Thermal Stability and Functionality of Whey Proteins

Thermal Stability and Functionality of Whey Proteins

Thennal Stability and Functionality of Whey Proteins J. N. DE WIT DMVlCAMPINA, Division IndustrtaJ Products, 5460 BA Veghel, The Netherlands ABSTRACT...

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Thennal Stability and Functionality of Whey Proteins J. N. DE WIT DMVlCAMPINA, Division IndustrtaJ Products, 5460 BA Veghel, The Netherlands


Functional properties of whey proteins are often impaired by inevitable heat treatments during processing of whey protein products and preservation of food products. The extent of deterioration depends on the relation between the rate of protein denaturation and the heat load applied. In a new kinetic approach, whey protein denaturation is split into two successive processes: 1) unfolding, determined by differential scanning calorimetry; and 2) aggregation, derived from high perfonnance gel permeation chromatography. A short review is presented of industrial recovery processes of whey protein products and their related functional properties. The effect of heat treatments between 70 and 9O"C is analyzed in terms of the kinetics of whey protein unfolding and aggregation. The results reveal that thermal denaturation of whey proteins during industrial heating processes is predictable. An important determinant for functional properties is the salt composition of whey protein products during heat treatments at temperatures above 7S"C. (Key words: functional properties, industrial whey protein concentrates, denaturation kinetics) INTRODUCTION

Whey proteins are often used to improve food products because of their high nutritional quality and their versatile functional properties. However, the behavior of whey proteins during food processing is very complex and is governed by their heat sensitivity (6). Many attempts have been made to predict desired

Received September 30, 1989. Accepted Februaxy 20, 1990. 1990 ] Dairy Sci 73:3602-3612

functional characteristics in food products on the basis of functional properties of whey proteins (7, 18). The term "functional properties" generally is used in relation to physicochemical parameters of proteins in aqueous solutions or in simple model systems. Functional properties primarily reveal information on the processing history and composition of the whey protein product. The usefulness of these parameters in predicting corresponding functional characteristics in food products is limited, mainly because of the complex behavior of whey proteins with other ingredients of any food product during food processing. Differences are noted between functional properties in aqueous solution and food products (Figure 1). The solubility of whey proteins in aqueous solution may differ widely from their solubility in a pasteurized fruit drink as a consequence of interactions with fruit colloids in this beverage (8). Similar discrepancies may be observed between foaming properties (of whey proteins in water) and aeration (of food products), between emulsifying properties and fat binding, between gelation properties and structure forming, and so forth. To predict the functional performance of whey proteins in many food products, these proteins must be in the undenatured state before food processing. The inevitable heat treatments used during processing and preservation of whey protein products can seriously affect the native state and stability of whey proteins. Consequently, a better understanding of the behavior of whey proteins during heat treatments is essential for the control of their functional properties during recovery and application of whey protein products. In this paper, protein denaturation and related functional properties are discussed in terms of the kinetics of industrial heating processes. The following topics are considered: 1) survey of some industrial recovery processes for whey protein products and their effects on the stability of whey proteins; 2) effect of






solubility aeration fat binding water binding structure formi ng

foamabilily emulsifiCiltion water uptake gelation

Figure 1. Functional characteristics of whey protein products.

denaturation on some functional properties of whey proteins; 3) kinetic analysis of heat-induced unfolding and aggregation of whey proteins under various conditions; and 4) feedback of these kinetic data to observations made on thennal denaturation during industrial recovery processes of whey protein products. EXPERIMENTAL METHODS

Functional Properties

The techniques used for the functional characterization of the whey protein products were selected arbitrarily and were based only on experience and equipment available (Figure 2). Methods have been devised for the evaluation of solubility, dispersibility, viscosity, foaming, emulsifying, and gelation properties of whey protein products (9). Protein solubility is determined according to an adapted procedure of the nitrogen solubility index. The basic steps of this test involve dispersing the sample in water under standardized conditions, followed by centrifugation at 20,000 X g for 10 min, and determination of the nitrogen content in the supernatant relative to that of the initial protein solution. Dispersion rate is based on the turbidity change of an aqueous dispersion of powder, immediately after a forced contact with water,


as described by De Wit and Klarenbeek (12). Viscosity as a ftmction of time or temperature up to 140"C is measured using a modified HOppler falling-ball viscosimeter called the Klarograph (13). Protein foaming properties are determined using a Hobart N-SO blender (Hobart Manufacturing Co., Troy, OH), in which 7S ml of a 3% (wt/vol) protein solution are whipped for S min at maximum speed (9). Emulsifying properties are determined after high pressure homogenization (20 MPa, SO"C) of a 4% (wt/wt) oil-in-water preemulsion, as described by De Wit and Klarenbeek (10). The emulsion stability (ES) is determined immediately after homogenizing and 1 wk later, using a centrifuge at 400 x g. The ES is defined as the fat content in the bottom layer relative to the initial fat content of the emulsion. Gelation properties are characterized, using an Instron Universal Testing machine, Model 1122 (Instron Instruments Ltd., Buckinghamshire, England), equipped with 5 N tension load cell operating in the oscillating mode for measuring the gelation temperature and gelation rate. The gel strength is measured at the gelation temperature by changing the operation mode from oscillation to tension (9). KInetIcs of ProteIn UnfoldIng

The kinetics of protein unfolding were studied using a Dupont 990 Thermal Analyzer with a heat-flux DSC cell, which operates on the measuring principle shown in Figure 3. The cell consists of a small heating box (oven), in which hermetically sealed sample pans are placed on a constantan plate provided with chromeJlalumel thermOCOl,..,les, fitted close to the samples. The sample 3nd an inert reference sample with a similar heat capacity (usually water or solvent) are heated at a programmed heating rate of SOC/min. The observed differences in heat flow (AY) between sample and reference sample are recorded as a function of the sample temperature (or time, since constant heating rates are used) (Figure 3). From the shape of the heat absorption curve, information on the kinetics of the unfolding process can be obtained by the method of Borchardt and Daniels (1). This method is based on the assumptions that 1) the rate of heat uptake Journal of Daily Science Vol. 73,

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(i.e., protein unfolding) is proportional to the baseline deflection (dH/dt) at a given temperature, and 2) the extent of unfolding is proportional to the heat evolved (Mi) at that temperature. If protein unfolding is a first-order reaction, the rate constant can be calculated from the following equation (24): k = dH/dt X 1/MIR.

-dC/dt where: n


= order of the reaction, obtained by us=



= fIrst-order rate constant at a selected temperature, = deflection from the baseline at that temperature (Figure 3), = total area below the curve (Mi) mi-


nus the area up to that temperature, and time.

k dH/dt


The quantities dHldt and MIR are taken from the DSC curve at successive temperature. Kinetics of Protein Aggregation

To determine the rate of protein aggregation the following procedure was followed. Aliquots (10 ml) of a .5% protein solution were heated in small tubes in a water bath at temperatures between 70 and 90°C. The heating rate was 5°C/min; the holding times at the selected temperature varied between 30 and 300 s, after which the samples were cooled rapidly by immersingthe tubes in ice. Subsequently, the turbid solutions were centrifuged for 60 min at 90,000 x g, and the supernatant was analyzed in .25 M phosphate at pH 6.7 by high performance gel permeation chromatography. A Dupont column, Type OF 250, was used at a flow rate of 1.5 ml/min, and the detection was performed at 280 om. The monomer concentrations of ~-lactoglobulin (P-Lg) and a-lactalbumin (a-La) were determined by measuring the peak heights. These results were compared with those obtained using standard solutions of P-Lg and a-La (Figure 4). The reaction order was determined by the differential method described by Hill and Grieger-Block (16). This method is based on differentiation of the experimental concentration versus time data, starting from the general equation: Journal of Daily Science Vol. 73,

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= ken

ing the logarithmic form of this equation, and rate constant derived from the integral of the general equation, giving: CJCo = exp (-kt) for a fust-order reaction, and Cr/Ct = [1 + kCo t] for secondorder reactions, with Co = initial monomer concentration, and Ct = monomer concentration after a heating time t.

The temperature dependence of the rate constants was derived from the Arrltenius equation according to: k = 1co·exp(-EAlRT) where: EA = energy of activation in joules per mole, R = universal gas constant (8.314 J/mol per K), and T = absolute temperature. DISCUSSION Effects of Processing on Thermal Stability of Whey Proteins

The principles of some industrial recovery processes for whey protein products are shown in Figure 5. This diagram indicates the main process treatments applied before evaporation and spray-drying, together with some information on the protein content of the products obtained. The starting whey (indicated in the center) is usually pasteurized [i.e., noc for 15 s and kept below the denaturation temperature of whey proteins so far as possible during the recovery of whey protein concentrate (WPC)]. Evaporation and drying of the WPC may cause serious whey protein denaturation, especially when the pH of the WPC deviates too much from 6.5 (4). Fractionation of whey is generally performed by diafiltration (OF) and ultrafiltration (UP) of whey at pH 3.0 (acid UF/DF WPC) or


pH 6.5 (nonna! UP WPC and neutral UP/DF wpc). The resulting WPC have protein contents expressed as the percentage of total solids (pm that vary from 35 to 85% (19). Lactose recovery is achieved by evaporation of whey, followed by crystallization and separation of lactose. The remaining liquid may be desalted by ionic exchange or electrodialysis, resulting in a WPC having protein contents from 30 to 40% of total solids (25). Dming these evaporation processes, temperatures should not be too high (<72'C), so that heat denaturation can be controlled.


Clarification refers to a specific defatting process of desalted whey, followed by UP or DF up to protein contents of 60 to 80% of total solids (defatted UP WPC) (11). During this process, any preViously denatured whey proteins are removed by the fat separation step (at pH 4.6). Two major ion-exchange processes have been developed for the recovery of charged whey proteins from the whey, ie., the Vistec stirred-bed and the Spherosil fixed-bed ionic exchange processes. The Vistec process is based on a dispersion of crosslinked cellulose

Characterization of milk proteins

Figure 2. Techniques used far fuDctionaI characterization of whey protein products.

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DSC cell (schematic) DSC thermogram

r - - - - - - - - - - - - - - - - - - - --1




"'-------time (s) r - - - - - - - - _. temp. (oC) I


LlT ~Ll Y 1----1-1

]-----"'T"""-i convertur


cold junction


Figure 3. Differential scaDDiog calorimetric (DSC) procedure for the kiDctic analysis of whey protein unfolding. kt first order rate constant at temperature T; dH/dt deflection from the baseline at temperature T; MlR total area below the curbe (MI) minus the area up to temperature T; t time; lIDd IiY differences in heat flow.



particles provided with negatively charged exchange (eoa-) groups (21). At pH close to 3, positively charged whey proteins are adsorbed in a stirred-bed reactor, and they are desorbed by increasing the pH to about 9. These pH adjustments increase the salt concentration, and so an additional UP step is needed. The resultant WPC has a high protein content (>90% PI 1'), containing whey proteins that are either native (WPI) or modified (Vistee), depending on whether of not the pH is corrected from 9 to 6.5 before UF and drying (15). Two fixed-bed silica-based column ion-exchange processes have been developed for the recovery of whey proteins (20). These Spherosil processes use either anionic (Spherosil QMA) or cationic (Spherosil S) ion exchangers. The proteins in sweet cheese whey are directly adsorbed by the positively charged Journal of Dairy Science Vol. 73,

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quaternary methyl ammonium groups of Spherosil QMA. Desorption takes place at pH below 4.0, immediately followed by evaporation and spray drying. Spherosil S is provided with sulfonic acid groups, which bind whey proteins at pH 4.5. Desorption is performed at pH above 8.0 by using ammonia. During the subsequent evaporation and drying stage, the ammonia will disappear, resulting in a pH drop to about 6.0, thus masking a very harmful high pH step for whey proteins in the heating stage. Effects of Protein DenaturaUon on the FuncUonal ProperUes

The functional properties determined for 13Lg and seven industrially prepared WPC are shown in Figure 6. Functional properties are presented comparatively on a scale between 0


is dried under acid pH conditions, causing pHinduced denatumtion of both a-lactalbumin (23) and serum albumin (5). Figure 6 clearly shows that the solubility at pH 4.6 (representing the percentage of undenalured whey proteins) is reflected in the other functional properties. This stresses the need for a controlled heat effect of whey protein products during processing and drying in order to keep the whey proteins in their native state. To be able to optimize industrially performed heating processes, for optimum functional use of whey proteins and for reduced fouling in heating equipment, detailed information is needed about the kinetics of unfolding and aggregation of whey proteins under various environmental conditions.







11 ~






.1 'I I '\












time (min) Figure 4. High performance gel permeation cbromatograms of whey in phosphate buffer at pH 6.7, before (-) and after (-) beat treatment for 3 min at SO'C. ~Lg ~ Lactoglobulin, a-La = a-1acta1bumin.


and 100%, so that the results obtained would be easier to discuss. At this scale, 100% refers to the best functional chaJ:acteristics ever obtained by that particular technique, using a freezedried laboratory sample of f3-Lg. Four functional properties are presented in a cumulative way, i.e., 400% should reflect the most functional protein product. The products indicated as 2, 3, and 4 were obtained by ion-exchange processes and 5, 6, 7, and 8 by membrane processes. The solubility at pH 4.6 appears to be 100% only for f3-Lg. Most of the industrially prepared samples show solubility characteristics between 80 and 90%. Reduced solubility (determined at pH 4.6) reflects whey protein denaturation, which usually is caused by evapomtion and drying steps of whey protein products. The Spherosil S, and the acid UF/DF WPC (products 4 and 8) exhibit serious protein denatumtion. This is explained by a combined pH and heat denaturation effect: Spherosil S is heated at alkaline pH during drying, which seriously denatures f3-Lg, through thiol and disulfide interactions (27), and acid UF/DF WPC

Kinetics of Unfolding and Aggregation of ~Lactoglobullns

Unfolding of globular proteins is accompanied by an endothermal heat effect (heat uptake). Figure 7A shows a thermogmm of a WPC and, dmwn in the same plot, one of f3-Lg in whey permeate (dotted line). Both curves overlap in the tempemture range between 71 and sooC, which indicates that kinetic data on the unfolding of f3-Lg may be derived from this part of the WPC curve. Figure 7B shows firstorder Arrhenius plots of the WPC curves analyzed between the onset (65°C) and the tmnsition temperature (76°C). The Arrhenius plot becomes nonlinear above the transition temperature, which may be caused by heat effects resulting from protein aggregation and expressed in this part of the CUIVe. As shown in Figure 7B, the Arrhenius plot of WPC reflects the kinetic data of /3-Lg from ?l to 76°C, whereas a lower activation energy (slope of the CUIVe) is shown from 65 to ?loC. This lower activatim energy may result from the unfolding of other whey proteins (a-lactalbumin and serum albumin) (5). Based m this observation, we decided to select a tempemture range of maximum 5°C just before the tmnsitim temperature in WPC thermograms, for the kinetic analysis of unfolding of /3-Lg. Some of the kinetic data of unfolding and aggregation of f3-Lg in water, whey permeate, exhaustively desalted WPC, whey, skim milk, and whole milk. are summarized in Table 1. As mentioned earlier, unfolding was observed by Journal of Dairy Science Vol. 73,

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UF/DF WPC 135-85." PITS)

lactose recovery Spherosil'C1I\A' WPCr====:::" 1>'lO'ro PITSI


Spherosil'S' WPC




• de-lactose

de-mineralized de-lactose
de-fatled UF WPC (6(}-70" PITS)

Figure 5. Processes for tbe recovery of whey proteins from wiley. DF = DiafiJlration, WPC = whey protein conceutrate. P/fS

= percentage total solids, QMA =

quaternary methyl ammonium.

means of differential scanning calorimetry as a first-order reaction up to the transition temperature. However, the reaction order of the aggregation process varied, depending on composition, temperature, and duration of heating. Most aggregation reactions appeared to follow (nearly) second-order kinetics, as expected (3), except those taking place in permeate and desalted WPC. The higher reaction order in WPC may be explained by the participation of other protein species that are present in the product. The aggregation of ~Lg in permeate followed a (pseudo) first-order reaction at all temperatures studied. This may be explained by the effect of a mechanism of two competing reactions (e.g., P-Lg and calcium for aggregate formation). Pseudo first-order conditions occur only if the molar concenttations of calcium or of other participating cations are much higher than that of ~Lg. Under those conditions, the Journal of Dairy Science VoL 73,

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participating cation concentration remains approximately constant during aggregation (2). Table 1 shows that at 7S·C the rate constants of unfolding are about two times higher than those of aggregation. Thus, under these conditions, protein unfolding is not a rate-detennining process for aggregation of whey proteins. The aggregation of ~Lg in water (and desalted WPC) proceeds at a much lower rate than in salt-<:Ontaining solutions. Also, the temperature coefficients (EA> of the rate constants of ~Lg aggregation in these media are lower. TIlese observations confirm the results obtained by Pantaloni (22) and Vreeman and Poll (26), who reported a time lag of seveml minutes in this temperature range before aggregation started. The mechanism responsible for this lag phase on aggregation is still unknown. However, the dominating effects of (Ca-) salts on the aggregation of P-Lg should be stressed. TIle kinetic



parameters for aggregation of (3-Lg in skim milk (fable 1) confinn those reported by Dannenberg and Kessler (3) for irreversible denaturation of (3-Lg in this medium. Some Functional and TechnologIcal Consequences

The kinetic information on unfolding and aggregation of whey proteins thus obtained under various conditions may explain a number of features of functional and technological nature. Figure 8 shows calculated results of unfolding and aggregation of (3-Lg, during a continuous heating process of whey at two extreme heating rates. A heating rate (1\) of I"Cls may occur in the heating section of industrial pasteurizers, and an Hr of I"C/min in the regenerative sections of heat exchangers with extensive facilities for heat recovery. The lines in Figure 8 indicate the net availability of unfolded (3-Lg, and the bars indicate that of the aggregated

% contriootion

protein over the indicated temperature range. At both heating rates, aggregation (Le., irreversible denaturation) appears to start after 60% unfolding has occurred. This implies that an Hr of I"Cls does not show aggregation below 83"C, whereas with an Hr of I"CImin, aggregation has started already at 73"C. Elsewhere (14), we argued that increased fouling of pasteurizers may be expected at temperatures of 8S"C and higher, by using the highest Hr. At lower heating rates and temperatures below 8S"C, both the limited availability of unfolded protein and the still moderate rate of aggregation appears to favor the deposition of (3-Lg on (milk) proteins, (colloidal) salts, and fat globules. Moreover, heat exchangers using different heating rates may have a significant effect on the extent of whey protein denaturation during heating to, or cooling from, the selected holding temperature. Therefore, to control thermal denaturation in heat exchangers, replacing the holding time at a selected temperature (as a characteristic

heat flow (mJIsl





300 .2

.3 200 - In k


90 temperature lOCI


o 2







prod.lct rurt>er



E!m F _ l I t y

P"l 4J5

3 tt. p-otWl


-tvra 4 " P'oUtIn

IZ2l ......on 10 "


Figure 6. Comparison of some functional properties of industrially prepared whey protein products with those of purified ~lactoglobulin: 1 ~lactog1obu1in, 2 Spberosil quaternary methyl ammonium, 3 native whey proteins, 4 Sphcrosil S, 5 defatted UP whey protein concentrate (WPC), 6 = neutral UP/diafiltration (OF) WPC, 7 = normal UP WPC, 8 = acid UPIDF WPC.










lIT x 103

Figure 7. Kinetic analysis of diffcnntial scanning calorimetric thennograms from whey protein concentrate (-) and ~lactoglobulin in whey permeate (--) as a function of temperature at pH 6.5. A: Thermognuns, B: Arrhenius plots. Protein concentration 10% (wt/vol). E a Activation energy.


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TABLE 1. Kinetic data of ~Iactoglobulin (.5%. wt/Vol) in various media at pH 6.5.

UDfoldiDg data Reaction

k 75 .-1




Water Permeate WPC

1.0 1.0 1.0 1.0 1.0 1.0

17.25 21.53 12.72 13.64 13.65 ND

Wbey Skim milk M.ilk, 3% fat


AgRgation data EA 70--75 (k1/mol)


k 75 (1./g••-1



367 338 346 294 261 ND

1.8 1.0 2.4 I.B 1.8 1.8


BA 7s--85 (kI/mo1)


214 275 3 IBS 300 307 374


.08 .12 .19 .19


Activation enctBY. lEA 2Detcrmined at B5'C. 3nerived from first-order reaction kinetics (rate constlDt k, in s-I).

parameter) by the equivalent heating time at a selected temperature (F value) appears to be a more effective method. The F value is calculated according to the equation (17):

Ig F

= [(f -

Trer)/Z] x dt,

where: Z

= 2.303

x R x 'f2/EA'

The F value is calculated by numerical integration for small time intervals (dt) and accessory temperature differences (T - T ref) to and from the (selected) holding temperature (free). The kinetic parameter (Z) in this equation expresses

the increase in temperature rC) required to achieve the same denaturation effect in onetenth of the time. The Z value for protein aggregation (irreversible denaturation) is calculated from the activation energy (EA) data shown in Table I, the universal gas constant (R), and the temperature (f) relevant for EA· This study supplies sufficient kinetic data for calculating the F value from temperature and time curves above the onset temperature for denaturation of P-Lg. Elsewhere (S), we showed that the denaturation of P-Lg is representative for that of WPC, Figure 9 shows temperature and time curves of two industrial pasteurization processes that are both characterized by a holding time of lOs at 9O"C and heat recoveries of 80 and 9S%, respectively. The arrows reveal the F value, when 7S, 80, or 8S"C


-----. eo ..



- - ...." - ¥


90 /






-E--', 15.9




Figure B. Pcrceotage of unfolding and of eggregation of duriDs heatiDg processes between 70 and 9O'C at heating rates of I'CI. and 1'C/min, as indicated. Aggregation of 1'C/miD (solid bar) or I'Cls (patterned bar) and unfoldiDI at 1'C/min (solid line) or I'CI. (broken line). ~lactog1obulin in whey

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70 I....:...-~____L.~_..L._.J~___'__~_'__~_____'_'~~







60 time


Figure 9. Temperatnre and time curves of pastewizers (25,000 lib) with 10. boldiDl time, using heat recoveries of BO and 95%, as indicated.



acD'egatlon (%)



25 20 15






wn.y. heat recov.-y 95%

80 _

WPI. heet recovery 80%

Figure 10. Effect of beatiog, during 10 • at tbe lDdieated temperaturel, on tbe ~tion of P-Iactoglobulin in wbey aDd Dative wbcy proteins (WPI), uing plllteOrizerll with heat recoveries of 80 aDd 9S%, as indicated.

are used as reference temperatures. The pasteurizer using 80% heat recovery operates with the effect of a holding time of more than 12 s at the indicated reference temperatures, whereas the pasteurizer using 95% heat recovery shows an F value of more than 16 s at 85·C or higher (for 10 s adjusted holding time). The effects of these different heating processes on the denaturation of ~Lg in whey and WPI are shown in Figure 10. Significant differences in protein aggregation (using heat recoveries of 80 and 95%) appear at 10 s holding time above 75·C. In WPI, this irreversible ~ tein denaturation increases to 7 and 10% on heating process adjusted to 10 s at 9O"C. The same heat treatments, perfonned at identical concentrations of ~Lg in whey, result in a denaturation at 9O·C of as much as 23 and 28% with heat recoveries of 80 and 95%, respectively. This feature is caused by the dominant

effect of salts, particularly calcium ions, on the denaturation of whey proteins. The heat treatments shown in Figure 10 are representative for most heating processes used during evaporation and drying of whey protein products. Also, the calculated (irreversible) denaturation effects of P-Lg [expressed as (100 - NSI pH 4.6)] are of the same magnitude as those observed for most whey protein products, such as those shown in Figure 6 and discussed by De Wit et at (9). This confirms the usefulness of this new kinetic approach for predicting the thermal stability and functionality of whey proteins after inevitable heating processes during the recovery of whey protein products. CONCLUSIONS

Some functional and technological aspects of whey proteins during and after heat treatments were studied on the basis of a new Joumal of Dairy Scieoce VoL 73,

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kinetic approach. In this approach, the kinetics of whey protein denaturation is split up into two successive processes: unfolding and aggregation. Within the temperature range between 70 and 9O o e, the rate of unfolding is several times higher than that of aggregation and does not appear to be rate detennining. The kinetics of aggregation appears to be representative for the irreversible denaturation of whey proteins. This infonnation may be useful predicting and controlling the functional properties of whey protein products during heat treatments. This study also shows that the thennal stability of whey proteins is significantly affected by environmental conditions, such as pH and ionic strength. REFERENCES 1 Borchardt, H. I, and F. Daniels. 1957. The application of differential thermal analysis to the study of reaction kinetics. J. Am. Chem. Soc. 79:41. 2 Capellos, C, and B.HJ. Bielski. 1972. Determination of order of reaction. Page 4 in Kinetic systems. C. Capellos and B.H.J. Bielski, ed. Wiley-Interscience, London, Engl. 3 Dannenberg, F., and H. G. Kessler. 1988. Reaction kinetics of the denatuIation of whey proteins in milk. J. Food Sci. 53:258. 4 De Wit, I. N. 1981. Structure and functional behaviour of whey proteins. Neth. Milk Dairy J. 35:47. 5 De Wit, I. N. 1984. Effects of various heat treatments on structure and solubility of whey proteins, J. Dairy Sci. 67:2701. 6 De Wit, J. N. 1984. Functional properties of whey proteins in food systems. Neth. Milk Dairy J. 38:71. 7 De Wit, J. N. 1988. Functiooal properties of whey proteins. A review. NIZO Rep. V-281, NlZO, PO Box 20, 6710 BA Ede, Neth. 8 De Wit, J. N. 1989. The use of whey protein products. A review. NIZO Rep. V-295, NIZO, PO Box 20, 6710 BA Ede, Neth.. 9 De Wit, I. N., E. Honte1ez-Backx, and M. Adamse. 1988. Evaluation of functional properties of whey protein concentrates and whey protein isolates. 3. Functional properties in aqueous solution. Neth. Milk Dairy J. 42:155. lODe Wit, I. N., and G. Klarenbeek. 1976. The emulsifying properties of whey protein concentrates. Zuive1zicht 68:442.

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11 De Wit, J. N., G. Klarenbeek, and R. de Boer. 1978. A simple method for the clarification of whey. Page 919 in Proc. 20th Int Dairy Congr., Paris, France. 12 De Wit, J. N., and G. Klarenbeek. 1986. Method for the determination of rehydration and solubility of powdered protein-rich milk products. Milcbwissenschaft 41(8):463. 13 De Wit, J. N., and G. Klarenbeek. 1986. The Klarograph: an automatic device for measuring viscosity, density and heat stability of milk and milk concentrates at temperatures up to 140·C. Zuivelzicht 78:168. 14De Wit, J. N., and G. Klarenbeek. 1989. Technological and functional aspects of milk proteins. Page 211 in Milk proteins. C. A. Barth and B. Schlimme, ed. Steinkopff Verlag Darmstadt, Gc:rmany. 15 Hannigan. K. I. 1982. Super protein from acid whey. Food Eng. 54(3):96. 16 Hill, C. C., and R. A. Grieger-Block. 1980. Kinetic data: generation, interpretation and use. Food Technol. 94:56. 17 Kessler, H. G., R. Fink, and F. P. Horak. 1984. The analysis and assessment of heat-induced changes in food contents with the aid of reaction kinetics. Z. Lebensm. Verfahrenstechnik 5:372. 18 Kinsella, I. E. 1984. Milk proteins: physicochemical and functional properties. Crit. Rev. Food Sci. Nutr. 21(3):197. 19 Marshall, K. R. 1982. Industrial isolation of milk: proteins: whey proteins. Page 339 ill Developments in dairy chemistry-I. Proteins P. F. Fox, ed. Appl. Sci. Publ., New York, NY. 20 Mirabel, B. 1978. Nouveau proc6d6 d'extraction des prot6ines du lactoserum. Ann. Nutr. Aliment. 32:243. 21 Palmer, D. B. 1977. High purity protein recovery. Process Biocbem. 12(6):24. 22 Pantaloni M. D. 1964. Etude de Ia transition R ~ S de la p-lactog10buline par spectropolarimetrie de diff6rences C. R. Acad. Sci. (Paris) 258:5753. 23 Pearce, R. 1.1983. Thermal separation of P-1actoglobulin and a-lactalbumin in bovine Cheddar cheese whey. Aust J. Dairy Techno\. 38(4):144. 24 Sharp, I. H. 1979. Differential thermal analysis reaction kinetics. R. C. McKenzie, ed. Academic Press, New York, NY. 25 Short, I. L. 1978. Prospects for the utilization of deproteinated whey in New Zealand. A review. NZ. J. Dairy Sci. Technol. 13:181. 26 Vreeman, H. I., and J. K. Poll. 1985. Het gedrag van P-Iactoglobuline tijdens verhitten tot 150'C zoaIs dat voIgt nit spectrofluorescentiemetingen. Int. NIZO Report NOV-1077, NIZO, PO Box 20, 6710 BA Ede, Neth.. 27 Watanaba K., and H. Klostermeyer. 1976. Heat-induced changes in sulphydryl and disulphide levels of P-1actoglobulin A and the formation of polymers. I. Dairy Res. 43:411.