ISFRS 3 Proceedings Lecture.qxd 17.01.

2003 9:09 Uhr Seite 163

3rd International Symposium on Food Rheology and Structure

Whey Proteins Polymers and Gels Through Enzymatic

Crosslinking: A Rheological Study
Ahmed Eissa*, Saad Khan*
*

Department of Chemical Engineering, North Carolina State University, Raleigh, NC, 27695

ABSTRACT
A promising and facile approach to develop low pH
whey proteins gels with tailored properties has been
conducted
using
enzymatic
modification.
Transglutaminase enzyme induced chemical
crosslinks when incubated with whey protein
solution at pH 8. Rheology, size exclusion
chromatography
along
with
electrophoresis
experiments showed large aggregate formation.
Acidification of the whey protein polymers to pH 4
induced gelation. Rheological properties of the
modified gels (prepared via Transglutaminase
crosslinking then slow acidification) showed higher
elasticity than the conventional heat-treated gel
(prepared via heating to 85 oC for 1 hr). Preheating
the whey protein solution before Transglutaminase
crosslinking caused even higher elasticity. Similar
trends were observed in water holding capacity. TG
incubated preheated gels exhibited the largest
capacity and conventional gels the lowest,
suggesting the presence of finest structure in the
former.

1 INTRODUCTION
Whey proteins (WP) are important food ingredients
that are used in a number of food products that
include dairy products, confectionary, and desserts.
WP compose about 20% of bovine milk and are
generally produced as a co-product of the cheese
industry. The utilization of WP has been an
important research focus over the past few decades
because of its abundance and excellent nutritional
value [1-3].
WP gelation is of considerable interest because it
can provide new food products with unique
functional performance, favorable texture, as well as
flavor enhancement properties [4, 5]. Whey protein
gels are most commonly produced using heat
treatment [6-8]. However, gelation of WP can be
produced with or without heating through salt
addition [9], pH adjustment [10], and enzyme
treatment [11-16].
WP gels in acidic conditions (pH < 4.6) remain
largely unutilized because of their weak and brittle
nature in contrast to the favorable elastic gels
produced at neutral or basic conditions. This is
mainly due to the absence of the strong covalent
chemical disulphide bonds at these acidic conditions

and pH-associated effects on the denaturation and

aggregation reactions [17]. The creation of chemical
crosslinks (bonds) in acidic whey protein gels could
improve their brittle and weak nature.
An alternative to creating chemical crosslinks is
enzyme-catalyzed crosslinking. Transglutaminase
enzyme (EC 2.3.2.13) (TG) has been used to
crosslink a wide range of food protein types [18].
The advantage of enzymatic crosslinking is that it
introduces permanent and strong chemical bonds.
Such chemical crosslinks bring favorable textural
and rheological properties increasing the rubbery
behavior of the food products [18], [19]. Enzymatic
crosslinking of food proteins have been reported in
literature [11-16].
2.1. Transglutaminase as a protein crosslinker
Microbial TG consists of 331 amino acids, with
cysteine residue as the active site. TG has an
optimum temperature of 50- 55 oC, and a pH of 6 to
7. This enzyme belongs to a class of enzymes,
which catalyzes the acyl transfer reaction between
the J-carboxamine group of a peptide-bound
glutamine residue and a primary amino group
(lysine) of various proteins. Both of these residues
are exposed to the surface due to their hydrophilic
nature. Values of free-energy change for transfer
are 2.9 kJ/mol and -4.6 kJ/mol for glutamine and
lysine, respectively [20]. The result of this reaction is
the formation of the irreversible crosslinks.
TG has been used to crosslink food proteins [15, 16,
21]. Enhanced physical and rheological properties
have been obtained upon using TG for crosslinking
E-lactoglobulin [16], [26-29]. Faergemand et al. [15]
studied the crosslinking of WP using TG. They
confirmed the formation of covalently linked
polymers by gel electrophoresis (SDS-PAGE) under
reducing conditions. They also found the apparent
viscosity of E-lactoglobulin solution incubated with
TG to increase with reaction time. Sharma et al [22]
treated skim milk with TG and proved the formation
of HJglutamyl)lysine bonds. Wilcox and
Swaisgood [21] used immobilized TG to crosslink
WP. They found an increase in the intrinsic
viscosity, gel strength, and brittleness upon enzyme
treatment. Most recently, Han et al. [23] used TG to
produce cream cheese so that the nutrients typically
lost as whey during processing are utilized in the
final cream cheese. The resulting cream cheese has

163

ISFRS 3 Proceedings Lecture.qxd 17.01.2003 9:10 Uhr Seite 164

3rd International Symposium on Food Rheology and Structure

the body, texture, and taste of conventional cream

cheese.

was used in measuring the rheological properties of

the gels.

In the previous citations, solution pH was almost

close to the neutral conditions with the presence of
a denaturant, which is typically dithiotheritol (DTT).

3.4 Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis (SDS-PAGE)

This research focuses on utilizing Transglutaminase

along with the conventional heat treatment
processes to tailor the rheological properties of the
acidic (pH 4) whey protein gels. Enzyme treatment
is done at slightly alkaline medium (pH 8) to make
use of the partially denatured structure of the WP at
this pH. Subsequently, gels are produced through
slow acidification. Properties of these enzymemodified gels are compared with conventional gels.

3 MATERIALS AND METHODS

Whey protein isolate (WPI) were obtained from
Davisco Foods International, LeSueur, MN. A
commercial version of Transglutaminase enzyme
was provided by Ajinomoto Co., Japan (1% Enzyme
and 99% Maltodextrin, by weight). Glocono-GLactone (GDL) was obtained from Sigma Chemical
Co., (St.Louis, MO). De-ionized water was used in
all the experiments (>15M:).
3.1 Preparation of protein solution
WPI powder was dissolved in de-ionized water to
obtain a final concentration of 7.7% w/w and stirred
for about 1 hr to ensure complete solubility. Protein
concentration was measured by the method of
Kjeldahl (6.38 x %N). Adjustment of pH was done
by NaOH (1 N), HCl (1 and 0.1 N), and GDL.
Solutions were de-aerated for about 15 minutes
under vacuum of 20 in Hg at room temperature to
remove the visible air bubbles.

A BIO-RAD Mini PROTEAN II unit was used for

SDS-PAGE in this work. SDS-PAGE under reducing
conditions has been used to estimate the relative
molecular weight to evaluate the polymerization
extent of the protein chains.
3.5 Size Exclusion Chromatography (SEC)
A Waters 9600 SEC, equipped with Photo Diod
Array (PDA) detector (Waters, PDA 996) was used.
The column used was Pharmacia (Superdex 75
HR10/30) separating in the range of 1-100,000 Da
approximately.
3.6 Water Holding Capacity (WHC)
WHC is an important property of food gels. Fine
stranded networks usually keeps water in and
minimize syneresis [24, 25]. WHC measurements
are all empirical methods. In this work, we used the
method of Kocher and Foegeding [26]. Samples
were cut into cylinders (1 cm height x 0.48 cm
diameter) and centrifuged at 153 g (2000 rpm).
WHC can be determined from the following relation:
WHC

Total wt of water in sample  wt of water released

Total wt of protein in sample

4 RESULTS AND DISCUSSIONS

The effect of enzyme treatment of WP solution
(7.7% w/w) at pH 7 and pH 8 was analyzed using
SDS-PAGE and SEC analysis. Fig. 1 represents
results of electrophoresis experiment on various
samples obtained under different conditions.

3.2 Preparation of gels

Conventional gels were obtained by heating WP
solution for 85 oC for 1 hr at pH 4. Modified WP gels
at pH 4 were obtained by adding 2% GDL to the WP
solution (previously treated with TG) and stirring for
about 15 minutes or until a clear solution was
obtained. The solution was then poured into
polymethylpentene wide mouth Nalgene jars (to
prevent gels from sticking to the container).
Solutions were left for 24 hrs at room temperature to
reach pH 4. Gels were cut into 20 mm diameter
discs with a metal punch. The thickness of the cut
gels ranged from 2 to 4 mm.
3.3 Rheological measurements
In this study we used a Rheometrics ARES - and a
TA Advanced Rheometer (AR 2000) - to investigate
the rheological behavior of our protein system.
Couette geometry has been used in measuring the
rheological properties of protein solutions. A thin
layer of n-hexadecane oil was maintained at all the
times on the surface of the protein sample to
prevent sample evaporation. On the other hand,
parallel plate geometry with roughened upper plate

164

Figure 1: SDS-PAGE gel. Lane 1 is the Molecular weight

marker. Lane 2 and 3 are native WP. Lane 4 is whey
protein solution at pH 7 treated with TG. Lane 5 is whey
protein solution at pH 8 treated with TG.

ISFRS 3 Proceedings Lecture.qxd 17.01.2003 9:10 Uhr Seite 165

3rd International Symposium on Food Rheology and Structure

crosslinking due to the more open or denatured

structure of WP.

e) 5 hrs
d) 9 hrs
c) 5 hrs

b) 15 min
a) 0 min

Figure 2. Chromatograms of whey protein solutions at pH

8 treated with TG enzyme (a,b,c and d). Chromatogram e
represents whey protein solution at pH 7 treated with TG
enzyme.

Comparison of WP solution (pH 7) incubated with

TG (lane 4) with that of WP solution (pH 8)
incubated with TG (lane 5) indicates the presence of
larger molecular weight, vis a vis, larger aggregates
at pH 8 rather than pH 7 .The same conclusions can
be extracted from chromatograms in Fig. 2, as we
notice development of protein aggregates in (b), (c)
and (d) at pH 8 as a function of TG incubation time.
In contrast, smaller aggregates are formed at pH 7
(e). This indicates higher ability of TG to crosslink
the WP at pH 8 rather than at pH 7. Such behavior
is believed to be due to the partially denatured
structure of the E-lactoglobulin at pH 8 [27].

pH 8 with Enzyme

pH 8 with No Enzyme

Our results taken together indicate that denaturation

is a necessity for crosslinking. This was first thought
to be due to inaccessibility of the residues to be
linked at the native (or folded) state. However,
glutamine and lysine residues are found to be
exposed on the surface due to their highly
hydrophilic nature in the hydropathy scale for amino
acid resides [20]. In addition, Schien [28] has shown
that only 4.2% of lysine residues and 6.3% of
glutamine residues tend to be buried in the interior
of the protein globules. Consequently we can
surmise that the reaction is not limited by residues
availability or accessibility; instead, the orientation of
the residues in the native state does not allow the
lock and key position of both enzyme and
substrate (i.e., WP).
While the partial denaturation of the WP at pH 8 is
useful in promoting enzymatic crosslinking due to
the more open structure, it is necessary to study the
effect of pH on enzyme activity at 50 oC, which is
the incubation temperature in all the enzyme-treated
samples. Enzyme activity was determined using the
hydroxamate method as described by Fold [29]
using Benzyloxycarbonyl-L-glutaminylglycine as a
substrate. It is obvious from Fig. 4 that the enzyme
activity is decaying more rapidly at pH 8 rather than
pH 7. This may restrict the practical enzyme
incubation time to 4 or 5 hours at pH 8. However, at
pH 7, the enzyme shows higher stability.

pH 7

pH 8

pH 7 with Enzyme

Figure 3. Newtonian viscosity of whey protein solutions

(7.7 wt%) as function of incubating time. Temperature of
incubation is 50 oC. Shear rate is 50 s-1.

The enhanced polymerization of whey protein at pH

8 compared with pH 7 is also supported by
rheological measurements. Fig. 3 shows no
increase in viscosity when incubating the WP
solution with enzyme at pH 7 at 50 oC. This
indicates that only very few cross-links have formed
that have almost no effect on solution viscosity. On
the other hand, at pH 8, there is a significant
increase in viscosity indicating a higher degree of

Figure 4. TG enzyme activity as function of time at pH 7

and pH 8. Incubation temperature is 50 oC.

Another experiment has been done in which WP

solution was preheated at 80 oC for 1 hr prior to
incubation with TG. The resulting overall viscosity
was higher than the sample treated only with TG as
shown in Fig. 5. This increase in viscosity is
believed to be due to the combined effects of
disulphide and HJ glutamyl)lysine crosslinks. No
gelation occurs at this stage due to the strong
electrostatic repulsion under low ionic strength
conditions.

165

ISFRS 3 Proceedings Lecture.qxd 17.01.2003 9:10 Uhr Seite 166

3rd International Symposium on Food Rheology and Structure

Preheated then enzyme treated

Enzyme treated

Enzyme treated

No enzyme

Conventional

Figure 5. Viscosity of whey protein solutions (7.7 wt %) ,

pH 8 after 5 hrs of incubation at 50 oC.

After WP polymerization by TG at pH 8, acidification

to pH 4 using 2% GDL was done to induce gelation.
Samples were poured (just after GDL was added
and dissolved completely in the vial) in
polymethylpentene jars and left at room temperature
for 24 hrs to form gels. Table 1 summarizes the
physical appearance of the gels obtained. Since gel
appearance provides information on their structure
[19], we can draw some qualitative conclusion
based on our results from Table 1. Turbid
particulate gels have an opaque milky white
appearance due to large aggregates scattering light
[30] while fine stranded gels are transparent or
translucent. As such, Table 1 suggests particulate
structure for conventional and enzyme treated gels,
and fine stranded structure for preheated then
enzyme treated gel [30].
Table 1. Gels Appearance
Gel
Conventional
Enzyme Treated
Preheated then Enzyme Treated

Appearance
Opaque
Opaque
Translucent

It was necessary to compare the gel elasticity of the

modified gels (enzyme treated, and preheated then
enzyme treated) with the conventional gel. In Fig. 6,
we notice that there is a sharp increase in elastic
modulus and dynamic yield stress upon using the
enzyme. We also observe that preheating the
sample before enzyme incubation leads to a further
increase in the elastic modulus.
Another important characteristic of a gel besides
rheology is its water holding capacity (WHC). It has
been found that a higher WHC corresponds to a
finer gel microstructure [25]. We find from Table 2
that the WHC of enzyme treated sample is higher
than the conventional gel. In addition, the
preheating step for the modified gels - further
enhances (increases) the WHC as seen in Table 2.

166

Preheated then enzyme treated

Figure 6. Elastic modulus of the acidic gels at 25 oC.

We believe that the enhancement in both the

rheological properties and water holding capacity is
due to the finer structure induced by both disulphide
and HJglutamyl)lysine crosslinks.
Table. 2. WHC for various gels
Gel
Conventional
Enzyme Treated
Preheated then Enzyme
Treated

WHC (g water/g protein)

6.19r0.05
8.39r0.01
9.38r0.13

4 CONCLUSIONS
In this study, we present a two-step approach to
develop low pH whey protein gels with tailored
properties. This involves enzymatic modification of
WP using TG at pH 8 followed by slow acidification
to pH 4. In addition, our studies at pH 8 as well as
that of TG treatment of preheated WP insights into
the role of chemical (enzyme catalyzed and
disulfide) crosslinks in WP gelation.
Acidic gels produced by TG treatment had higher
elastic modulus, higher dynamic yield stress, and
higher water holding capacity than the conventional
heat-treated gels. These properties are further
improved by preheating the whey protein prior to TG
treatment.

ACKNOWLEDGMENT
The authors acknowledge Southeast Dairy Food
Research Center for funding the projects, Davisco
Foods Int. for supplying the WP, and Ajinomoto co.
for supplying the TG enzyme.

REFERENCES
1.
DeWit, J.N. and G. Klarenbeek, Effects of
various heat treatments on structure and solubility of
whey proteins. Journal of Dairy Science, 1984.
67(11): p. 2701-10.
2.
Tziboula, A., et al., Microfiltration of milk
with ceramic membranes. Influence on casein

ISFRS 3 Proceedings Lecture.qxd 17.01.2003 9:10 Uhr Seite 167

3rd International Symposium on Food Rheology and Structure

composition and heat stability. Milchwissenschaft,

1998. 53(1): p. 8-11.
3.
Muir, D.D. and A.W.M. Sweetsur,
Optimization of the heat stability of protein-rich
concentrates prepared by ultrafiltration of skim-milk.
Journal of Food Technology, 1984. 19(2): p. 263-71.
4.
Gezimati, J., L.K. Creamer, and H. Singh,
Heat-Induced Interactions and Gelation of Mixtures
of .beta.-Lactoglobulin and .alpha.-Lactalbumin.
Journal of Agricultural and Food Chemistry, 1997.
45(4): p. 1130-1136.
5.
Mleko, S., Rheological properties of milk
and whey protein desserts. Milchwissenschaft,
1997. 52(5): p. 262-266.
6.
Gezimati, J., H. Singh, and L.K. Creamer,
Aggregation and gelation of bovine .beta.lactoglobulin, .alpha.-lactalbumin, and serum
albumin. ACS Symposium Series, 1996.
650(Macromolecular Interactions in Food
Technology): p. 113-123.
7.
Foegeding, E.A., H. Li, and S.R. Bottcher,
Gelation of globular proteins. IFT Basic Symposium
Series, 1998. 13(Phase/State Transitions in Foods):
p. 253-271.
8.
Mleko, S., Studies on permeability and
rheology of heat and sodium ions-induced whey
protein gels. Journal of Food Science and
Technology, 2000. 37(3): p. 307-310.
9.
Hongsprabhas, P., S. Barbut, and A.G.
Marangoni, The structure of cold-set whey protein
isolate gels prepared with Ca++. LebensmittelWissenschaft und -Technologie, 1999. 32(4): p.
196-202.
10.
Mleko, S. and E.A. Foegeding, pH induced
aggregation and weak gel formation of whey protein
polymers. Journal of Food Science, 2000. 65(1): p.
139-143.
11.
Frgemand, M., J. Otte, and K.B. Qvist,
Crosslinking of Whey Proteins by Enzymic
Oxidation. Journal of Agricultural and Food
Chemistry, 1998. 46(4): p. 1326-1333.
12.
Soeda, T., et al., Processed food containing
powder of milk whey proteins, in Jpn. Kokai Tokkyo
Koho. 1998, (Ajinomoto Co., Inc., Japan). Jp. p. 6
pp.
13.
Swaisgood, H.E., X.L. Huang, and G.L.
Catignani, Enzymic modification of proteins to
improve functionality. Book of Abstracts, 214th ACS
National Meeting, Las Vegas, NV, September 7-11,
1997: p. AGFD-020.
14.
Yildirim, M. and N.S. Hettiarachchy,
Biopolymers produced by crosslinking soybean 11S
globulin with whey proteins using transglutaminase.
Journal of Food Science, 1997. 62(2): p. 270-275.
15.
Faergemand, M., J. Otte, and K.B. Qvist,
Enzymic crosslinking of whey proteins by a Ca2+independent microbial transglutaminase from
streptomyces lydicus. Food Hydrocolloids, 1997.
11(1): p. 19-25.

16.
Aboumahmoud, R. and P. Savello,
Crosslinking of whey protein by transglutaminase.
Journal of Dairy Science, 1990. 73(2): p. 256-63.
17.
Errington, A.D. and E.A. Foegeding, Factors
Determining Fracture Stress and Strain of FineStranded Whey Protein Gels. Journal of Agricultural
and Food Chemistry, 1998. 46(8): p. 2963-2967.
18.
Dickinson, E., Enzymic crosslinking as a
tool for food colloid rheology control and interfacial
stabilization. Trends in Food Science & Technology,
1997. 8(10): p. 334-339.
19.
Anon, Use of a transglutaminase modified
protein as a fat replacer in foods. Research
Disclosure, 1995. 369: p. 16.
20.
Eisenberg, D., et al., Hydrophobic moments
in protein structure. Faraday Symp. Chem. Soc.,
1982. 17: p. 109-120.
21.
Wilcox, C.P. and H.E. Swaisgood,
Modification of the Rheological Properties of Whey
Protein Isolate through the Use of an Immobilized
Microbial Transglutaminase. Journal of Agricultural
and Food Chemistry, 2002. 50(20): p. 5546-5551.
22.
Sharma, R., P.C. Lorenzen, and K.B. Qvist,
Influence of transglutaminase treatment of skim milk
on the formation of .vepsiln.-(.gamma.glutamyl)lysine and the susceptibility of individual
proteins towards crosslinking. International Dairy
Journal, 2001. 11(10): p. 785-793.
23.
Han, X.-Q., J.K. Pfeifer, and R.H. Lincourt,
Process for making a wheyless cream cheese using
transglutaminase, in U.S. 2002, (Kraft Foods
Holdings, Inc., USA). Us. p. 16 pp.
24.
Verheul, M. and S.P.F.M. Roefs, Structure
of whey protein gels, studied by permeability,
scanning electron microscopy and rheology. Food
Hydrocolloids, 1998. 12(1): p. 17-24.
25.
Bowland, E.L. and E.A. Foegeding, Effects
of anions on thermally induced whey protein isolate
gels. Food Hydrocolloids, 1995. 9(1): p. 47-56.
26.
Foegeding, E.A., The effect of cations on
rheological properties of whey protein gels. Food
Proteins: Struct. Funct., Symp. Food Proteins
\"Struct.-Funct. Relat.\", 4th, 1993: p. 341-3.
27.
Faergemand, M., J. Otte, and K.B. Qvist,
Emulsifying properties of milk proteins cross-linked
with microbial transglutaminase. International Dairy
Journal, 1998. 8(8): p. 715-723.
28.
Schein, C.H., Solubility as a function of
protein structure and solvent components. Biotech.,
1990. 8(4): p. 308-17.
29.
Folk, J.E. and P.W. Cole,
Transglutaminase: mechanistic features of the
active site as determined by kinetic and inhibitor
studies. Biochim. Biophys. Acta, 1966. 122(2): p.
244-64.
30.
Hudson, H.M., C.R. Daubert, and E.A.
Foegeding, Rheological and Physical Properties of
Derivatized Whey Protein Isolate Powders. Journal
of Agricultural and Food Chemistry, 2000. 48(8): p.
3112-3119.

167