Enzymatic Cross-linking of Milk Proteins

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3rd International Symposium on Food Rheology and Structure

163

Whey Proteins Polymers and Gels Through EnzymaticCrosslinking: 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 pHwhey proteins gels with tailored properties has beenconducted using enzymatic modification.Transglutaminase enzyme induced chemicalcrosslinks when incubated with whey proteinsolution at pH 8. Rheology, size exclusionchromatography along with electrophoresis

experiments showed large aggregate formation. Acidification of the whey protein polymers to pH 4induced gelation. Rheological properties of themodified gels (prepared via Transglutaminasecrosslinking then slow acidification) showed higherelasticity than the conventional heat-treated gel(prepared via heating to 85

oC for 1 hr). Preheating

the whey protein solution before Transglutaminasecrosslinking caused even higher elasticity. Similartrends were observed in water holding capacity. TGincubated preheated gels exhibited the largestcapacity and conventional gels the lowest,

suggesting the presence of finest structure in theformer.

1 INTRODUCTION

Whey proteins (WP) are important food ingredientsthat are used in a number of food products thatinclude dairy products, confectionary, and desserts.WP compose about 20% of bovine milk and aregenerally produced as a co-product of the cheeseindustry. The utilization of WP has been animportant research focus over the past few decadesbecause of its abundance and excellent nutritional

value [1-3].WP gelation is of considerable interest because itcan provide new food products with uniquefunctional performance, favorable texture, as well asflavor enhancement properties [4, 5]. Whey proteingels are most commonly produced using heattreatment [6-8]. However, gelation of WP can beproduced with or without heating through saltaddition [9], pH adjustment [10], and enzymetreatment [11-16].

WP gels in acidic conditions (pH < 4.6) remainlargely unutilized because of their weak and brittle

nature in contrast to the favorable elastic gelsproduced at neutral or basic conditions. This ismainly due to the absence of the strong covalentchemical disulphide bonds at these acidic conditions

and pH-associated effects on the denaturation andaggregation reactions [17]. The creation of chemicalcrosslinks (bonds) in acidic whey protein gels couldimprove their brittle and weak nature.

An alternative to creating chemical crosslinks isenzyme-catalyzed crosslinking. Transglutaminaseenzyme (EC 2.3.2.13) (TG) has been used tocrosslink a wide range of food protein types [18].

The advantage of enzymatic crosslinking is that itintroduces permanent and strong chemical bonds.Such chemical crosslinks bring favorable texturaland rheological properties increasing the rubberybehavior of the food products [18], [19]. Enzymaticcrosslinking of food proteins have been reported inliterature [11-16].

2.1. Transglutaminase as a protein crosslinker

Microbial TG consists of 331 amino acids, withcysteine residue as the active site. TG has anoptimum 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 betweenthe -carboxamine group of a peptide-boundglutamine residue and a primary amino group(lysine) of various proteins. Both of these residuesare exposed to the surface due to their hydrophilicnature. Values of free-energy change for transferare –2.9 kJ/mol and -4.6 kJ/mol for glutamine andlysine, respectively [20]. The result of this reaction isthe formation of the irreversible crosslinks.

TG has been used to crosslink food proteins [15, 16,21]. Enhanced physical and rheological propertieshave been obtained upon using TG for crosslinking

-lactoglobulin [16], [26-29]. Faergemand et al. [15]studied the crosslinking of WP using TG. Theyconfirmed the formation of covalently linkedpolymers by gel electrophoresis (SDS-PAGE) underreducing conditions. They also found the apparent

viscosity of -lactoglobulin solution incubated withTG to increase with reaction time. Sharma et al [22]treated skim milk with TG and proved the formation

of glutamyl)lysine bonds. Wilcox andSwaisgood [21] used immobilized TG to crosslinkWP. They found an increase in the intrinsicviscosity, gel strength, and brittleness upon enzyme

treatment. Most recently, Han et al. [23] used TG toproduce cream cheese so that the nutrients typicallylost as whey during processing are utilized in thefinal cream cheese. The resulting cream cheese has




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the body, texture, and taste of conventional creamcheese.

In the previous citations, solution pH was almostclose to the neutral conditions with the presence ofa denaturant, which is typically dithiotheritol (DTT).

This research focuses on utilizing Transglutaminase

along with the conventional heat treatmentprocesses to tailor the rheological properties of theacidic (pH 4) whey protein gels. Enzyme treatmentis done at slightly alkaline medium (pH 8) to makeuse of the partially denatured structure of the WP atthis pH. Subsequently, gels are produced throughslow acidification. Properties of these enzyme-modified gels are compared with conventional gels.

3 MATERIALS AND METHODS

Whey protein isolate (WPI) were obtained fromDavisco Foods International, LeSueur, MN. A

commercial version of Transglutaminase enzymewas provided by Ajinomoto Co., Japan (1% Enzyme

and 99% Maltodextrin, by weight). Glocono--Lactone (GDL) was obtained from Sigma ChemicalCo., (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 toobtain a final concentration of 7.7% w/w and stirredfor about 1 hr to ensure complete solubility. Proteinconcentration was measured by the method ofKjeldahl (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 minutesunder vacuum of 20 in Hg at room temperature toremove the visible air bubbles.

3.2 Preparation of gels

Conventional gels were obtained by heating WPsolution for 85

oC for 1 hr at pH 4. Modified WP gels

at pH 4 were obtained by adding 2% GDL to the WPsolution (previously treated with TG) and stirring forabout 15 minutes or until a clear solution wasobtained. The solution was then poured intopolymethylpentene wide mouth Nalgene® jars (to

prevent gels from sticking to the container).Solutions were left for 24 hrs at room temperature toreach pH 4. Gels were cut into 20 mm diameterdiscs with a metal punch. The thickness of the cutgels ranged from 2 to 4 mm.

3.3 Rheological measurements

In this study we used a Rheometrics ARES - and aTA Advanced Rheometer (AR 2000) - to investigatethe rheological behavior of our protein system.Couette geometry has been used in measuring therheological properties of protein solutions. A thinlayer of n-hexadecane oil was maintained at all the

times on the surface of the protein sample toprevent sample evaporation. On the other hand,parallel plate geometry with roughened upper plate

was used in measuring the rheological properties ofthe gels.

3.4 Sodium Dodecyl Sulfate Polyacrylamide GelElectrophoresis (SDS-PAGE)

A BIO-RAD Mini PROTEAN II unit was used forSDS-PAGE in this work. SDS-PAGE under reducing

conditions has been used to estimate the relativemolecular weight to evaluate the polymerizationextent 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 75HR10/30) separating in the range of 1-100,000 Daapproximately.

3.6 Water Holding Capacity (WHC)

WHC is an important property of food gels. Fine

stranded networks usually keeps water in andminimize syneresis [24, 25]. WHC measurementsare all empirical methods. In this work, we used themethod of Kocher and Foegeding [26]. Sampleswere cut into cylinders (1 cm height x 0.48 cmdiameter) and centrifuged at 153 g (2000 rpm).WHC can be determined from the following relation:

samplein proteinof wt Total

released water of wt sampleinwater of wt Total WHC

4 RESULTS AND DISCUSSIONS

The effect of enzyme treatment of WP solution(7.7% w/w) at pH 7 and pH 8 was analyzed usingSDS-PAGE and SEC analysis. Fig. 1 representsresults of electrophoresis experiment on varioussamples obtained under different conditions.

Figure 1: SDS-PAGE gel. Lane 1 is the Molecular weightmarker. Lane 2 and 3 are native WP. Lane 4 is wheyprotein solution at pH 7 treated with TG. Lane 5 is wheyprotein solution at pH 8 treated with TG.




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Figure 2. Chromatograms of whey protein solutions at pH8 treated with TG enzyme (a,b,c and d). Chromatogram erepresents whey protein solution at pH 7 treated with TGenzyme.

Comparison of WP solution (pH 7) incubated withTG (lane 4) with that of WP solution (pH 8)incubated with TG (lane 5) indicates the presence oflarger molecular weight, vis a vis, larger aggregatesat pH 8 rather than pH 7 .The same conclusions canbe extracted from chromatograms in Fig. 2, as wenotice 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 crosslinkthe WP at pH 8 rather than at pH 7. Such behavioris believed to be due to the partially denatured

structure of the -lactoglobulin at pH 8 [27].

Figure 3. Newtonian viscosity of whey protein solutions(7.7 wt%) as function of incubating time. Temperature ofincubation is 50

oC. Shear rate is 50 s

-1.

The enhanced polymerization of whey protein at pH8 compared with pH 7 is also supported byrheological measurements. Fig. 3 shows noincrease in viscosity when incubating the WPsolution with enzyme at pH 7 at 50

oC. This

indicates that only very few cross-links have formedthat have almost no effect on solution viscosity. Onthe other hand, at pH 8, there is a significantincrease in viscosity indicating a higher degree of

crosslinking due to the more open or denaturedstructure of WP.

Our results taken together indicate that denaturationis a necessity for crosslinking. This was first thoughtto be due to inaccessibility of the residues to belinked at the native (or folded) state. However,glutamine and lysine residues are found to beexposed on the surface due to their highlyhydrophilic nature in the hydropathy scale for aminoacid resides [20]. In addition, Schien [28] has shownthat only 4.2% of lysine residues and 6.3% ofglutamine residues tend to be buried in the interiorof the protein globules. Consequently we cansurmise that the reaction is not limited by residues’availability or accessibility; instead, the orientation ofthe residues in the native state does not allow the“lock and key” position of both enzyme andsubstrate (i.e., WP).

While the partial denaturation of the WP at pH 8 is

useful in promoting enzymatic crosslinking due tothe more open structure, it is necessary to study theeffect of pH on enzyme activity at 50

oC, which is

the incubation temperature in all the enzyme-treatedsamples. Enzyme activity was determined using thehydroxamate method as described by Fold [29]using Benzyloxycarbonyl-L-glutaminylglycine as asubstrate. It is obvious from Fig. 4 that the enzymeactivity is decaying more rapidly at pH 8 rather thanpH 7. This may restrict the practical enzymeincubation time to 4 or 5 hours at pH 8. However, atpH 7, the enzyme shows higher stability.

Figure 4. TG enzyme activity as function of time at pH 7and pH 8. Incubation temperature is 50

oC.

Another experiment has been done in which WPsolution was preheated at 80

oC for 1 hr prior to

incubation with TG. The resulting overall viscositywas higher than the sample treated only with TG asshown in Fig. 5. This increase in viscosity isbelieved to be due to the combined effects of

disulphide and glutamyl)lysine crosslinks. No

gelation occurs at this stage due to the strongelectrostatic repulsion under low ionic strengthconditions.

pH 8 with Enzyme

pH 8 with No Enzyme

pH 7 with Enzyme

a) 0 min

b 15 min

c) 5 hrs

d) 9 hrs

e) 5 hrs

pH 7

pH 8




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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, acidificationto pH 4 using 2% GDL was done to induce gelation.Samples were poured (just after GDL was addedand dissolved completely in the vial) inpolymethylpentene jars and left at room temperaturefor 24 hrs to form gels. Table 1 summarizes thephysical appearance of the gels obtained. Since gelappearance provides information on their structure[19], we can draw some qualitative conclusionbased on our results from Table 1. Turbidparticulate gels have an opaque milky whiteappearance due to large aggregates scattering light

[30] while fine stranded gels are transparent ortranslucent. As such, Table 1 suggests particulatestructure for conventional and enzyme treated gels,and fine stranded structure for preheated thenenzyme treated gel [30].

Table 1. Gels Appearance

Gel Appearance

Conventional Opaque

Enzyme Treated Opaque

Preheated then Enzyme Treated Translucent

It was necessary to compare the gel elasticity of themodified gels (enzyme treated, and preheated then

enzyme treated) with the conventional gel. In Fig. 6,we notice that there is a sharp increase in elasticmodulus and dynamic yield stress upon using theenzyme. We also observe that preheating thesample before enzyme incubation leads to a furtherincrease in the elastic modulus.

Another important characteristic of a gel besidesrheology is its water holding capacity (WHC). It hasbeen found that a higher WHC corresponds to afiner gel microstructure [25]. We find from Table 2that the WHC of enzyme treated sample is higherthan the conventional gel. In addition, the

preheating step – for the modified gels - furtherenhances (increases) the WHC as seen in Table 2.

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

We believe that the enhancement in both therheological properties and water holding capacity isdue to the finer structure induced by both disulphide

and glutamyl)lysine crosslinks.

Table. 2. WHC for various gels

Gel WHC (g water/g protein)

Conventional 6.190.05

Enzyme Treated 8.390.01

Preheated then EnzymeTreated

9.380.13

4 CONCLUSIONS

In this study, we present a two-step approach todevelop low pH whey protein gels with tailoredproperties. This involves enzymatic modification ofWP using TG at pH 8 followed by slow acidificationto pH 4. In addition, our studies at pH 8 as well asthat of TG treatment of preheated WP insights intothe role of chemical (enzyme catalyzed anddisulfide) crosslinks in WP gelation.

Acidic gels produced by TG treatment had higherelastic modulus, higher dynamic yield stress, andhigher water holding capacity than the conventionalheat-treated gels. These properties are furtherimproved by preheating the whey protein prior to TGtreatment.

ACKNOWLEDGMENT

The authors acknowledge Southeast Dairy FoodResearch Center for funding the projects, DaviscoFoods Int. for supplying the WP, and Ajinomoto co.for supplying the TG enzyme.

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Preheated then enzyme treated

Enzyme treated

Conventional




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Enzymatic Cross-linking of Milk Proteins