are the target of the enzyme when the
amount of monomers is approximately <10%. The outcome of this
is consequently an enlargement of and/or additional cross-links
formed within the aggregates. Since caseins that were longer
incubated with mTGase always caused lower stiffness of their acid
gels at comparable polymerisation degree, the importance of
aggregate size is strongly underlined.
Acknowledgement
We kindly thank Ajinomoto Foods Europe SAS for providing the
microbial transglutaminase used in this study.
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Please cite this article in press as: Jaros, D., et al., Cross-linking with microbial transglutaminase: Relationship between polymerisation degree
and stiffness of acid casein gels, International Dairy Journal (2013), http://dx.doi.org/10.1016/j.idairyj.2013.10.011