Effect of preheat treatment on the transglutaminase-catalyzed

cross-linking of goat milk proteins

Jose Manuel Rodriguez-Nogales *

Chemistry Research Centre, Autonomous University of Hidalgo State, Carretera Pachuca-Tulancingo,

Km 4.5, Pachuca, 42070 Hidalgo, Mexico

Received 14 June 2005; received in revised form 14 July 2005; accepted 15 July 2005

Abstract

The susceptibility of the individual goat’s milk proteins to cross-linking with transglutaminase was investigated. Results from capillary gel

electrophoresis showed that a heat treatment of milk before reaction with transglutaminase enhanced the reactivity of milk proteins towards

protein cross-linking. The results of this paper suggest that the specificity of transglutaminase varied with the type of milk proteins. k-casein

was more susceptible to cross-linking than a- and b-casein. However, no significant differences between both caseins were observed.

Furthermore, only in heated milk, b-lactoglobulin was significant cross-linked by transglutaminase, while preheated and unheated a-

lactalbumin was susceptible to enzymatic cross-linking. Finally, an optimization strategy based on desirability functions together with

experimental design was used to optimize the preheating conditions (temperature and time) of goat’s milk that maximized the cross-linking

reactions catalyzed by transglutaminase.

# 2005 Elsevier Ltd. All rights reserved.

Keywords: Goat’s milk; Protein cross-linking; Transglutaminase; Heat treatment; Response surface methodology

www.elsevier.com/locate/procbio

Process Biochemistry 41 (2006) 430–437

1. Introduction

Transglutaminase (EC 2.3.2.13) is an enzyme which

forms inter- and intramolecular isopepetide bonds between

protein-bound glutamine and lysine residues [1] with the

formation of e-(g-glutamyl)lysine crosslinks [2]. This

enzyme is a tool to improve the rheological properties of

food including milk proteins. However, to have a better

knowledge of the individual reactivity of the caseins and

whey proteins in a micellar system, it would be necessary to

separate and quantify these proteins from milk. Classical gel

electrophoresis methods, immunologic techniques, iso-

electric focusing, and HPLC are usually used for protein

separation and determination [3]. On the other hand, over the

* Present address: c/Malaga, 36, 2A, 09007 Burgos, Spain.

Tel.: +52 771 71 72000x6501; fax: +52 771 71 72000x6502.

E-mail addresses: rjosem@uaeh.reduaeh.mx,

manolomexico@hotmail.com.

1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2005.07.009

past decade, capillary electrophoresis has been used

successfully to analyze milk proteins, and indeed its speed

and ease of use make it a highly competitive technique for the

study of dairy products [3–6].

The effects of transglutaminase on milk proteins have been

extensively investigated [7–14]. However, there are no studies

about the reactivity of the individual protein from goat’s milk

towards transglutaminase-mediated cross-linking in spite of

the great potential for the food technology that involves the

enzymatic modification of functional properties of the goat’s

milk proteins. This potential is reflected by the fact that the

production and consumption of goat’s milk and its by-

products are increasing worldwide [15]. The structure of

goat’s milk casein is sufficiently different from cow’s milk and

easily to be differentiated in the laboratory.

The objective of this work was to study the extent of the

goat’s milk protein cross-linking through the action of

transglutaminase and to investigate if a preheat treatment of

goat’s milk before the reaction with transglutaminase could be

used to enhance the enzymatic cross-linking.

J.M. Rodriguez-Nogales / Process Biochemistry 41 (2006) 430–437 431

2. Materials and methods

2.1. Materials

Sodium hydroxide and phosphoric acid were of analytical

grade. Hydroxylpropyl cellulose (HPMC), dithiothreitol

(DTT) and urea were obtained from Sigma (St. Louis, MO,

USA). All solutions were prepared with purified water

(Milli-Q system, Millipore Corp., Bedford, MA, USA).

Microbial Transglutaminase from Stretoverticillium mobar-

aense (Activa1 TI) was a gift from Ajinomoto Co. Ltd.,

Japan.

2.2. Transglutaminase activity measurement

The colorimetric hydroxamate procedure described by

Grossowicz et al. [16] was used for transglutaminase activity

measurement of commercial preparation. One unit of

transglutaminase was defined as the amount which causes

the formation of 1.0 mmol of hydroxamic acid/min at 37 8C.

The enzyme preparation was used without any further

purification and showed a transglutaminase activity of

98 U g�1.

2.3. Cross-linking conditions of proteins in goat’s milk

Preliminary studies of the effect of treating milk with

transglutaminase were carried out with raw goat’s milk

preheated at 85 8C for 15 min [17]. Before heating, the

milk was skimmed by low-speed centrifugation (3000 � g

for 20 min) [18]. A mixture of milk and enzyme (final

enzyme/substrate ratio of 0.02 (w/w)) was incubated at

40 8C for 15, 30, 60 and 120 min. The required quantity of

transglutaminase was added directly to the milk. Parallely,

samples were monitored using unheated raw goat’s milk

with transglutaminase. In order to optimize the preheating

conditions, the effect of the preheat treatment of milk on

cross-linking behaviour was studied with goat’s milk

heated at different temperatures (70–90 8C) for different

lengths of time (15–60 min) before incubation with

transglutaminase at 40 8C, and at an enzyme to substrate

ratio of 0.02% (w/w). Samples were heated in test tubes in

a temperature controlled water bath, and after heat

treatment the samples were rapidly cooled at 4 8C. The

enzymatic reaction was terminated by heating the mixture

at 80 8C for 2 min [17]. Control samples were prepared

following the procedure described above, except that

enzyme was omitted. All experiments were realized in

triplicate. A variance analysis of the data (ANOVA) was

performed (a = 0.05) and the comparison of means was

realized by using the Tukey test.

2.4. Capillary electrophoresis

Capillary electrophoresis was carried out using a

Beckman P/ACETM system MDQ, equipped with a UV

detector, a temperature-controlled capillary compartment

and an autosampler. Separations were performed using a

fused-silica capillary column eCapTM (Beckman Instru-

ments, Fullerton, CA, USA) of 60 cm � 50 mm i.d. (50 cm

to the detector window). Sample solutions were injected for

5 s at 0.5 psi. The separations were conducted at 20 kV and

the separation temperature was kept constant at 30 8C. UV-

detection was performed at 214 nm. The running buffer was

14.7 M H3PO4, 6 M urea and 0.05% HPMC. The pH was

adjusted at 3.0 with 2 M NaOH. Sample buffer (pH 8)

consisted of 10 mM H3PO4, 8 M urea, 10 mM DTT and

1 mM Lys-Try-Lys. Before each injection, the capillary was

washed with 0.1 M NaOH (5 min), deionised water (5 min),

and 1 M HCl (5 min) and equilibrated with the running

buffer (5 min) [19].

Sample solutions of milk treated with transglutaminase

were prepared dissolving 150 mL of reaction mixture

(milk–enzyme) in 1 mL of sample buffer [20]. The samples

were filtered through 0.45 mm filters (Millex-GV13,

Millipore, Molsheim, France) before analysis by capillary

electrophoresis. Each sample was analyzed three times

(n = 3) and the average of the relative area of each peak was

calculated.

2.5. Factorial design and the desirability function

A central composite design with two variables (32 + cen-

tre points) was applied to find the optimum conditions and to

analyze how sensitive the responses were to variations in

the settings of the experimental variables. This design is

useful for estimating the coefficients in a second degree

polynomial. A total of 12 experiments were performed and

the centre point of the design was repeated four times in

order to allow a better estimation of the experimental error

and provide extra information about the response surface

curvature [21]. The variables were modified and the peak

areas of the individual milk proteins were determined.

Relative peak area (%) of individual protein for goat’s milk

(a-lactalbumin, b-lactoglubulin, as1-casein (I, II and III), k-

casein, b2-casein and b1-casein) and total as-caseins, total

b-caseins and whey proteins (sum of a-lactalbumin and b-

lactoglubulin) were the responses selected for this study. The

relative peak areas (%) were defined as the percentages of

peak areas of proteins obtained from milk treated with

transglutaminase in relation to the peak areas of proteins

obtained from milk without enzymatic treatment. The levels

of the factors are shown in Table 1. The experiments were

produced in random order and triplicate measurements of

responses were run on each experiment. The desirability

function was used for the optimization of the preheating

conditions which maximize the transglutaminase-induced

cross-linking. Statistical analysis was performed using the

computer program Statgraphics1 Plus for Windows 4.0

(Statistical Graphics Corp., Rockville, MD 20852-4999,

USA).

J.M. Rodriguez-Nogales / Process Biochemistry 41 (2006) 430–437432

Table 1

Design matrix and responses of the central composite design (32 + centre points)

Run number Factor Relative peak area (%)

Time (min) Temperature (8C) a-La b-Lg as1-CN I as1-CN II as1-CN III k-CN b2-CN b1-CN

1 15.0 70.0 62.0 64.6 22.1 31.7 33.0 18.3 26.6 25.9

2 37.5 70.0 52.7 22.5 22.9 34.6 42.6 25.5 30.4 27.3

3 60.0 70.0 47.3 12.2 23.0 26.2 29.3 29.0 25.5 23.5

4 15.0 80.0 49.0 62.0 33.1 53.3 65.4 24.1 44.5 42.6

5 37.5 80.0 27.7 46.1 33.9 47.8 49.4 23.0 43.8 34.1

6 60.0 80.0 24.4 24.1 31.0 40.9 41.5 17.3 29.7 32.7

7 15.0 90.0 40.5 48.9 22.1 63.4 75.4 35.4 8.7 6.1

8 37.5 90.0 30.8 31.7 22.0 42.0 50.9 21.0 8.4 9.4

9 60.0 90.0 34.4 31.2 13.4 27.4 42.9 14.1 7.8 7.4

10 37.5 80.0 27.4 45.6 33.0 46.9 48.7 23.4 44.0 34.5

11 37.5 80.0 28.0 46.3 34.6 44.3 49.5 23.1 43.5 33.6

12 37.5 80.0 27.5 46.9 34.3 47.9 49.0 22.8 44.1 34.2

Fig. 1. Effect of the incubation time in the capillary electrophoresis profiles

of unheated goat’s milk treated with transglutaminase (final enzyme/sub-

strate ratio of 0.02% at 40 8C). (1) a-lactalbumin; (2) b-lactoglobulin; (3)

as1-casein I; (4) as1-casein II; (5) as1-casein III; (6) k- casein; (7) b2-casein;

(8) b1-casein.

3. Results and discussion

3.1. Preliminary studies

Unheated and heated milk (85 8C for 15 min) were used

to study the susceptibly of goat’s milk proteins to the

enzymatic cross-linking with transglutaminase. The mon-

itoring of the decrease of the monomeric forms of the main

milk proteins is a way to measure the protein cross-linking

catalyzed by transglutaminase. The high resolution of the

capillary electrophoresis technique allowed the identifica-

tion of the main caseins and whey proteins from goat’s milk

[as1-(I, II and III), k-, b2-, b1-casein, a-lactalbumin and b-

lactoglubulin] and, therefore, the monitoring of enzymatic

cross-linking was possible.

Capillary electrophoresis profiles of unheated and heated

goat’s milk after incubation with transglutaminase are

shown in Figs. 1 and 2, respectively. Unfortunately, there are

not commercially available standard of pure goat’s milk

proteins, therefore, the identification of goat’s caseins and

whey proteins was established by comparing the electro-

pherograms from previous reports [3,22]. In unheated

milk, goat’s milk proteins were poorly susceptible to the

polymerization by transglutaminase, except for k-casein

which showed a notable reduction in its peak area with the

increase of the reaction time (Fig. 1). In the case of preheated

goat’s milk electropherograms, an increase of the reaction

time induced a reduction in the peak area for all main goat’s

milk proteins. At 120 min of reaction time, b1-, b2-, k-,

as1(III)-caseins appear as defined peaks, however, as1-

casein I and II could not be resolved well because both peaks

were at top of several wider and poorly defined peaks

appeared at similar elution times (Fig. 2). The presence of

these broad, not well-resolved peaks could be due to the

polymerization of the monomeric forms of the milk proteins

catalyzed by transglutaminase.

On the other hand, in capillary electrophoresis profiles of

unheated milk, well-defined peaks and stable baselines were

achieved. However, in heated milk, the detection baselines

of the electropherograms were less stable and the peaks were

wider and poorly defined. These results could be explained

by the increase of protein cross-linking, which causes an

increase in size of the molecules and a stronger interaction

with capillary walls.

The capillary electrophoresis data for caseins (from

unheated and preheated milk) incubated with transglutami-

nase up to 120 min are shown in Fig. 3, while the relative

peak areas of whey proteins are shown in Fig. 4. In unheated

milk, the relative peak areas of monomeric forms of b1- and

J.M. Rodriguez-Nogales / Process Biochemistry 41 (2006) 430–437 433

Fig. 2. Effect of the incubation time in the capillary electrophoresis profiles

of preheated goat’s milk (85 8C/15 min) treated with transglutaminase (final

enzyme/substrate ratio of 0.02% at 40 8C). (1) a-lactalbumin; (2) b-

lactoglobulin; (3) as1-casein I; (4) as1-casein II; (5) as1-casein III; (6) k-

casein; (7) b2-casein; (8) b1-casein.

b2-caseins were decreased with the reaction time to around

85% (at 120 min of reaction), and of the monomeric form of

k-casein was significantly decreased to 55% (at 120 min of

reaction). On the other hand, there were slight changes

during incubation in the relative area of as1-caseins with a

reduction of only 11–18% of the initial peak areas at

120 min of reaction. Regarding the whey proteins, the

enzymatic treatment of the milk caused a slight reduction of

the peak area for whey proteins, although it’s quite clear that

a-lactalbumin was significant reactive towards enzymatic

cross-linking without a preheating treatment. The reduction

achieved for this globular protein was around 30% of its

initial peak area (Fig. 4).

After transglutaminase cross-linking, milk proteins from

preheated milk showed a greater decrease of the relative area

than for unheated milk. The incubation time up 120 min

caused a decrease of the peak area of b-caseins about 55%

and, for k-casein the decrease was more intense with a

reduction of the 82% of the initial peak area. At 60 min of

reaction, only the 58, 34 and 51% of as1-caseins III, II and I

were evaluated, respectively. As evidence from Fig. 4, the

largest reduction in the peak area due to transglutaminase

reaction was observed for a-lactalbumin from preheated

milk. At 120 min of reaction, nearly 65% of b-lactoglobulin

was converted from the monomeric form and only a 15% of

the total a-lactalbumin was left as monomer.

Based on these results, it can be concluded that the

transglutaminase-mediated cross-linking has successfully

been realized for goat’s milk proteins. The degree of

enzymatic reaction was more important when a preheating

step of the milk was realized. It is well established that heat

treatment of milk modifies several of its physiochemical

properties. In general, increasing temperature progressively

disorders both protein and water by disrupting the hydrogen

bonding which stabilises the protein structure and causes

unfolding that lead to protein–protein interaction [9]. Heat-

denatured b-lactoglobulin can form a complex k-casein

[23,24], whereas a-lactalbumin forms a heat-induced b-

lactoglobulin/a-lactalbumin complex, which then interacts

with k-casein [24]. These reactions appear to enhance the

susceptibility of milk proteins to transglutaminase reactions

by means of a closer approach of the proteins at the active

site of the enzyme transglutaminase and the subsequent

formation of polymers [9,16].

Regarding the susceptibility of b- and as1-caseins from

heated goat’s milk towards transglutaminase-mediated

cross-linking, similar results were observed for both type

of caseins. However, some studies realized with cow’s milk

pointed out that the specificity of transglutaminase varies

with the type of casein and that the reactivity of as1-caseins

was found great reduced with respect to that of b-casein

[16]. This behaviour has been explained by the fact that the

a-casein forms the backbone structure whereas b-casein is

in a dynamic state with a disordered, flexible and open

structure that facilitates the enzymatic cross-linking [17].

However, these results obtained with cow’s milk can not be

easily extrapolated to goat’s milk, because the micellar

system of goat’s milk differs noticeably from that of cow’s

milk by several properties, more specially micelle composi-

tion, size, mineralization and hydration [25]. Furthermore,

goat’s milk is more sensitive to heat treatment than cow’s

milk and is characterized by a lower stability of their

micelles [25]. This could be the reason for the different

behaviour found between both types of milk during the

enzymatic cross-linking of b- and as1-caseins. In goat’s

milk, heat-induced changes in micellar system could have

improved the accessibility of the transglutaminase to a-

caseins compared to cow’s milk.

3.2. Optimization of the preheating conditions

In this study, a 32 central composite design [26] was used

to determine the effect of the preheating temperature and

time of the goat’s milk on the reactivity of caseins and whey

proteins towards a cross-linking reaction catalyzed by

transglutaminase. The different trials of the factorial design

consisted of all possible combinations of both factors at

three levels. The factors, their levels and the design matrix of

the study together with the relative peak areas obtained from

each goat’s milk proteins are shown in Table 1. For this

J.M. Rodriguez-Nogales / Process Biochemistry 41 (2006) 430–437434

Fig. 3. Relative peak areas (%) of individual caseins from goat’s milk treated with transglutaminase (final enzyme/substrate ratio of 0.2% at 40 8C for 30 min) at

different reaction time. Open circles represent unheated goat’s milk and black circles represent preheated goat’s milk (85 8C for 15 min).

experimental design, the preheating time was fixed at 15.0,

37.5 and 60.0 min and the preheating temperature was

assayed at 70, 80 and 90 8C (which are temperatures

commonly used in the dairy technology). To determine the

experimental error and the curvature of the systems, the

centre point of the experiments were replicated four times

(80 8C for 37.5 min). A quadratic model was selected for

this analysis and the statistical evaluation of the results was

carried out by analysis of variance (ANOVA). From this

analysis, it can be concluded that the linear factors

Fig. 4. Relative peak areas (%) of b-lactoglobulin and a-lactalbumin from goat’s

40 8C for 30 min) at different reaction time. Open circles represent unheated goat’

(preheating temperature and time) showed more influence

on the responses that the interaction between the factors and

the quadratic factors. Regarding the main factors, the

preheating temperature had a higher effect on the responses

than the preheating time. The analysis of the results of

Table 1 by multiple regression leads to second order

polynomial equations that describe the influence of both

factors on the percentage of individual goat’s milk proteins

which was not susceptible to the enzymatic cross-linking

(Table 2). The coefficients of determination indicated that

milk treated with transglutaminase (final enzyme/substrate ratio of 0.2% at

s milk and black circles represent preheated goat’s milk (85 8C for 15 min).

J.M. Rodriguez-Nogales / Process Biochemistry 41 (2006) 430–437 435

Table 2

Regression coefficients of the second order polynomial for the relative peak area (%) of individual and total goat’s milk proteins

a-La b-Lg as1-CN I as1-CN II as1-CN III k-CN b2-CN b1-CN a-Lg + b-Lg Total as-CN Total b-CN

Ca 860.98 �944.62 �739.25 �720.07 �525.52 79.35 �1261.25 �1148.01 �620.31 �663.96 �1201.65

Tb �1.98 �5.39 1.09 2.46 2.05 2.73 0.44 �0.34 �4.79 1.88 0.05

Tc �18.75 27.35 19.03 17.56 12.60 �2.63 33.39 30.71 19.07 16.45 31.95

t2 0.01 0.02 0.00 0.00 0.00 0.00 �0.01 0.00 0.02 0.00 0.00

T2 0.01 0.04 �0.01 �0.03 �0.03 �0.04 0.00 0.01 0.03 �0.03 0.00

T � t 0.11 �0.18 �0.12 �0.10 �0.06 0.02 �0.22 �0.20 �0.12 �0.09 �0.21

R2 93.45 91.54 95.13 95.85 91.10 95.22 95.31 96.54 93.97 96.69 96.92

a Model constant.b Preheating time (min).c Preheating temperature (8C).

these models are workable and can be applied in the

subsequent optimization stage.

In Fig. 5, the response surface plots that describe the

influence of both factors on the relative peak area for b-

caseins (A); as-caseins (B); k-casein (C) and whey proteins

(D) from goat’s milk are presented. A rather flat response

surface indicates that the enzymatic cross-linking milk

protein can tolerate variation in the processing conditions

without responses being seriously affected, whereas a very

pointed surface indicates that the response would be sensitive

to the processing conditions used [24]. The study of these

plots showed that the lowest values for the percentages of

Fig. 5. Effect of the preheating temperature and time of goat’s milk treated with t

caseins; (C) k-casein and (D) whey proteins (sum of b-lactoglubulin and a-lacta

Table 3

Optimization of the preheating parameters for response variables

a-La b-Lg as1-CN I as1-CN II as1-CN III k-

ta 50.3 59.0 60.0 60.0 60.0 60

Tb 83.7 70.0 90.0 70.0 90.0 90

Optimum value 25.8 14.8 14.4 27.3 32.2 12

a Preheating time (min).b Preheating temperature (8C).

relative peak areas ofb-caseins,as-caseins and k-casein were

achieved at the highest temperature/time combination of

preheat treatment of goat’s milk (90 8C for 60 min). However,

for whey proteins the optimum combination of the factors

where observed at the lowest preheating temperature (70 8Cfor 59 min). The optimum conditions (preheating time and

temperature) and the minimum values of percent relative peak

area for all responses are shown in Table 3.

Finally, the desirability function was used for the

optimization process. Using the desirability function, all

the responses were combined in one overall response: the

overall desirability [20]. The combination of the responses

ransglutaminase on relative peak areas for (A) total b-caseins; (B) total as-

lbumin).

CN b2-CN b1-CN a-Lg + b-Lg Total as-CN Total b-CN

.0 60.0 60.0 58.7 60.0 60.0

.0 90.0 90.0 70.0 90.0 90.0

.5 4.0 6.5 20.4 27.0 5.29

J.M. Rodriguez-Nogales / Process Biochemistry 41 (2006) 430–437436

Fig. 6. Influence of preheating temperature and time on the overall desir-

ability for the preheated goat’s milk treated with transglutaminase.

in one desirability function requires the calculation of the

individual functions. In this case, the responses have to be

minimized in order to find the temperature–time combination

that simultaneously minimizes the percentage of peak area for

all milk proteins. In Fig. 6, the graphic that describes the

influence of the factors on the overall desirability are

presented. The analysis of the overall desirability function

with the previously mentioned statistical software package

resulted in the optimum combination of the both variables

where the maximum enhancement of cross-linking reaction

by transglutaminase was achieved. The results of this analysis

show that the optimum preheating treatment was obtained at

90 8C for 60 min. Under these conditions, the relative peak

areas (%) for as-caseins, b-caseins, k-casein and whey

proteins were 27.3, 5.4, 12.7 and 35.2%, respectively.

4. Conclusions

Goat’s milk proteins were considerably susceptible to

cross-linking by transglutaminase and significant improve-

ments in the enzymatic cross-linking were observed when a

preheating step of the milk was realized. Likely, the thermal

denaturalization of whey proteins and the rearrangement of

casein components in the micellar structure through a series

of aggregation and dissociation reactions, that take place

during the heating of milk, improve the reactivity of protein

substrate towards protein cross-linking. On the other hand,

the optimization of the preheating conditions using

quadratic response surface methodology and the desirability

function resulted to the optimum values of the factors at

which maximum reduction of the monomeric forms of the

goat’s milk proteins through an enzymatic cross-linking

could be achieved.

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