Studies on Whey Protein Concentrates. 1. Compositional and ThermalProperties1

MAYANK T. PATEL and ARUN KILARADepartment of Food Science

The Pennsylvania State UniversityUniversity Park 16802

LEE M. HUFFMAN and SHEELAGH A. HEWITTNew Zealand Dairy Research Institute

Palmerston North, New Zealand

AVIS V. HOULIHANQueensland Food Research laboratories

Queensland, Australia

ABSTRACT

Cheddar-cheese-type whey proteinconcentrates were studied for their com­positional and thermal attributes. Thesamples were prepared from three milksystems, namely, skim milk:, whole milk:,and skim milk enriched with buttermilk.The concentrates from skim milk werelower in all fat components and higher inproteins, except for the membrane-associ­ated protein. The buttermilk-enrichedsamples had the most membrane-associ­ated components. The concentrates fromwhole milk and buttermilk-enriched, skimmilk were similar in protein composition,except for membrane-associated protein.The whole milk samples had the highestconcentrations of total and free fat com­ponents. Lactose content and mineralcomposition were similar for the threetypes of concentrates.

Thermal properties and denaturationkinetics were examined by differentialscanning calorimetIy. The samples exhib­ited a single broad endothermic peak withthe denaturation temperature near 76"Cand the enthalpy ranging from 1.86 to2.16 caVg. The concentrates from skimmilk had higher denaturation enthalpy,

Received July 5, 1989.Accepted Decembc% 4, 1989.lPublished as Paper Number 8196 of the Journal Series of

the Pennsylvania State Agricultural Experiment Station, Uni­versity Part:.

whereas the concentrates from butter­milk-enriched, skim milk had slightlyhigher thermal stability. The overall de­naturation process for whey proteins fol­lowed the reaction order n = 1.5 with anactivation energy ranging from 217 to251 kl/moI. The thermal properties wereobserved to be related to a number ofcompositional factors. The denaturationenthalpy was positively correlated with /3­lactoglobulin and protein content, andnegatively correlated with bound fat,membrane protein, and membrane-associ­ated lipid components. The denaturationtemperature correlated positively withphospholipid content, and the onset dena­turation temperature correlated positivelywith iron content.(Key words: whey protein, composition,thermal properties)

INTRODUCTION

The proteins remaining in milk serum afterall the caseins have been removed are termedwhey proteins. Wheys obtained as by-productduring the manufacture of various cheeses andcaseins are sources of recoverable proteins,which can be concentrated by various physico­chemical methods. The resulting whey proteinconcentrates (WPC) can be used as functionalingredients in many formulated foods. Twocritical aspects of WPC utilization in food ap­plications are the inherent compositional vari­ability and thermosensitivity of whey proteins.Commercial WPC manufactured under similarconditions often have large variations in their

1990 J Daily Sci 73:1439-1449 1439

1440 PATEL ET AL.

physicochemical and fWlctional properties (22).The thennal stability of whey proteins is ofmajor importance because heat treatments areinevitably used in the processing of dairy prod­ucts and in the manufacture of whey proteinproducts. Heating induces denaturation, aggre­gation, and precipitation in whey proteins. This,in tum, impairs ftmctional properties and bringsabout reduction in solubility, emulsifying,foaming, and gelation properties (32, 35). Ther­mal behaviors of individual whey proteins inmodel, whey, and milk systems have been stud­ied extensively by classical methods involvingseries of heat treatments with various time andtemperature combinations (10, 13) and usingdifferential scanning calorimetry (12, 18, 23,36). Only a few studies have addressed thethermal behaviors of all the whey proteinstaken together (I, 5, 11, 13).

The purposes of the present work. were: 1) tocharacterize the compositional and thermalproperties of Cheddar-<:heese-type WPC pro­duced from different types of fat-modifiedmilks, 2) to determine whether any relationshipexists between the thermal properties and com­position of WPC, and 3) to study the kinetics ofwhey protein denaturation.

MATERIALS AND METHODS

Whey Protein Concentrates

Six WPC were prepared from different lotsof Cheddar-cheese-type whey at the New Zea­land Dairy Research Institute, PalmerstonNorth, New Zealand Fresh, refrigerated wholemilk, comprising evening and moming bulkcollections, was obtained from the same supplydistrict during the period of November throughDecember and was used as starting material.Wheys were recovered from cheeses producedfrom three types of milk: whole milk, skimmilk, and a 90:10 (wt:wt) blend of skim milkand buttermilk. The trials were replicated sothat six batches of whey, two for each of thethree milk types, were produced Figure 1shows the manufacturing process for thecheeses and the WPC.

Compositional Analyses

Moisture was determined by air oven dryingat 105 ± 2'C for 16 h (4). Total N (fN) was

10urnal of Dairy Science Vol. 73, No.6, 1990

determined by the Kjeldahl method (24) using aKjel-Foss automatic 16200 analyzer (AIS N.Foss Electric, Hillerod, Denmark). NonproteinN was determined as soluble N in 12% TCAfiltrate of WPC (41). True protein was esti­mated from TN after correction for NPN usinga protein conversion factor of 6.38. Total fatwas determined using the Mojonnier mixedsolvent extraction procedure after hydrolysis ofWPC with heat and concentrated HCl (4). Freefat was determined (without preacid digestion)by fat extraction with anhydrous ethyl etherusing Tecator Soxtex lIT solvent extractionsystem (fecator Inc., Hemdon, VA) (4). Boundfat was estimated as the difference betweentotal and free fat. Lactose was determined by acolorimetric method (45) at 420 om using theChemLab autoanalyzer system (ChemLab in­struments Ltd, Homchurch, Engl.). Ash con­tent was measured by ashing the samples at550'C in an electric muffle furnace. The WPCwere analyzed for P, K, Ca, Fe, AI, and Zn byinductively coupled plasma atomic emissionspectroscopy using a method described byDahlquist and Knoll (9). Inorganic phosphate(8) and chloride (7) contents were determinedusing the ChemLab autoanalyzer by colorimet­ric procedures. Sodium was determined byflame emission spectrometry at 589 om and Mgby atomic absorption spectrometry at 422.7 om.

Milk fat globule membrane (MFGM) frac­tion was isolated from the WPC using a proce­dure (31), which involved centrifugation of re­constituted WPC (10% wt/vol) through aconcentrated sucrose solution and recovery ofmembrane material at the sample and sucroseinterface. The isolated membrane fractions andthe WPC were analyzed for protein, lipid, phos­pholipid, and triglyceride. Protein was mea­sured by modified Lowry procedure (19). lip­ids were extracted and analyzed by the methoddescribed by Folch et at. (20). Phospholipidcontent of the lipid extracts was calculated bymultiplying the phosphorus level by 25; phos­phorus level was determined by the method ofAmes and Dublin (2). Triglyceride concentra­tion was determined by subtracting the phos­pholipid content from the total lipid material.The free protein, lipid, triglyceride, and phos­pholipid concentrations were estimated as thedifferences in composition between the totaland the membrane components.

The WPC were analyzed for a-lactalbuminand ~-lactoglobulin by high performance gel

PHYSICOCHBMICAL PROPERTIES OF WHEY PROTEIN CONCENTRATES 1441

Figure 1. Flow diagram for the IIIlIIlUfacture of the experimental whey protein collCelltrateS from skim milk (SWPC),whole milk (WWPC) aJJd bultermilk-emiched, skim milk (BSwpc).

penneation chromatography (21). A Waters As­sociates (Milford, MA) liquid chromatographsystem consisting of M6000A dual pumps, aWISP 710B autoinjecter, an M490 multi­wavelength detector (operated at 280 om), andan 840 data and chromatography control stationwas used for sample analysis. The WPC sam­ples were dispersed (3 to 5 mg/ml) in .1 Mphosphate buffer (pH 6.8) containing 5% SOSand .1% dithioerythrltol, followed by heatingthe dispersions at 85"C for 15 min. Fifty micro­liters of filtered samples were injected andeluted through a Bio Sil TSK guard column(Bio ROO. Richmond, CA) and a Bio Sil TSK250 column (Bio Rad) linked in series. Sampleswere eluted in a.l M phosphate buffer (pH 6.8)containing .1% SOS at a flow rate of .5 mJlmin.1be content of a-lactalbumin and ~lactoglobu-

lin were determined using extinction coeffi­cients of 20.1 (26) and 9.7 (46), respectively.

Differential Scanning calorimetry

Thermal properties of the WPC were mea­sured using a Perkin-Elmer (Norwalk, CT)OSC-4 differential scanning calorimeter (DSC)equipped with a thermal analysis data station(TADS) software program. The instrument wascalibrated for temperature (1) and denaturationenthalpy (MI) using indium (T = 156.6"C, .MI= 6.8 cal/g) as a standard. Calorimetric experi­ments were conducted using 10% (wt/wt) WPCdispersion in distilled water at natural pH (6.34to 6.38). About 50 J1l of hydrated (40 to 60min) WPC dispersions were accurately weighedinto large volume capsules (O-ring stainless

Journal of Dairy ScieDCe Vol. 73, No.6, 1990

1442 PATEL ET AL.

k = Ko[exp(-EafRT)] [2]

A k__ > B + MI

where a. is the degree of denaturation and n isthe order of reaction.

The reaction rate is assumed to have a tem­perature dependence of:

where Ko = preexponential factor (lis), Ea =activation energy of denaturation (J/mol); R =gas constant, and T =absolute temperature (K).

Combination of equations [1] and [2] yields:

Compositional Attributes

Table 1 shows the mean values of the majorand the minor compositional attributes of theWPC manufactured from the three types ofcheese milk. The composition of the WPC wascomparable to that of membrane-processedWPC. Moisture and ash contents were in goodagreement with the results of previous studies(29, 30). The true protein contents of the WPCranged from 66 to 73%, which was in thenormal range for UP-processed concentrates(33). Protein contents of the WPC were slightlylower than those reported in other studies (29,30, 34). This could be due to the different fatconcentration in the WPC as a result of differ­ent milk types and modified method of cheese

RESULTS AND DISCUSSION

The equation [3] can be reduced to linearfonn as follows:

dO/dt = Ko[exp(-EJRT)](1 - o.)D [3]

Here, the reaction rate (dO/dt) at any temper­ature T can be calculated as the ratio of peak.height to total area, and the fraction of dena­tured protein (a.) can be calculated as the ratioof partial area to total peak area.

The values of Ko, Ea, arid n are obtainedfrom the multilinear regression perfonned usingIn (dO/dt) as dependent variable and Iff andIn (1 - a.) as two independent variables.

Statistical Analyses

The experimental trials were carried out as arandomized complete block design, where thethree milk types were randomized within eachblock (replicate). The WPC samples were ana­lyzed in triplicate for the various compositionaland thermal properties. The results were ana­lyzed by two-way analysis of variance, and thesignificance of difference between treatmentmeans was tested with least squares means testat 10% significance level (44). Simple correla­tion coefficients (r) between the compositionaland the thennal properties of the WPC (n == 6)were calculated using the SAS (43) program.

In(dO/dt) = 1nKo - EafRT + n[ln(1 - a.)][4]

[1]dO/dt = k(1 - o.)!1

where A is the protein before denaturation, B isthe protein after denaturation, and k is the rateof reaction.

In kinetic calculations, the rate of denatur­ation of A with time and its relationship withtemperature is calculated from a rate equationshown as:

steel pan), and the capsules were henneticallysealed using a crimper. The capsules wereweighed before and after the DSC scan toensure the effectiveness of sealing. The sampleswere scanned from 25 to 125·C at the scanningrate of 10·C/min against a previously denaturedWPC dispersion as a reference. The scannedsamples, after cooling to room temperature,were rescanned to detennine whether the wheyproteins had been denatured extensively andirreversibly. Onset denaturation temperature(f0)' denaturation temperature (fD), and AlIwere computed from the thennogram by theTADS system. The peak. maximum temperaturewas taken as TD, and the temperature at theextrapolation of maximum deflection of thecurve onto the baseline was taken as To. En­thalpy of denaturation was expressed as calo­ries per gram of WPC. The AlI was also calcu­lated and expressed as calories per gram ofprotein from the measured protein content ofthe WPC.

Kinetic parameters for denaturation of wheyproteins were computed from the DSC thenno­grams using Perkin-Elmer TADS, DSC-4 kinet­ics software. The basis of the computation is asfollows: The denaturation process is repre­sented by:

Journal of Dairy Science Vol. 73, No.6, 1990

PHYSICOCHEMICAL PROPERTIES OF WHEY PROTEIN CONCENTRATES 1443

TABLE 1. Compositionl of whey protein concentrates (WPC) prepared from fat-modified cheddar cheese wheys.

Whey protein concentrates2

Components SWPC WWPC BSWPC

X SD

Moisture, % 5.03 .16ToW N, % 12.35 .14NPN, % .73 .01True protein, % 73.31 .00a-Lactalbumin, % 10.7 .1/3-Lactoglobulin, % 32.4a .6Protein (Lowry), % 76.5a 2.5

Membrane protein, % 1.85a .00Free protein, % 74.7a 2.5

Lipid, % 4.9aa .68Membrane lipid, % 1.19 .06Free lipid, % 3.71a .62

Phospholipid, % l.86a .04Membrane phospholipid, % .86 .01Free phospholipid, % 1.00"" .03

Triglyceride, % 3.04a .64Membrane triglyceride, % .33 .OSFree triglyceride, % 2.na .60

Total fat, % 4.55a .77Free fat, % .50 .29Bound fat, % 4.04a .48

Lactose monohydrate, % 7.6 .6Ash, % 2.94 .14Phosphorus, % .34 .02Sodium, % .18 .01Potassium, % .65 .02Calcium, % .49 .02Magnesium, % .06 .00Chloride, % .08 .00Inorganic phosphate, % .23 .01Iron, mg/kg 12.5 3.5Aluminum, mg/kg 5.3 .4Zinc, mg/kg 6.2 .2

X

4.4311.39

.6668.499.7

29.5b

67.0b

2.12a

64.9b

11.59b

1.4010.19b

2.05a

1.00I.OSa

9.56b

.419.15b

11.80"3.728.09b

7.32.66

.32

.17

.61

.43

.05

.07

.1416.75.25.9

SD

.24

.39

.032.26

.4

.4

.0

.15

.22.21

.042.25

.03

.02

.042.15

.032.172.482.15

.34

.7

.12

.02

.01

.04

.01

.00

.00

.035.4

.0

.2

X

4.7711.57

.7169.2610.028.6b

63.0b

2.80b

6O.2b

9.37b

1.647.74ab

2.98b

1.031.96b

6.37ab

.615.76ab

8.35b

.557.80b

7.12.87

.38

.20

.63

.46

.05

.08

.2016.46.06.5

SD

.06

.16

.001.05

.2

.31.0.07

1.1.23.16.08.19.06.12.06.09.03.00.05.05.6.06.02.01.01.01.00.01.02

3.61.0

.5

a,~eans within a row having different superscripts differ (P<.I).

IData expressed as mean of two replication and standard deviation.

2SWPC =The WPC from skim milk Cheddar cheese whey, WWPC =WPC from whole milk: Cheddar cheese whey,and BSWPC = WPC from buttermilk-emiched, skim milk Cheddar cheese whey.

making. ~-Lactoglobulin, the major componentof whey proteins, constituted 41 to 44% of thetrue protein in the WPC, whereas the a-lactal­bumin constituted 14.2 to 14.6% of the trueprotein in the WPC. The observed inconsis­tency between the values of protein contentdetermined by Kjeldahl and Lowry methodscould be due to the inherent differences be­tween whey proteins and bovine serum albu­min, which was used as a standard (27). Morespecifically, lipids and phospholipids are re­ported to interfere with the Lowry method (37),although SDS was added in these detennina­tions to minimize that effect (19). The lactose

content of the WPC ranged from 6.5 to 8.2%,which was higher than that reported by Huf­fman (1989, unpublished data). Potassium, cal­cium. phosphorus, and sodium were the pre­dominant minerals in the WPC. Theconcentrations of these four minerals deter­mined for the WPC in the present study werehigher than those reported by Mangino et al.(30) for UF-processed WPC. Calcium and so­dium contents of the WPC were similar tothose reported by Huffman (1989. unpub­lished).

Among the various compositional attributesstudied for the WPC; ~-laetoglobulin; total,

Journal of Dairy Science Vol. 73, No.6, 1990

1444 PATEL ET AL.

Differential Scanning Calorimetric Studyof Thermal Denaturation of Whey Proteins

Figure 2. Differential scauning calorimetric thenno­grams of whey protein COIlCCIltratel (WPC) from vmouamilk systems (SWPC-l &. 2 =skim milk, WWPC-l & 2 =whole milk, BSWPC-l &. 2 = bIlttenni1k-cmiched, skimmilk); scauning rate 10'C/min; 10% (wt/wt) dispersion indistilled water at pH 6.34 to 6.38.

total and free fat components, and the butter­milk-enriched, WPC had the highest concentra­tion of membrane-associated constituents.There were few obvious trends among lactoseor mineral contents of the WPC from the threetypes of milk.

BSWPC·1

SWPC·2

WWPC·2

BSWPC·2

110

SWPC·l__~~ WWPC·l

70

TEMPERATURE (OC)

50JO

I.~~/s

Thermal denaturation of whey proteins wasexamined using DSC, which generates two im­portant attributes of the protein, namely, ther­mal stability as revealed by TD and the state ofTD as revealed by Mi. In this study, mixturesof whey proteins in the form of WPC wereevaluated for thermal characteristics rather thanthe isolated individual whey proteins studied byother workers (15, 36, 42).

All the WPC exhibited more or less similarpattern of peaks in the DSC thermograms (Fig­ure 2), reflecting the similar WPC composition

free, and membrane protein; total and free lipid;total and free triglyceride; total and free phos­pholipid; and bound fat contents were signifi­cantly (P<.l) affected by the types of milk. Forsome constituents, namely, troe protein, freefat, lactose, and iron, the variation between thereplicate samples was greater than the variationamong WPC from different types of milk; how­ever, iron was the only component with signifi­cant block effect. Such variations are oftenquite large for WPC samples that have beenprepared under nearly identical conditions (22).

The WPC from skim milk had significantly(P<.l) higher contents of ~-lactoglobulin, totalprotein, and free protein, and lower contents oftotal fat and bound fat as compared with theWPC from whole milk and buttennilk-en­riched, skim milk; differences in those attri­butes between the later two WPC were nonsig­nificant (P>.l). The WPC from whole milk hadsignificantly (P<.I) higher contents of free lip­id, total triglyceride, and free triglyceride ascompared with the WPC from skim milk buthad similar (P>.I) contents of those attributeswhen compared with the WPC from buttennilk­enriched, skim milk. The WPC from butter­milk-enriched, skim milk consistently had moremembrane-associated constituents (i.e., protein,lipid, triglyceride, and phospholipid) than theWPC from whole milk or skim milk had Thiscould be due to the phospholipid componentspresent in MFGM material in buttermilk. Even A

though the phospholipid level was substantially ~

increased in the WPC from buttennilk-en- ~riched, skim milk, the level of this component ~isolated in the membrane fraction did not in- 0

crease to the same extent. It is possible that !o'"

some of these membrane components could I­

exist in a "free" form and not as the high ffidensity complexes typical of MFGM and skimmilk membrane material. The lowest concentra­tions of membrane-associated constituents werein the WPC from skim milk, although therelative differences were small.

In general, the results in Table 1 indicatethat the WPC from skim milk were signifi­cantly lower in all fat components and higher inproteins (except for the membrane-associatedprotein). The WPC from whole milk and but­termilk-enriched, skim milk were similar inprotein composition except for membrane pro­tein, which was higher in the later. The wholemilk WPC had the highest concentration of

Journal of Dairy Science Vol. 73, No.6. 1990

PHYSICOCHEMICAL PROPERTIES OF WHEY PROlEIN CONCENTRATES 1445

TABLE 2. Thermal properties1 of whey protein concentrates (WPC) prepared from fat-modified Cheddar cheese wheys.

WPC

Denaturation enthalpy

ProteinDenaturationtemperature

Onsetdenaturationtemperature

SD.4.5.3

SD.2.2.1

X75.675.776.1

----- ('C) -----X69.769.870.0

----- (caIIg) -----

X SD X SDSWPC 2.l4a .02 2.92.04WWPC l.97b .02 2.88 .12BSWPC 1.9Ob .04 2.75 .01

a,~eans within a column having different superscripts differ (P<.1).

1Data expressed as mean of two replication and standard deviation.

2SWPC = The WPC from skim milk Cheddar cheese whey, WWPC = WPC from whole milk Cheddar cheese whey,and BSWPC = WPC from buttennilk-eoriched, skim milk Cheddar cheese whey.

of 13-lactoglobulin and a-lactalbumin observedin a number of studies. All the samples showedonly one broad endothennic peak, with TDranging from 75.4 to 76.2·C, which may corre­spond to the denaturation of 13-lactoglobulin (5,15, 42). The lack of sharpness of the peakcould be attributed to differences in the heatstability of the individual components of wheyproteins or to the lack of cooperativity in thedenaturation process (39) or to the differentthennal stability of specific domains of theprotein molecules (38). de Wit (11) has re­ported two distinct peaks for whey proteins:one near 70·C and other near 130·C. In thepresent study, no peak near 130·C was ob­served when the WPC were scanned from 25 to14O·C. The denaturation process in the hetero­geneous whey protein system seemed to begoverned by the dominating 13-lactoglobulinfraction, since the denaturation temperaturesobserved for isolated 13-1actoglobulin in numberof studies (5, 14, 15, 42) are comparable withthe denaturation temperatures observed for theWPC in this and other studies (5, 11, 14). InFigure 2, a typical shoulder on the left side ofthe peaks between 62 and n·c reveals theoverlapping of two endothermic peaks. It maybe due to denaturation of a-lactalbumin, whichis less heat stable than li-Iactoglobulin (5, 14,42). The denaturation temperature for a-lactal­bumin, as determined by DSC, has been re­ported to be 63·C (14), 65·C (42), or 61·C (5).

Rescanning of the cooled WPC samplesshowed no denaturation peak, indicating exten­sive and irreversible denaturation of the wheyproteins. de Wit et al. (14) and Ruegg et al.

(42) have reported 80 to 90% renaturation ofisolated a-lactalbumin in calorimetric studies.Such renaturation has not been observed for 13­lactoglobulin and serum albumin (13, 42).However, in this study, proteins in the WPCwere not renatured following the thermal dena­turation, which is in agreement with observa­tions of others (5, 13). The inability of (X­

lactalbumin to renature upon cooling in theheterogeneous whey protein system could beascribed to the formation of coaggregates fol­lowing denaturation of the whey proteins.

Thermal Properties of theWhey Protein Concentrates

Mean values of the thennal properties of theWPC manufactured from various milk systemsare shown in Table 2. It is well known that theheating (scanning) rate of samples has amarked effect on the results of Mi and TD

obtained by DSC (47); however, the resultshave not been significant when the scanningrate is between 2 and 10·C/min (15, 36, 40). Ascanning rate of 10·C/min was used in thisstudy, because it represents the common condi­tion used in many studies and, hence, shouldprovide a basis for comparison of results.

The Mi values ranged from 1.86 to 2.16 cal!g WPC with the WPC manufactured from skimmilk having significantly higher Mi values thanthose of the other WPC. This difference pre­sumably reflects a higher concentration of un­denatured protein as the WPC from skim milkhad about 4 to 5% more protein than the otherWPC had The Mi values have been correlated

Jomnal of Dairy Science Vol. 73, No.6, 1990

1446 PATEL ET AL.

TABLE 3. Kinetic parametenl of whey protein concentrates (WPC) prepared from fat-modified Cheddar cheese wheys

Activationenergy

Reactionorder

Preexponentialfaclors

(kJ/moI)

X SDSWPC 238ab 1WWPC 246& SBSWPC 221b 4

X1.41.51.3

SD.1.0.0

X81.0­83.4­74.4b

(1/s)

SD.4

1.71.4

""Means within a column having different superscripts differ (P<.l).

lOata expressed as mean of two replication and standard deviation.

2SWPC ='!be WPC from skim milk Cheddar cheese whey, WWPC =WPC from whole milk Cheddar cheese whey,and BSWPC = WPC from buttermilk-enriched, skim milk Cheddar cheese whey.

with the content of ordered secondary structureof protein (25) and could be used to monitorthe proportion of protein in concentrates that isnot denatured during processing (3). When theMI values were expressed on an equivalentprotein basis, there was no significant differ­ence for the three types of WPC.

The MI, when expressed as calories pergram of protein, ranged from 2.74 to 2.96, andthe values were comparable with the MI re­ported for a-lactalbumin and ~-laetoglobulin

(12, 14, 15, 42). Such arithmetic conversion ofthe MI values from calories per gram of WPCto calories per gram of protein, using the valuesfor protein content of the WPC, assumes thatthe nonprotein components in the WPC have noeffect on MI. However, the effect could besignificant in the case of WPC that have rela­tively a lower concentration of protein and ahigher concentration of nonprotein components.de Wit (11) and de Wit et al. (14) have demon­strated the effect of lactose and residual fat onMI values for WPC.

Variation was slight for TD and To amongthe WPC from the three types of milk; howev­er, the variation among the samples was non­significant (P>.I). As mentioned earlier, thetransition temperatures observed for the WPCwere in fair agreement with the values observedin other studies (5, 11, 14). The WPC manufac­tured from the buttennilk-enriched, skim milkhad slightly higher thermal stability (higher TD)than that of the other two types of WPC. Thedifference could be ascribed to a possible sta­bilizing effect of the higher levels of phospho­lipid (amphiphilic molecules) on whey proteinsin the WPC from buttermilk-enriched, skimmilk. The stabilizing effect of amphiphiJes,such as SDS, on whey proteins against heat

Journal of Dairy Science Vol. 73, No.6, 1990

denaturation has been demonstrated byDonovan and Mulvihill (17).

Denaturation Kinetics of Whey Proteins

The kinetic parameters for the denaturationof whey proteins in WPC dispersions, as stud­ied by DSC, are shown in Table 3. As de­scribed earlier, the kinetic measures were ob­tained from the endothermic peak area for thetemperature range of 60 to 9O"C. The linearrelationship between partial peak area and con­centration of undenatured whey proteins is es­sential for obtaining the kinetic constants. Parkand lAmd (36) have demonstrated such a rela­tionship for 13-lactoglobulin over a concentra­tion range of 0 to 15%. Also, Ma and Har­walkar (28) have demonstrated that the heatingrates ranging from 5 to 20·C/min and proteinconcentration ranging from 5 to 20% have noeffect on the kinetic parameters obtained fromDSC thermograms of oat globulin. de Wit andSwinkels (15) and Park and Lund (36) haveused DSC to obtain kinetic measures of 13­lactoglobulin denaturation.

In this study, we used the mixtures of wheyproteins in the form of WPC to study thekinetics of the overall denaturation process. Theactivation energy for the WPC ranged from 217to 251 kl/mot The reaction order for the over­all whey protein denaturation process for theWPC dispersions (10% wt/vol) in distilled wa­ter at pH 6.34 to 6.38 ranged from 1.3 to 1.5.The Ea and the reaction order for whey proteindenaturation were lower for the WPC manufac­tured from buttennilk.-enriched, skim milk thanthose for the other WPC. No reported work: isavailable for direct comparison of the kinetic

PHYSICOCHEMICAL PROPERTIES OF WHEY PROTEIN CONCENTRATES 1447

TABLE 4. Signif'lcant correlation coefficients (r) between compositional and thermal properties of whey proteinconcentrates prepared from fat-modified Cheddar cheese wheys.

Thermal properties

Compositionalproperties

DenaturationenthaIpyl

Denaturationtemperature

Onsetdenaturationtemperature

!3-Lactoglobu1inProtein (Lowry)Free proteinMembrane proteinMembrane lipidMembrane triglyceridePhospholipidFree phospholipidMembrane phospholipidBound fatIron

.87*

.98***

.99***-.86*-.93**-.87*

-.95**-.91*

.83*

.82*

.93**

IBased on enthalpy per gram of whey protein concentrate.

*P<.05, n = 6.

**P<.OI, n = 6.

***P<.OOI, n = 6

up to the peak temperature, and the subsequentnonlinearity has been attributed to secondaryreactions such as aggregation.

Figure 3. Arrhenius plots for the thermal denaturationof whey proteins for various concentrates (WPC); k = rateconstant; T = absolute temperature; SWPC = WPC fromskim milk, WWPC =WPC from whole milk and BSWPC =WPC from buttermilk-enriched, skim milk. R2:>.99.

3.1.,

- WWPC

•••••. BSWPC

.......... SWPC

2.9

l/T (10-3 11K)

2.8

: r,-2

UJ

"- -3

-4.YC

-5 ~--.J

-6

-7 r-8

2.7

measures obtained in this study for the overallwhey protein denaturation. de Wit andSwinkels (15) have reported that the denatur­ation of ~-lactoglobulin between 65 and 70'Cin phosphate buffer (pH 6.75) follows first­order kinetics with an Ea of 306 kllmol; how­ever, Park and Lund (36) have reported thedenaturation of l3-lactoglobulin between pH 6.0and 9.0 follows second-order kinetics with anEa of 523 kllmol. The kinetic parameters ob­tained using DSC are comparable with the ki­netic parameters obtained using a classicalmethod involving heating samples at varioustime-temperature combinations (10). Dannen­berg and Kessler (10) have found that the dena­turation of ~-lactoglobulin in the temperaturerange of 70 to 9O'C follows a reaction order of1.5 with an Ea of 265 to 280 kllmol and that ofa-lactalbumin follows a reaction order of 1.0with an Ea of 269 kJ/mol.

Arrhenius plots of the TD of whey proteinsfor the various WPC are shown in Figure 3.The rate constants at various temperatures wereobtained from DSC thermograms using a kinet­ics program. The straight line for each WPCreveals a good fit (R~.99) of the thermogramswith the Borchardt and Daniels kinetic model(6). de Wit and Swinkels (15) have obtainedstraight line Arrhenius plot for 13-lactoglobulin

Journal of Dairy Science Vol. 73, No.6, 1990

1448 PATEL ET AL.

RelatIonship Between Compositionaland Thermal Properties

Table 4 shows the relationship between thecompositional attributes and the thennal prop­erties of the WPC in tenns of Pearson correla­tion coefficients (r) (n = 6). The .6H (caloriesper gram of WPC) was positively correlatedwith p-Iactoglobulin, Lowry protein, and freeprotein contents, and was negatively correlatedwith bound fat and membrane-associated com­ponents of protein, lipid, triglyceride, and phos­pholipid.. The TD correlated positively withtotal and free phospholipid; whereas, the Tocorrelated positively with iron content in theWPc.

The positive correlation between proteincontent and .6H is obvious when the later isexpressed in calories per gram of WPC, but thepositive correlation between p-lactoglobulinand .6H could be interpreted in tenus of itsdominating role in governing the denaturationprocess in the heterogeneous whey protein sys­tem. As discussed earlier, the similarity be­tween TD observed for isolated p-Iactoglobulinand the TD observed for WPC in this study andother studies, further accentuate the dominatingrole of P-Iactoglobulin in overall denaturationprocess of whey proteins. The negative rela­tionship of .6H with the bound fat and themembrane-associated lipid components couldbe ascribed to a possible requirement for therupturing of more hydrophobic interactions dur­ing heat denaturation of the WPC having higherproportions of bound fat and membrane-associ­ated lipid components. Because rupturing ofhydrophobic bonds is an exothermic process(28), it might lead to a decrease in the .6H ofthe endothenns of protein denaturation. Thepositive correlation of TD with phospholipidcould be interpreted as the stabilizing effect ofphospholipid on whey proteins against heat de­naturation. Such stabilizing effect of lipid onserum albumin has been demonstrated by Ber­nal and Jelen (5) and de Wit et al. (14), usingthe DSC. Also, as mentioned earlier, an in­crease in the thennal stability of protein bySDS binding has been demonstrated (17). Thepositive correlation of iron with To could beinterpreted in tenus of stabilizing effect of ironon the more heat-sensitive components of wheyproteins against heat denaturation. An increasein the thennal stability of conalbumin by iron

Journal of Dairy Science Vol. 73, No.6, 1990

binding has been known for some time (16).Even though correlation analysis does not

explain a cause-and-effect relationship, it pro­vides useful infonnation on the interactions ofvarious physicochemical attributes. However, afurther investigation, encompassing differenttypes and more number of WPC, will be re­quired to generalize the relationship betweencompositional and thennal properties of WPC.

ACKNOWLEDGMENTS

The authors wish to thank. the New ZealandDairy Research Institute (NZDRI) for thepreparation of the WPC samples; and G. C.Patel and M. N. Vaghela (The PennsylvaniaState University), and M. S. Roberts and E. F.Conaghan (NZDRI) for their help in sampleanalyses. This research was a. part of a coopera­tive research effort with the CommonwealthScientific Industrial Research Organization(CSIRO) Dairy Research Laboratory, Australia;Queensland Food Research Laboratories, Aus­tralia; the New Zealand Dairy Research Insti­tute, New Zealand; Ohio State University, andThe Pennsylvania State University; and wassupported in part by NSF Grant !NT 8211388.

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