Rheological analysis of anion-induced matrix transformations in thermally induced whey protein isolate gels

Food Hydrocolloids

Vol. 9 no. 1 pp.57-64, 1995

Rheological analysis of anion-induced matrix transformations in thermally induced whey protein isolate gels* Eilene L.Bowland, KAllen Foegeding! and Donald D.Hamann Department of Food Science, North Carolina State University, Raleigh, NC 27695-7624, USA 1 To

whom correspondence should be addressed at Box 7624, Raleigh, NC, USA

Abstract The formation of thermally induced whey protein isolate (WPI) gels exhibiting fine-stranded, mixed and particulate matrices was investigated. Various concentrations ofstabilizing salts (sodium sulfate and sodium phosphate) or a chaotropic salt (sodium thiocyanate) were used to form the different types of thermally induced gel matrices. Gelation under all conditions showed similar trends in small-strain viscoelastic properties. Storage modulus (G') increased during heating to and hold at 800 e followed by an additional increase in cooling to 25°C. Mixed matrix gels had proportionately more hydrophobic interactions (large t1G'hea/t1G'coo/) and had greater initial gelation rates than fine-stranded and particulate matrix gels. The proportion of hydrophobic interactions in the gels correlated with the anions' influences on the hydrophobic effect as indicated by their positions in the Hofmeister series.

Introduction Gelation of whey proteins contributes to the appearance, water binding capacity and texture of foods. Changes in texture are reflected by changes in fracture properties. Previous research has shown that monovalent and divalent cation salts have valence-specific effects on fracture properties of thermally induced whey protein isolate (WPI) gels (1). Changes in fracture properties, as well as water-holding capacities and appearance of gels, reflect microstructural alterations (2). In monovalent cation salt-containing gels, these microstructural alterations consist of a gradual change in final gel microstructure from fine-stranded to mixed to particulate matrices. This change occurs as salt concentrations increase from 25 to 500 mmol/drrr'. The extent of the change depends on the anion's position in the Hofmeister series and its calcium chelating ability (2). Understanding the mechanism by which anions mediate the microstructural transition will facilitate effective manipulation of functional properties of whey proteins in food systems. Accomplishing this objective requires investigation of the gelation mechanism and of how various protein-protein interactions involved in gelation translate .:. Paper no. FSR94-9 of the Journal Series of the North Carolina Agricultural Research Service. Raleigh. NC 27695-7624. The use of trade names does not imply endorsement by the North Carolina Agricultural Research Service of products named. nor criticisms of similar ones not mentioned.

© Oxford University Press

into different matrices. In this study small strain rheological analysis was used to investigate fine-stranded, mixed and particulate matrix formation in thermally induced WPI gels. These gels were produced by altering the concentration of two divalent, stabilizing anions (sulfate and phosphate) or one monovalent chaotropic anion (thiocyanate) .

Materials and methods Whey protein isolate suspensions Protein content of the WPI was determined using proximate analysis data from protein determination (N x 6.38, macroKjeldahl) (3). Protein suspensions were prepared by hydrating WPI (Davisco International, Inc., Le Sueur, MN, USA) in either 25,75 or 500 rnmol/drrr' Na2S04; 25, 150 or 500 mmol/drrr' sodium phosphate buffer at pH 7.0; 25,200 or 500 mmol/dm ' NaSCN; or 75 mmol/dm ' Na2S04 and 10 mrnol/dm' sodium phosphate buffer at pH 7.0 under constant stirring at 25 ± 3°C for 30 min. These salt concentrations were chosen based on the type (finestranded, mixed or particulate) of thermally induced gel matrix formed (Table 1). Suspensions were degassed under vacuum for 2 h, adjusted to pH 7.0 with 0.75 mol/drrr' NaOH or 0.75 mol/drrr' HCl and then diluted to 10% protein (w/v) with the appropriate salt solution.

E.L.Bowiand, E.A.Foegeding and D.D.Hamann

58

Table 1 Relationship between salt type and concentration , and gel microstructure I. Concentration (mrnol/drrr')

Salt

Na ZS0 4 Na zSOisodium Phosphate buffer Sodium phosphate buffer NaSCN

1 Recorded

Fine -stranded

Mixed

Particulate

25

75 75/10

500

25 25

150 a-200 b-500

500

from Bowland and Foegcding (2).

Dynamic rheological analysis A Bohlin VOR rheometer (Bohlin Reologi , Inc ., Cranbury, NJ , USA) was used to determine rheological transitions which occurred during gelation of WPI suspensions. Th e Bohlin C 14 concentric cylinder measuring system was used in all experiments. The measuring system consisted of a rotating cup and a fixed bob attached to a 11.14 g ern torque bar. Gels were formed by heating the samples from 25 to 80°C at 1°C/min, holding the temperature at 80°C for 3 h and then cooling the samples from 80 to 25°C at l OCI min. Testing conditions were est ablished using results from frequency and strain sweeps of cooled gels. A frequency of 0.05 Hz and a maximum strain of 0.00827 were used to determine storage moduli (G', elastic rigidity), loss moduli (G", viscous rigidity) and phase angles of samples before , during and after gelation. Frequency sweeps produced G' -frequency relationships that were described by the power law.

G'

= A (Frequency)"

Values of A and N were determined by performing a linear on log G' versus log frequency . A was the ymtercept and N was the slop e of the resulting line . Gel points (G P) were determined according to the method of Foegeding et ai. (4). The first ten G' data points with values ~1 % of the torque bar 's range were plotted against time . Linear regression analysis of these points produced an equation of a line which permitted the determination of the GP and initial gelation rate. The GP was the intercept of the regression line of G' versus time or temperature. The slope of the line represented the initial gelation rate. ~egression

Statistical analysis Differences in gel point times and initial gelation rates were determined using the 'pdiff' procedure under least squared means (5) . Differences between NazS04-containing samples with and without sodium phosphate buffer were

determined using the least significant difference (Isd) procedure (5).

Results and discussion Small strain rheological analysis In. gels containing 25 to 500 mmol/drrr' salt , a change in the nucrostructures from fine-stranded to mixed to particulate occurs. The extent of the change at any concentration depends on the anion's position in the Hofmeister series and its ability to chelate calcium (2). As a result , different concentrations of NaZS04' sodium phosphate buffer or NaSCN were used in this study to produce fine-stranded, mixed or particulate matrices (Table 1). Gels containing 25 mmol/drrr' Na ZS04' sodium phosphate buffer or NaSCN exhibit fine-stranded matrices (2). The mixed nature of mixed matrices varies greatly with the intermediate concentrations of the salts . However, changes in microstructure correspond to the shear strain at fracture trend . As a result , salt concentrations producing mixed matrices were chosen based on minimum shear strain at fracture values . Gels containing 75 mmol/drrr' Na ZS04 ' 150 mmol/dm? sod ium phosphate buffer and 200 mmol/drrr' NaSCN exhibit mixed matrices and produce minimum strain values. Gels containing 500 mmol/drrr' Na ZS04 or sodium phosphate buffer exhibit particulate matrices , and gels containing 500 mmol / dm' NaSCN exhibit mixed matrices (2). Each salt at the above concentrations was used to investigate the effects of anions on the gelation process of fine-stranded, mixed and particulate matrix gels. Rheological testing conditions were selected to minimize gel matrix damage and thus allowed for dynamic me asurement of gelation. Changes in the rheological properties of the samples were reflected in changes in storage moduli (G'), loss moduli (G") and phase angles. For all treatments, b.ot? G' (Figures 1-3) and G" (not shown) developed similarly. However , the predominantly elastic gels exhibited final G' values 10 to 100 times greater in magnitude than Gil values . As a result , only G' values are discussed. G' increased during holding and a dramatic increase occurred during cooling. Comparable G' development patterns have previously been reported for WPI , 13lactoglobulin and BSA gels (6-8). Average G' values at the end of cooling are seen in Table 2. Both sulfate and phosphate stabilize proteins but their effects on G' varied . The G' values of sulfate-containing gels were larger for mixed matrices and smaller for particulate matrices when compared to fine-stranded matrices. Conversely, the G' values of phosphate-containing gels were largest for particulate gels and smallest for fine stranded gels. The effects of the destabilizing anion thiocyanate on G' values were also unique. Gels containing thiocyanate exhibited a decrease in G' as the salt concentration increased to 200 mmol/dm' and the matrix transformed from fine-stranded to mixed (a-mixed) . When the thiocyanate salt concentration was further increased to 500

Rheolo gical analysis of anion-indu ced matrix transformations

59

Cool

Heat

20 . - - - - - - - - : : - - - - - - - - - - - - - - - - - - .

15

Time Figure 1

(mln.)

G' development in WPI samples containing 25 mrnol/drrr' Na2S0 4 (square), sodium phosphate buffer (circle) or NaSCN

(triangle).

20

Cool

Heat

.-----r-------------------.

15

5

oMH!!H~~~-..,.....--..,.....--..,......---L....._-____I 300 200 100 o

Time

(min.)

Figure 2 G' developm ent in WPI sample s cont aining 75 mrnol/drrr' Na2S04 (square) , 150 rnrnol/drrr' sodium phosphate buffer (circle), 200 mrnol/drrr' NaSCN (triangle) , or 7S mmol/drrr' Na2S04 and 10 rnmol/dm' sodium phosph ate buffer (X) .

E.L.Bow/and, E.A.Foegeding and D.D.Hamann

60

Cool

Heat

20 r----~-----------------.

15

-e 5

otJi!ltH!tIH~-.,...--~----,r--L..,.----1 300 200 100 o

Time

(min.)

Figure 3 G' development in WPI samples containing 500 mrnol/dm! Na2S04 (square), sodium phosphate buffer (circle) or NaSCN (triangle) .

Table 2

Th e relat ionsh ip among salt. matrix and final Gil and Gf2 values

Salt

Na 2S04 Na 2S04/sodium phosphate buffer Sod ium phosphate buffer NaSCN

I

Average of two replications ±

Mixed (kPa)

Find-str anded (kPa) G'

Gf

G'

11.9± 0.1

11.3 ± 1.0

0.6 ± 0. 1 7.1 ± 1.1

3.6 ± 1.3

16.3 ± 16.6 ± 6. 1 ± a-35.4 ± b-B.7 ±

Particulate (kPa) Gf

0.4 0.2 0.1 0.7 1.1

13.8 18.3 19.0 14.7 11.1

± ± ± ± ±

0.2 0.6 2. 1 1.5 1.8

G'

Gf

5.9 ± 0.2

5.4 ± 0.6

6.9 ± 0.7

9.4 ± 0.2

so .

~ Fracture moduli (Gf, shea r stress at fracture/shear strain at fracture ) values were recorded from Bowland and Foegedin g (2).

JValues for a- and b- are for gels contain ing 200 and 500 mrnol/drrr' NaSCN respectively.

mrnol/dm", the G' values of the resulting mixed (b-mixed) matrix gels increased . No associations between final G' values and microstructure were apparent. A comparison of small strain moduli (G') with fracture moduli (Gf) previously reported (2) (Table 2) allowed for a comparison of gel rigidity at small and large strains respectively . G' values were greater than or the same as Gf values for gels containing sulfate only, and for fine- stranded and b-mixed matrix, thiocyanate-containing gels . Gels with G' values greater than Gf values exhib it a decrease in

rigidit y with increasing strain. Mixed or particulate matrix-phosphate-containing gels , and fine-stranded matrix-thiocyanate-containing gels, exhibited lower G' than Gf values. This relat ionship indicated that rigidity increased with strain (sometimes called strain hardening) . The different stress/strain relationships exhib ited by whey protein gels at low and at fracture str ains cont rasts with the stress/str ain response of the ela stic hydrog el acrylamide . Acryl amide gels exhibit linear stress/strain relationships up to fracture (9).

Rheological analysis of anion-induced matrix transformations

Comparing rigidity moduli at small and large strains provided an indication of the mechanism of gel stabilization . A lack of change in moduli indicates stabilization via interactions that are linear in their response to strain energy. The difference between G' and Gf values of finestranded and particulate matrix , sulfate-containing gels was minimal (Table 2) . This relationship indicated that these gels were largely stabilized by interactions that were linear in their response to strain energy. The decrease between G' and Gf exhibited by some gels (mixed matrix, sulfatecontaining gels or fine stranded and b-mixed matrix, thiocyanate-containing gels) suggested that interactions maintaining rigidity at low strains were lost at high strains . The increase between G' and Gf exhibited by all phosphate-containing and a-mixed matrix, thiocyanate-containing gels indicated a transition to more rigid interactions at large strains. The molecular mechanisms responsible for these complexities are unknown ; however, they appear to be key factors determining textural properties of foods (i.e . fracture causing strains) . According to rubber elastic theory, G' should be large at high temperatures and small at low temperatures (10). The WPI gels in this study did not exhibit such a relationship . G' increased as temperature decreased (Figures 1-3) . Other researchers have reported increases in G' as temperature decreases (6-8). This behavior is attributed to the stabilization of these gels by a combination of bonds and interactions. G' increases with temperature if entropy elasticity and hydrophobic interactions are the dominant forces contributing to matrix rigidity . Conversely, an increase in rigidity during cooling is indicative of hydrogen bonds contributing to matrix rigidity. Thus the increase in G' during heating and holding was attributed mainly to hydrophobic interactions and the increase in G' during cooling was attributed to the formation of hydrogen bonds. The relative contribution of hydrophobic interactions and hydrogen bonds to G' was determined by comparing the change in G' during heating to the change in G' during cooling (LlG'hea/.1.G'cool) (Table 3). Fine-stranded and

Table 3 Salt

Relationship among salt , matrix and 6.C' hcacl6.C'coolI Fine-stranded (kPa)

0.8 ± 0.0 Na2S04 Na2S04!sodium phosphate buffer Sodium phosphate buffer 0.4 ± 0.0 NaSCN 0.3 ± 0.0

Mixed (kPa)

Particulate (kPa)

1.0 ± 0.0 1.0 ± 0.0

0.3 ± 0.0 -

0.6 ± 0.0 a- 20.9 ± 0.0 b-1.4 ± 0.0

0.4 ± 0.0 -

1 Average of two replications ± SD. 2Values for a- and b- are for gels containing 200 and 500 rnrnol/drrr' NaSCN respectively.

61

particulate matrices (2) exhibited low .1.G'heat/.1.G' cool ratios. Mixed matrices exhibited high ratios . The LlG' hea/.1.G'cool trends within each salt correlated with the known effects of salt s on proteins. At low ionic strengths, all salts tend to 'salt-in' (i.e. destabilize) proteins. At f.l. > 0.15, salts begin to 'salt-out' (i.e. stabilize) or 'saltin' proteins by increasing or decreasing the hydrophobic effect (11). According to the Hofmeister series , anions saltout proteins in the following order SOi->HPoi-» > SCN- (12,13). The increase in LlG'hea/.1.G' cool ratios between 25 rnrnol/drrr' of either Na ZS04 or sodium phosphate buffer (fine-stranded matrix) and 75 mmol/drrr' (mixed matrix) Na ZS04 or ISO mrnol/dm' (mixed matrix) sodium phosphate buffer respectively (Table 3) reflected increased salting-in or destabilization. This destabilization increased the regions available for hydrophobic interactions during gelation and thus increased the relative contribution of hydrophobic interactions to final G' values. As a result, .1.G'hea/G'cool increased. The decrease in .1.G\ea/.1.G'cool between gels containing 75 mrnol/dm? (mixed matrix) Na ZS04 or 150 mmol/drrr' (mixed matrix) sodium phosphate buffer and 500 mrnol/drrr' (particulate matrix) of either salt reflected increased salting-out or stabilization. This stabilization decreased the regions available for hydrophobic interactions during gelation and thus increased the contribution of hydrogen bonds to final G' values. As a result, .1.G'hea/G'cool decreased. The .1.G'hea/ G'coo) trend produced by NaSCN-containing gels supported these observations. The .1.G'hea/G'cool ratio increased for gels containing 25 mmol/dm? (fine-stranded matrix) to 500 mmol/drn? (mixed matrix) NaSCN. NaSCN destabilizes proteins. Consequently more hydrophobic regions were exposed for hydrophobic interactions during gelation, and .1.G\ea/G'cool increased. These results indicated that mixed matrices contained proportionately more hydrophobic interactions than fine-stranded or particulate matrices. None of the treatments produced large differences in phase angle development. However, variations in heating transitions existed (Figure 4). After cooling all the gels exhibited comparable phase angles of '''S. The phase angle provides an indication of the viscoelasticity of a sample . A phase angle of 90° denotes a completely viscous sample , and a phase angle of 0° denotes a completely elastic sample. Therefore, results indicated that all of the gels were predominantly elastic in nature. Changes in the phase angle during heating and holding reflected viscous relaxation which occurred within 20 s (i.e . 0.05 Hz). Phase angles are dependent not only on the visoelasticity of the sample, but also on the frequency with which they are measured. To investigate relaxations over a broader time span, frequency sweeps from 0.005 to 10.0 Hz were performed on final gels. Frequency sweeps indicated that G' was a function of frequency to the N power. N, obtained from a linear regression of log G' versus log frequency, provided another indication of the viscoelastic nature of the gels. The N value of a purely elastic gel is zero

£. L. Bowland, E.A.Foegeding and D.D.Hamann

62

Cool

Heat

90 . . - - - - - - - - - - - - - - - - - - - -......

60

30

O~--""T"""!...--__r---_--...,....--L..r--~

o

300

200

100

Time

(min.)

Figure 4 Phase angle development in WPI samples conta ining 25 rnmol/d rrr' Na2S04 (square), sodium phosphate buffer (circle) or NaSCN (triangle).

Table 4

Relationship among salt, matri x and power law exponent NI.2

Salt

Na2S04 Na2S04 and sodium phosphate buffer Sod ium phosphate buffer NaSCN

Fine-str anded (kPa) 3.9

X

10- 2 ± 0.00

4.2 x 10- 2 ± 0.004 4.0 x 10- 2 ± 0.001

Mixed (kPa)

Particulte

(kPa )

2. 1 x 10- 2 ± 0.001 2.3 x 10- 2 ± 0.001 4.5 x 10- 2 ± 0.004 a-34.5 x 10- 2 ± 0.001 b-3.2 x 10- 2 ± 0.001

5.8

X

10- 2 ± 0.001

4.8

X

10- 2 ± 0.003

I Average of two replications ± SD . l T he power law exponen t N was determined by linearly regre ssing on the data points of log G' versus log frequency. ~ Va l ues for a- and b- are for gels containing 200 and 500 mrnol/drrr NaSCN respectively.

because G' of a purely elastic gel is frequency independ ent. All N values were with in 10- 2 of zero (T able 4) , further ind icating that all of the gels were predominantly elastic in nature . Comparison of N values among microstructure types revealed no general relationships .

Gel points and gelation rates Gel points (GPs) were determined for each of th e gels except the fine-stranded matrix, pho sphate-containing gels. Fine-stranded matrix, phosphate-containing gels did not produce G' values ~1 % of the torque bar's range until the

cooling phase. Extr apolation to the GP from the cooling phase produced erroneous results . Consequently , the OP of fine-stranded matrix , phosphate-contain ing gels was not determined. Other phosphate-containing samples gelled between 76 and 78 (Table 5) . Samples cont aining sulfate or th iocyan ate gelled between 79 and 80°C. Gel point times indicated that the times required for gelation at 80°C varied . No simple relati onships among OPs, salt type and gel microstructure were derived from th e data. How ever, some general observations were mad e . In the gelation process , proteins denature before aggregatin g and finally forming a QC

Rheological analysis of anion-induced matrix transformations

Table 5

63

Effects of salt and matrix on WPI gel formation

Salt

Na2S0.j and sodium phosphate buffer Sodium phosphate buffer

NaSCN

Matrix

fine -stranded mixed particulate mixed find-stranded mixed particulate fine-stranded a- 2mixed b-mixed

Gel point times (min ) 56.2 ± O.I d 54.8 ± O.4c 60.2 ± O.I f 54.I±0.I 3 ND 51.0 ± 0.6" 53.8 ± O.I b 57.3 ± O.Oc 54.2 ± O.Ob 54.0 ± 0.2 b

Gel point (0C)

80.0 80.0 80.0 78.9

± 0.6

NO 76.2 ± 0.0 78.2 ± 0.9 80.0 79.2 ± 0.0 79.2 ± 0.7

Initial gelation rate (kPa/min)

9.2 3.7 77.2 3.3 79.9 11.9 18.0 17.8 14.4 3.5

± ± ± ± ± ± ± ± ± ±

0.8 b 0.2"

z.i0.3 1.3c 0.5 b .c 1.Sb .c .d 2.1 c .d

I.oc .d 0.4"

ND . not determined. "- '" Means within columns with different superscripts indicate significantly different times required to gel or significantly different initial gelation rates (P < 0.05). 1 Average of two replications ± SD . ~ Values for a- and b- are for gels containing 200 and 500 mmol/drrr' NaSCN respectively . JGel point times of Na~S04' and Na ~S04 and sodium phosphate buffer containing gels were determined to be not significantly different using the least significant difference procedure.

gel (10). Anions which stabilize proteins inhibit denaturation and thus inhibit gelation. As a result, stabilizing anions were expected to require longer times to gel and produce higher GPs than destabilizing anions . With the exception of fine-stranded matrix thiocyanate-containing gels, gels containing sulfate required significantly (P < 0.05) longer times to gel than samples containing thiocyanate or phosphate. Contrary to the stabilizing effect of phosphate, phosphate-containing samples began to gel at relatively low temperatures (Table 5). A relationship between initial gelation rate and microstructure was apparent. Gels containing sulfate or phosphate exhibited relatively high initial gelation rates when fine-stranded and particulate matrices were formed, and low initial gelation rates when mixed matrices were formed . The initial gelation rates of thiocyanate-containing gels supported this relationship . The initial gelation rates of thiocyanate-containing gels only decreased as gel matrices transformed from fine-stranded to mixed . No explanation for the magnitude differences in initial gelation rates among the salts was discerned.

Chelation effects of phosphate The stabilizing anions, phosphate and sulfate did not produce analogous results in the magnitude of G' values , the relationship between G' and Gf (Table 2), the viscoelastic nature of gels (Table 4), GPs or the magnitude of initial gelation rates (Table 5). This disparity may be due to the chelation of calcium by phosphate. Calcium is present in WPls at approximately ten times the concentration of the other dominant divalent cation magnesium (14). Consequently, the chelation of divalent cations by

phosphate was considered to be calcium chelation. Bowland and Foegeding (2) found that addition of 10 mrnol/dm ' sodium phosphate buffer, to a 75 mrnol/drrr' (mixed matrix) NazS04-containing sample chelated enough calcium to change the fracture properties and microstructure of the gel (2) . To determine if differences between sulfate- and phosphate-containing samples in the current study were due to calcium chelation by phosphate, samples containing 75 mrnol/drrr' Na ZS04 and 10 mmol/dnr' sodium phosphate buffer were characterized and compared to gels containing 75 mrnol/dm? Na ZS04 (mixed matrix). Addition of 10 mrnol/drrr' sodium phosphate buffer to 75 mrnol/drrr' NazS04-containing samples did not alter the gelation process from that of the 75 rnmol/drrr' Na ZS04containing samples (Figure 2). G' and G" (not shown) were comparable in trend and magnitude to the 75 mmol/drrr' NazSO-t-containing samples. These results were consistent with the findings that G' and G" exhibited no simple relationship to microstructure. These results also support Stading and Hermansson's findings that small strain analysis does not detect changes in microstructure while large strain analysis does (15). Large strain analysis primarily detects macro-properties of a matrix such as strand and/or particulate rigidity . Changes in microstructure greatly affect these macroproperties. As a result, large strain analysis detects changes in microstructure. Conversely, small strain analysis primarily detects micro-properties of a matrix such as rigidity of individual molecules composing the strands and/or particulates. Therefore, changes in gel matrices that resulted in changes in rigidity of strands and/or particulates were not reflected in small strain analysis. Although addition of 10 mmol/dm' sodium phosphate buffer to 75 mmol/drrr' NaZS04 containing samples did not

64

E.L.Bowland, E.A .Foegeding and D.D.Hamann

alter G' moduli, the relationship between G' and Gf was altered. The mixture of phosphate buffer and Na ZS04 produces no change in G' and an increase in Gf (Table 2) . Phosphate chelation of calcium appears to cause a shift from strain weakening to strain hardening mechanical properties. This shift was also reflected in changes in gel microstructure (2). The usefulness of small strain rheological properties in understanding matrix properties cannot be denied; however, the absence of a phosphate effect on G' strongly suggests that mechanical information relative to sensory properties is only determined at fracture-causing strains. Phase angle development and magnitude (not shown), and N values (Table 4) were not altered by addition of phosphate to the 75 mmol/dm' NazS04-containing samples. Thus the viscoelastic nature of the gels at small strains was not altered. In addition, the ratio of hydrophobic interactions to hydrogen bonds which determined gel network rigidity was not altered as shown by the lack of change in the tlG' hea/G' cool values (Table 3). Also, phosphate addition did not induce significant (P < 0.05) changes in GP or initial gelation rates (Table 5). Collectively these results indicated that calcium chelation by phosphate did not affect the microproperties of the matrix that were measured by small strain analysis. As a result, differences in small strain rheological properties of Na zS04- or sodium phosphate buffer-containing gels appeared to be due to the different anions (sulfate and phosphate) and not to calcium chelation . A lack of change in tlG' hea/G' cool and the initial gelation rate was surprising. Addition of 10 mmol/drrr' sodium phosphate buffer to a 75 mmol/drrr' NazS04-containing sample shifted the microstructure to a more fine-stranded matrix (2). This shift was expected to increase tlG\ea/ G' cool values and the initial gelation rate. It is unknown why these changes did not occur. Perhaps, the shift in macro-properties of the material was not accompanied by a significant shift in micro-properties .

Conclusions Matrix formation in each treatment showed similar trends in development of small strain viscoelastic properties. However, anion specific trends were seen in magnitudes of rheological moduli and GPs. Calcium chelation by phosphate did not influence these trends. No specific transitions indicative of a type of matrix existed. Mixed matrix gels had proportionately more hydrophobic interactions (large tlG' hea/G' cool) and had greater initial gelation rates than fine-stranded and particulate matrix gels . The proportion of hydrophobic interactions in the gels correlated with the anions' influences on the hydrophobic effect as indicated by their positions in the Hofmeister series.

Acknowledgements Funding for this project was partially supplied by Hershey

Foods Corporation and Southeast Dairy Foods Research Center. Davisco International, Inc . supplied the whey protein isolate used in this project.

References 1. Kuhn ,P .R. and Foegeding ,E .A . (1991) 1. Agric . Food Chern., 39, 1013. 2. Bowland ,E.L. and Foegeding,E.A . (1994) Food Hydrocoll., 9, 45-54. 3. AOAC (1984) Official Methods of Analysis, 14th edn. Association of Official Analytical Chemists, Arlington, VA. 4. Foegeding,E.A., Kuhn,P.R. and Harden,C.C. (1992) J. Agric. Food Chern., 40,2092. 5. SAS (1982) User's Guide: Statistics. SAS Institute, Inc. , North Carolina, USA. 6. Foegeding,E.A., Kuhn,P.R. and Hardin,C.C. (1992) 1. Agric. Food Chem . , 41, 341. 7. Hines ,M.E. and Foegeding,E.A. (1993) J. Agric. Food Chern. , 41, 341. 8. Paulsson,M., Desjmek ,P . and van Vliet,T. (1982) J . Dairy Sci., 73, 45. 9. Foegeding,E.A., Gonzalez ,C., Hamann,D.D. and Case,S . (1994) Food Hydrocoll., 8, 125. 10. Ferry ,J.D. (1980) In Viscoelastic Properties of Polymers. John Wiley and Sons, Inc., New York . 11. Eagland,D . (1975) In Duckworth,R.B. (ed.), Water Relations of Foods . Academic Press, Inc ., New York. 12. Creighton,T.E. (1983) In Proteins Structures and Molecular Properties. W.H.Freeman and Co., New York. 13. Damodaran,S. and Kinsella,J.E. (1982) In Cherry ,J.P . (ed.), Food Protein Deterioration Mechanisms and Functionality. American Chemical Society, Washington, DC. 14. Morr,C.V. and Foegeding,E.A. (1990) Food Tech., 44, 100. 15. Stading,M. and Hermansson,A .-M. (1991) Food Hydrocoll. , 4, 339.