8/12/2019 1994 Effect of Calcium and Magnesium Ions on Aggregation of Whey Protein Isolate and Its Effect on Foamng Properties

1/7

858 J. Agric. ood Chem. 1994, 42, 856-862

Effects of Calcium and Magnesium Ions on Aggregation of WheyProtein Isolate and Its Effect on Foaming PropertiesHaimin g Zhu an d Sr in iv asan Damo d ar an ;

De partm ent of Food Science, University of Wisconsin-Madison, 1605 Linden Drive,Madison, Wisconsin 53706

Th e effects of calcium an d magnesium ions on time -dep ende nt aggregation of whey protein isolate(WPI) and t he influence of the aggregates on the foaming properties of W P I have been studied. BothMg2+and Ca2+ n duced form ation of stable colloidal aggregates; th e ra te of aggregation was slow, andthe r ate a nd e xten t of aggregation were maximum a t 0.02-0.04 M concentration. Both foamabili ty andfoam sta bility of W P I foams were affected by th e con centration of the div alent ions, as well as incuba tiontime in salt solutions before foaming: Maxim um foam ability and foam stability were observed whenW P I was foamed immediately a fter the addition of the d ivalent salts, and th ey decreased progressivelywith incub ation time, indicating th at s olution-ph ase aggregation of W P I adversely affected its foamingproperties. In addit ion, evidence also is presente d which suggests tha t, during initial stages, th e proteinfilms formed a t he foam interface undergo contraction in the presence of Ca2+ nd Mg2+ ons, resultingin a decrease in pressure at plateau borders of the foam.

INTRODUCTIONWhey protein concentrates (WPC) prepared by ultra-filtration typically contain abo ut 0.5 calcium (Liao andMangino, 1987; Kim et al., 1989; Pa tel an d Kila ra, 1990).Lite rature repo rts on the effect of calcium on the foamingproperties of whey proteins are often contradictory. Somestudies have reported that addition of calcium to wheyproteins decreased th e overru n (Cooney, 1974; Richert e tal., 1974),whereas other studies have shown th at calciumenhanced th e foaming properties of whey proteins (Hans enand B lack, 1972; McDonough e t al., 1974). In a com par-ative study on the surface active properties of W PCprepared from several batches of skim m ilk whey, whole

milk whey, and buttermilk-enriched skim milk whey apositive correlation was found between the foamingproperties and calcium content of W P C samples (Pateland K ilara, 1990). However, since the calcium c onte nt ofthose WPC samples varied only narrowly from 0.42 to0 . 5 1 % , it is not clear whether the positive correlationobserved would be true at higher and at lower calciumconce ntrations as well. Since calcium is a major min eralcom ponen t of WPC, systematic studies based on a soundmethodology are needed to elucidate the effects of calciumon the film-forming and foaming properties of wheyproteins. In the present stu dy , the effects of divalentcalcium an d magnesium ions on the foaming properties ofwhey protein isolate prep ared by an ion-exchange processare presented.MATERIALS AND METHODS

Commercial whey protein isolate (BiPRO)prepared by anion -exchange process was obtained from Le Sueur Isolates Co., LeSueur,MN. All of the experimentsreported here were conductedon a single lot of whey protein isolate. According to theman ufacturer, the typical composition of this WPI on dry basiswas 95% protein,

8/12/2019 1994 Effect of Calcium and Magnesium Ions on Aggregation of Whey Protein Isolate and Its Effect on Foamng Properties

2/7

Effects of Ca and Mg Ions of WPI J. Agric. ood Chem., Vol. 42, No. 4, 1994 857

0 400 800 1200T h e ( m l n )

80

6000

40

040 400 6 0 0 1200Tlme (mln)

Figure 1. Effects of CaClz (A, top ) and MgClz (B, bottom ) onthe rate of aggregation of WPI ( 5 ) in water at pH 6.8 0 osalt; A,0.02 M; Q0.04 M; 0,O.M M; A,0.08My 0.10M, X0.15M; + 0.20 M.parametersof the two kinetic phases. Each foamingexpe rimen twas done at least in triplicate.RESULTS AND DISCUSSION

Effects of Divalent CationsonAggregation ofWPITh e effects of calcium an d magnesium ions on the r ate ofaggregation of W PI a re shown in Figure 1. Th e r a t e an dexte nt of aggregation of W PI progressively increased whenth e concen tration of either CaCl2 or MgCl2 was increas edfrom 0 t o 0.04M. A greater increase in the rate and extentof aggregation was observed between 0.01 an d 0.02 M inth e case of CaCl2and between 0.02 n d 0.04 M in the caseof MgC12 (F ig ure 1 .Above 0.04M, bo th CaC12 an d MgClzprogressively decreased th e rat e and ext ent of aggregationof WPI. A t allsaltconcentrations, th e rat e of aggregationof W PI was slow and reached an equilibrium value onlyafter about 200-400 min. Prolonged stan ding of th esolutions did not cause settling of th e aggregated particlesa t the bottom of the tube, and th e solutions remainedtranslucent in apppearance, indicating th at th e aggregateswere stable colloidal particles. Th e salt concentration usturbidity profiles of W PI inc ubat ed for 1400min in CaClzand MgCl2 solutions are presented in Figure 2. In bothCaClz an d MgClz solutions maxim um aggregation of WP Ioccurred a t about 0.04M salt concentration. However,

70

60

0. 00 0.05 0. 10 0.15 0.20 0.25Sa l t concen t ra t l on M)

Figure 2. Salt concentration u s turbidity profiles of WPI ( 5 )in CaClZ 0 ) nd n MgClz 0)olutions. The transmittance at500 nm of the protein solutions was taken after the solutionswere incubated for 24 h at room temperature. All solutionscontained 0.02% sodium azide.th e exte nt of aggregation of WPI was more in CaClz tha nin MgClz solution. This may be attr ib uted either toformation of larger colloidal aggregates or to a greateramount of protein aggregation in CaC12 than in MgClzsolutions.Previously, Zittle et al. (1957) reported that calciumwas bound very strongly to @-lactoglobulin ven below 10mM CaC12 Concentration. T he am ou nt of calcium boundwas stoichiometrically equivalent to th e net charge of theprotein. Although several studies relating to aggregationand precipitation of heated @-lactoglobulin n d WPC bycalcium ions have been reported (Zittle et al., 1957;Varunsatian et al., 1983; Patocka and Jelen, 19911, thetime -dep ende nt aggregation behavior of neither native&lactoglobulin nor W PI by calcium ion has been reported.Th e dat a presented here clearly indicate th at th e nativewhey proteins in WPI undergo t ime-dependent slowaggregation in divalent salt solutions, and the rate andextent of aggregation are depen den t both on salt concen-tration an d on the type of divalent cation. This aggregationpheno men on might be related to specific binding of Caz+and M g2+ ions to individual whey proteins. Underequilibrium conditions, that is, at long incubation time,the salt concentration us aggregation profile (Figure 2)exhibits a salting-in and salting-out solubility profilesimilar to those observed for other food proteins (Shen,1981; Kum osinski, 1990). At low salt concentration (0-40m M ), binding of Ca2+ an d M g2+ ions to whey proteinscauses aggregation (salting-out) of th e proteins; a t highersal t concentrations, how ever, additiona l binding of thesedivalent cations to the protein aggregates results inresolubilization (salting-in) of the aggregates. Th e initialaggregation a t ow salt concentrations might be attr ibu tedto two possible mechan isms: Firs t, binding of divale ntcations might cross-link the proteins via ionic bridges,leading to polymerization. However, Zittle e t al. (1957)had shown th at even at low concentrations (10 mM) th enum ber of moles of Ca2+ ions bound to &lactoglobu linwas directly related to th e net charge of the protein a ndwas independe nt of both protein concentration a nd thedenatured state of B-lactoglobulin. This could not bepossible if Ca2+was involved in forma tion of ionic bridgesbetween th e protein molecules. Second, binding of Caz+or Mg2+ ons to strong binding sites in whey proteins might

8/12/2019 1994 Effect of Calcium and Magnesium Ions on Aggregation of Whey Protein Isolate and Its Effect on Foamng Properties

3/7

858 J. Agric. ood Chem., Voi. 42, No. 4, 19940.157 J

Zhu and Damodaran

: A0.10

0.05anvQ

0.00

-0.051:

-0.10 0 5 0 1 0 0 1 5 0Time (min)

\

No salt.02, 0.04 M

---t 0 . 1 0 M

\

,\\\

\\ 20 M,

0.15

0.10

0.05-n0.00

0 5 0 1 0 0 1 5 0Time (min)

Figure 3. Changes in the pressure inside the foam apparatusas a function of time during decay of WPI foams. (A, top) WPIin CaC12: 0,no salt; A, .02M; Q0.04 M; 0 O . l O M; A, .20 M.(B, bottom) WPI in MgC12; 0, no salt; A, .02 M; 0,0.04 M; 0 ,0.06M;.,.10 M. WPI concentration was 5 ,and the pH was6.8.

Pr o t e in f l l mcon t lnuous phagedisperse phase

L a m e l l a

Figure 4. Schematic representatio n of initial contraction of theprotein film and expansion of plateau borders in WPI foams inthe presence of calcium and magnesium ions.induce conform ational changes in whey proteins, resultingin an increase in the hydrophobic area of the proteinsurface , which may facilitate protein-protein interactionand aggregation. It is not clear which of these twomechanisms is primarily responsible for the time-depend-en t aggregation of whey proteins. It is probable th at bothof these me chanisms might be op erative at varying degrees.T he salting-in of th e aggregates observed a t higher CaCl2and MgCl2 concentration might be due to additionalbinding of salt to th e whey proteins, which may increaseth e hydrophilicity a nd solvation of the proteins, resultingin dissociation and resolubilization of the aggregates.

10i