ELSEVIER

Int. Dairy Journal 6 (1996) 645-658

Copyright 0 1996 Elsevier Science Limited

Printed in Ireland. All rights reserved

0958-6946/96/$15.00 + 0.00

0958-6946(95)00054-2

Stability and Rheological Properties of Salad Dressing Containing Peptidic Fractions of Whey Proteins

Sylvie L. Turgeorf, Christian Sanchezb, Sylvie F. Gauthie?* & Paul Paquinb

%ervice des Technologies Alimentaires, Direction de la Recherche et du Developpement, Minis&e de l’Agriculture, des Pecheries et de 1’Alimentation du Quebec, 3600 Bd Casavant

Ouest, Saint-Hyacinthe, Quebec, Canada, J2S 8E3 ‘Centre de Recherche STELA, Departement de Sciences et Technologie des Aliments,

Pavillon Paul-Comtois, Universite Laval, Ste-Foy, Quebec, Canada, GlK 7P4

(Received 12 April 1995; revised version accepted 20 July 1995)

ABSTRACT

A whey protein concentrate (WPC), a heat-treated WPC (9O”C, pH 2.5, IO min) and peptidic fractions obtained by ultrafiltration of their tryptic and chymotryptic hydrolysates were incorporated in a salad dressing jbrmulation at 0.5, 1.0 or 1.5%

(w/w) protein. Emulsion stability was evaluated at 25 and 4°C during a h-month storage period and rheological properties were measured by dynamic oscillatory shear experiments. Peptidic fractions obtained from tryptic hydrolysates produced the most stable salad dressings (over 6 months at the 1.0% and 1.5% protein level) with rheological properties similar to a commercial mayonnaise. The most impor- tant ,fizctors for emulsion stability were the incorporation level and the nature of

peptides. Copyright 0 1996 Elsevier Science Limited

INTRODUCTION

Many food products, such as salad dressing and mayonnaise, are oil-in-water emulsions which may be stabilized by emulsifiers and stabilizers. Emulsifiers act generally by one or more mechanisms, including reduction of the interfacial tension between oil and water phases, or covering fat globules with a charged layer to create a physical barrier preventing flocculation (Walstra, 1986 and others). Stabilizers, mainly polysaccharides, usually stabilize emulsions by increasing the viscosity of the aqueous phase. Proteins are among the most widely

* To whom correspondence should be addressed.

645

646 S. L. Turgeon et al.

used emulsifiers because they can decrease the interfacial tension and form a protective layer around fat globules in emulsions (Phillips, 198 1; Halling, 1981; Dickinson & Stainsby, 1982).

The functional properties of whey proteins have been studied in great detail (among others, Morr et al., 1973; McDonough et al., 1975; Morr, 1985, 1987a, 1987b; Liao & Mangino, 1987; Morr & Foegeding, 1990; Britten & Giroux, 1991; Schmidt & McNeill, 1993; Taylor et al., 1994) and numerous review papers have been published (Leman & Kinsella, 1989; Tornberg et al., 1990; Gauthier et al., 1993; Robin et al., 1993). Many parameters such as whey origin, nature of pre- treatment and extent of protein denaturation affect these functional properties (Morr, 1979; de Wit et al., 1988; Hawks et al., 1993). Heat and enzymatic treatments have been used in order to increase the use of whey proteins in the food industry. Heat treatments under acidic conditions cause protein denaturation with minimal aggregation (Buchheim & Jelen, 1976) improving the foaming ability (Paquet et al., 1978) and modifying the solubility, the viscosity and the gelling properties (Modler & Harwalkar, 1981).

Enzymatic hydrolysis of proteins results in peptides with smaller molecular sizes and less secondary structure. Generally, the solubility, water absorption, emulsifying and foaming capacity are improved by enzymatic treatment of proteins. However, excessive hydrolysis may alter some functional properties affecting viscosity and gelation, and reduce foam and emulsion stability (Kuehler & Stine, 1974). The functional properties of hydrolysed proteins are governed mainly by their molecular weight (Adler-Nissen, 1986) and hydrophobicity (Chaplin, 1988; Chaplin & Andrews, 1989; Turgeon et al., 1992a).

An enzymatic process has been proposed (Turgeon & Gauthier, 1990) in order to produce peptidic fractions from whey protein concentrate (WPC) or heat- treated whey protein concentrate (H-WPC) using specific enzymes (trypsin, chymotrypsin). The peptidic fractions are obtained by a two-step ultrafiltration. The first step allows the separation of the hydrolysate from the enzyme and the second, with a smaller cut-off membrane (1000 Da), eliminates amino acids and small peptides from the total hydrolysate. This process results in peptide fractions with different ranges of molecular weight and varied potential use. The studies of interfacial properties and emulsifying capacities of these peptidic fractions have emphasized the great effect of thermal pretreatment, enzyme specificity, ranges of molecular weight, pH and ionic strength, on their functional properties (Turgeon et al., 1991a, 1991b, 1992b).

The aim of the present study was to incorporate the whey peptidic fractions into salad dressing-type concentrated emulsions, to study their stability and rheological properties. The influence of incorporation level and storage temperature was taken into consideration.

MATERIALS AND METHODS

Materials

Commercial whey protein concentrate (WPC, 35.3% w/w, protein) was obtained from a cheese plant (Saputo, St-Hyacinthe, Quebec, Canada). Trypsin (bovine pancreas, type III-S) and cc-chymotrypsin (bovine pancreas, type II) were

Peptide jiactions based salad dressing 647

purchased from Sigma Chemical Co. (St Louis, MO, USA). The commercially available reference mayonnaise-type salad dressing (Kraft, Montreal, Quebec, Canada) was stable at 25°C over the experiment duration (6 months).

Production of peptidic fractions

Peptidic fractions were obtained by ultrafiltration of tryptic or chymotryptic hydrolysates of WPC or H-WPC (90°C, pH 2.5, 10 min) produced according to Turgeon & Gauthier (1990). The substrate was suspended in distilled water (lo%, w/w; pH 8.0, 40°C) followed by enzymatic hydrolysis using an enzyme/substrate ratio of 1:200. After 45 min reaction, hydrolysed products were separated by ultrafiltration (UF) (30,000 Da cut-off; HF 15-43-PM30, Romicon Inc., Woburn, MA, USA). The UF retentate and permeate were denoted reaction mixture (RM) and total hydrolysate (TH), respectively. The TH was further fractionated by a second step UF (1000 Da cut-off; HF 1543-PMI, Romicon Inc., MA, USA). The second retentate was a mixture of polypeptides (MP) and the corresponding permeate, the amino acids and small peptides fraction (AA). The chemical composition of the different peptidic fractions was determined by Turgeon & Gauthier (1990). The protein yield was evaluated as 60% for TH (first ultra- filtration), 45% for MP and 55% for AA (second ultrafiltration). The degree of hydrolysis (percentage of peptide bonds cleaved by the protease) and the protein content of the hydrolysed fractions are shown in Table 1. Around 80-100% of MP and AA peptides had molecular weights ranging from 5000 to < 2000 Da (Turgeon & Gauthier, 1990).

Preparation of the salad dressing-type emulsion

Salad dressing contained 62.0% corn oil (Mazola, Best Foods, Etobicoke, Ontario, Canada), 32.8% vinegar (50 O.P., Steinberg), 2.4% sugar, 1.5% salt, 1.2% lactose and 1.3% spices (0.4% onion, 0.4% paprika, 0.5% garlic, Steinberg). Protein (WPC or H-WPC) or their peptidic fractions were added at three incorporation levels: 0.5, 1 .O and 1.5% (w/w) protein. Each formulation was triplicated. The blender container (Waring blender) was first filled with all ingredients but corn oil, then stirred at maximum speed for 30 sec. Oil was then rapidly added and the final mixture was stirred for 1 min. Concentrated emulsions were divided into three parts to evaluate storage stability at 2.5 and 4°C and rheological properties.

Evaluation of storage stability of emulsions

Typically, a 50 mL graduated disposable syringe was filled with 50 mL emulsion and stored at 25 and 4°C. The volume of aqueous phase separated from the emulsion was recorded at various time intervals within 6 months.

Evaluation of rheological properties of emulsions

Most emulsions exhibit both solid and viscous behaviour (elasticity and viscosity, respectively) when shear stress is applied (Sherman, 1970). Viscoelastic properties of emulsions can be satisfactorily measured by dynamic testing. Such

648 S. L. Turgeon et al

TABLE I Degree of Hydrolysis (%) and Protein Content (% in Dry Matter) of the Peptidic Frac-

tions Obtained by UF Fractionation’

Protein and

peptide,fkactions Degree of h Protein h Protein incorporation level (96) hydrolysis content

(Xl (Xi 0.5 1.0 I.5

Tan 6’ qed Tan 6 q* Tan 6 q*

WPC H-WPC WPC, TH-trypsinf WPC, MP-trypsin” H-WPC, TH-trypsin H-WPC, MP-trypsin WPC, TH-chymotrypsin WPC, MP-chymotrypsin H-WPC, TH-chymotrypsin H-WPC, MP-chymotrypsin

Commercial Mayonnaise

37.6 _e _ _ _ _ _ 35.9 0.22 117 0.47 249 0.51 343

12.4 29.7 - - 0.13 634 0.17 929 12.5 43.5 0.16 362 0.24 777 0.19 1292 13.2 29.1 0.46 255 0.19 847 0.29 1066 9.9 39.8 0.19 150 0.27 675 0.22 918

12.5 30.7 - - 0.70 25 0.58 58 10.8 43.2 - - - - 1.08 19 12.5 31.7 - - 0.77 25 0.80 52 10.8 41.4 - - 0.63 16 0.49 55

0.12 414

“Fractionation done on tryptic and chymotryptic hydrolysate of whey protein concentrate (WPC) or heat-treated WPC (H-WPC, 90°C pH 2.56, 10 min); dynamic viscoelastic parameters (tan 6 and rl*, where q* is the complex viscosity, Pa s) of emulsions made with three incorporation levels (0.5, 1.0 and 1.5%, w/w protein) of WPC, H-WPC or the peptidic fractions. Conditions of measurement were: temperature: 25°C; frequency: 50.3 rad s-‘; strain amplitude < 0.015. bObtained from Turgeon & Gauthier (1990). ‘Tan 6 is the ratio G/‘/G’ and represents the relative viscous characteristic of material. dn* is the complex viscosity (Pa.s) ‘(-): Emulsion too liquid and unstable to allow dynamic testing. fTH: Total hydrolysate mixture. sMP: Mixture of polypeptides (retentate of UF of TH, small fragments and free AA removed).

measurements were carried out on the day after emulsion preparation on a controlled shear stress rheometer (Carri-Med, Surrey, UK) fitted with a parallel plate geometry probe (gap: 1 mm). Emulsions were subjected to a frequency sweep from 0.63 to 63 rad s-l at a fixed sinusoidal shear stress of 3. 10m4 rad. Storage and loss moduli (G’ and G”, resp.), as well as dynamic viscosity v*, were recorded. G’ is a measure of the energy stored and released per oscillating cycle and G” is the energy dissipated or lost as heat. The G/‘/G’, namely loss tangent (tan S) was also calculated. This useful parameter characterizes the relative viscous behaviour of an emulsion at a fixed frequency. The dynamic viscosity 7: is defined by the ratio of complex shear stress to complex rate of strain, i.e. r /o (Barry, 1975). Each measurement was carried out in duplicate.

The limit of the linear viscoelastic behaviour was determined in preliminary studies by stepwise variation of shear stress at constant frequency (50.3 rad s-’ or 8 Hz). The linear viscoelasticity limit varied between emulsions; 3. lop4 rad (<

Peptide ,fractions bused salad dressing 649

0.015 strain amplitude) was found to be the lower limit and consequently used for all frequency sweeps. Within a linear viscoelastic region, mainly non-destructive measurements are performed and systems may be analysed effectively on a structural basis. The linear behaviour is related to the material ability to support stress (or strain) without any macroscopic structural changes. Therefore, small amplitude dynamic testing allows the characterization of the structure at rest (Elliott & Ganz, 1977).

Statistical analysis

The effects of heat treatment, nature of enzyme and UF fractionation on the storage stability and rheological properties of the experimental emulsions were analysed by variance analysis of a 2 (heat treatment) x 2 (enzymes) x 2 (TH and MP) factorial plan.

RESULTS

Storage stability of emulsions

Whey proteins and their peptidic fractions have generated two kinds of emulsions: either liquid, orange-coloured emulsions which were unstable; or more stable products which were colourless and showed mayonnaise-type consistency. The former emulsions seemed to destabilize by rapid creaming with coalescence leading to three phases: an aqueous phase at the bottom, oil in the middle and aerated coarse emulsion at the top. The mayonnaise-type emulsions destabilized very slowly and resulted in the draining of the aqueous phase at the bottom of the sample.

Figure 1 illustrates the storage stability of emulsions at 25°C and 4°C during a 6-month storage period. The results shown are the times at which emulsion destabilization (phase separation) was observed. The emulsions generally showed similar stability behaviour at 25°C and 4°C (Fig. 1) with higher values for the lower storage temperature (4°C). At 25°C six samples among the 30 emulsions prepared were stable over 6 months, while at 4°C eight emulsions remained stable over the same period. These stable samples were mainly obtained by incorporation of 1.0 and 1.5% peptidic fractions prepared from tryptic hydrolysis of WPC or H-WPC (1 l/14 stable emulsions).

Emulsions made with 0.5% of non-hydrolysed proteins (WPC and H-WPC) showed low stability (< 17 days) at both storage temperatures. With WPC, emulsions were unstable (< 4 days) regardless of storage temperature (25 or 4°C) or incorporation level (0.551.5%) while with H-WPC, a higher stability was obtained under the same conditions. In fact, a very stable emulsion was obtained by adding 1.5% of H-WPC, the sample remaining stable for 5 and 6 months at 25 and 4°C respectively. For peptidic fractions, no clear effects of the heat treatment applied to the WPC on the stability of resulting emulsions were observed.

The more stable emulsions at 4 and 25°C were prepared with peptidic fractions obtained from tryptic hydrolysates (P < O.OOOl), emphasizing the significance of enzyme specificity. The UF fractionation also influenced the storage stability of

650 S. L. Turgeon et al.

200

180

160

140

120

100

80

60

40

20

0

(a)

T 1.0% _ IM

180

160

140

120

100

80

60

40

20

0

12 3 45 67 8 9 10

Protein and peptide fractions

Fig. 1. Storage stability at 25°C (a, b, c) and 4°C (d, e, f) of emulsions made with three incorporation levels (0.5, 1.0 or 1.5%, w/w protein) of whey protein concentrate (WPC), heat-treated WPC (H-WPC, 9O”C, pH 2.5, 10 min.) and their total and UF-purified peptidic fractions (TH and MP) obtained by UF fractionation of tryptic and chymotryptic hydrolysates. (Number at top of bars represent days).Proteins and peptidic fractions are WPC (l), H-WPC (2), WPC, TH-trypsin (3), WPC, MP-trypsin (4), H-WPC, TH-trypsin (5), H-WPC, MP-trypsin (6), WPC, TH-chymotrypsin (7), WPC, MP-chymotrypsin (8), H-

WPC, TH- chymotrypsin (9), H-WPC, MP-chymotrypsin (10).

Tim

e fo

r em

ulsi

on d

esta

biliz

atio

n (d

ays)

0~:8

a~~z

~z~

1 .

I .

I .

I .

I I

. I

. I

. I

. -

P

i: “8

6 5 5 i 5

Tim

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ulsi

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esta

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atio

n (d

ays)

T

ime

for

emul

sion

des

tabi

lizat

ion

(day

s)

652 S. L. Turgeon et al.

used at 1.5% incorporation level gave a stable emulsion (6 months) at both storage temperatures (25 and 4°C).

Rheological properties

Since consistency of the product (liquid vs solid) seemed to influence the emulsion stability, viscoelastic properties of the samples have been evaluated by small amplitude oscillatory testing; this method gives valuable information on the structure of systems at rest and is useful to study emulsions.

Typical mechanical spectra of emulsions made with 1.5% proteins and peptidic fractions are given in Fig. 2; similar data were generated for 0.5 and 1.0% incorporation (not shown). Emulsions made with WPC have not been included in Fig. 2 since their very poor consistency prohibited measurement of their dynamic properties in the linear viscoelasticity range. The curves recorded with emulsions containing TH or MP tryptic hydrolysates of WPC or H-WPC were similar, and only the former are included in Fig. 2. Also, curves obtained for emulsions containing TH and MP chymotryptic hydrolysates of H-WPC (not shown) were similar to the one obtained for emulsion containing TH chymotryptic hydrolysates of WPC. Samples made with tryptic hydrolysates showed mechanical behaviour distinctly different from that of chymotryptic hydrolysates. Emulsions stabilized by tryptic hydrolysates (TH and MP fractions) showed dynamic moduli which were essentially frequency independent, over the frequency range considered, and G’ is always larger than G”. Such high viscoelasticity in concentrated emulsions is associated with a gel- type structure (Barry, 1975). The volume fraction of the emulsions was 0.62, which is near to the close packing of spherical fat globules (X 0.64). Thus, the formation of a flocculated emulsion network may be strongly expected. The network was probably made either by a three-dimensional structure created between particles or macromolecules, or by the formation of local networks between macromolecules adsorbed on adjacent globules (microcoagulation process). Further evidence of the gel-like structure is the slight increase of G’ at high frequency and its high value at very low frequency (Fig. 2a). This arises because the strong interactions mainly contributing to the elastic modulus need a long time to relax. At high frequency, the oscillation time is rapid and the viscoelastic properties approach those of a permanently cross-linked network. At long oscillation times (low frequency), gel-type materials still possess permanent interactions which give a predominantly solid behaviour. The storage modulus (G’) tends to level off when the frequency is close to zero.

Emulsions based on H-WPC showed viscoelastic properties similar to those based on tryptic hydrolysates. However, although their G” were the same, their G’ values were significantly lower (P < 0.001). The G” results obtained from the tryptic hydrofysates-based emulsions need to be considered with care because the calculated coefficients of variation were high, typically more than 30-50%. Gault & Fauquant (1992) reported also that the viscous modulus of individual whey protein gels were difficult to access when measured on a controlled shear stress rheometer type CARRI-MED (up to 100% coefficient of variation). They suggested that the rheometer was not sensitive enough to measure low viscous moduli in predominantly elastic gels. The comparison of results reported in Table 1 with those drawn in Fig. 2 support this view. Table 1 gives the complex

Peptide fractions based salad dressing 6.53

q H-WPC

l WPGTH-hyp.

0 WPC. MP-byp.

A WPC.THchym.

A WFC, MP-chym.

g 4-

m H-WPC

5 3_ l wlc,TH-uyp.

0 22 WPc,MP-byp. .

8 : ii8 A WPc.TH-dlym.

A wFc,MFkhym.

2- 0

A . .AAA

A 9

A AAA 1-

A

01 I I 1

-0.5 0,o 0,5 I,0 1.5 2.0

(4

(b)

log freauencv (rad s-l)

WPC, MP-chym.

0 0 14 28 42 56 70

frequency (rad s-f)

Fig. 2. Frequency dependence of (a) storage (G’) and (b) loss (G‘) moduli and (c) loss tangent (tan 6) of emulsions made with 1.5% (w/w) protein incorporation level of heat- treated whey protein concentrate (H-WPC, 9O’C, pH 2.5, IO min.) and peptidic fractions (TH and MP) obtained by UF fractionation of WPC and H-WPC tryptic and chymo- tryptic hydrolysates. Temperature: 25°C; Strain amplitude <O.OlS.Emulsions containing

TH and MP fractions from H-WPC were similar to those from WPC (data not shown).

654 S. L. Turgron et al

viscosity (n*) and the loss tangent (tan 6 = G”/G’), which represents the relative viscous characteristics of a viscoelastic material, while Fig. 2 shows the frequency dependency of the loss tangent. We may observe that emulsions made with tryptic hydrolysates give scattering of G” values (Fig. 2b) and had tan 6 within 0.2-0.3, characteristics of weak predominantly elastic gels (Table I and Fig. 2~). On the other hand, emulsions with H-WPC or chymotryptic hydrolysates have higher viscous component (tan 6 of 0.5-1.0) and no scattering of G” values (Fig. 2).

Unlike H-WPC and tryptic fraction-based emulsions, those made with chymotryptic hydrolysates had significantly lower G’ and G” moduli and tan b (Fig. 2). Clearly, this means that fewer interactions affect the structure of emulsions and that more temporary interactions favoured the viscous component. At low frequency, the G’ and G” moduli of the WPC, MP-chymotryptic-based emulsions approached zero and rose steeply at high frequency (Fig. 2a and b). At these frequencies, the loss tangent was less than unity but was larger than one over a wide frequency range (Fig. 2~). This mechanical behaviour is typical for concentrated dispersions (Ross-Murphy, 1986). Similarly, the dynamic moduli of salad dressings based on WPC-TH, H-WPC-TH and H-WPC-MP fractions do not plateau at high frequency but do not drop to zero at low frequency (Fig. 2). These emulsions, predominantly elastic, showed rheological behaviour between those of weak gels and concentrated dispersions.

It may be pointed out (see Table I) that the dynamic viscosity (n*) increased with the protein and peptide incorporation levels. Many other parameters influence emulsion rheological properties, such as globule diameter and size distribution, viscosity of the continuous phase, occurrence and type of emulsifiers, etc. (Sherman, 1970). Among these parameters, the thickness of the adsorbed protein layer affects the emulsion rheology by disturbing the flow around globules and by increasing the dispersion viscosity (Dickinson & Stainsby, 1982). Emulsions based on peptide fractions obtained from tryptic hydrolysates had higher viscosity in comparison to the chymotryptic fractions. Furthermore, the viscosity of emulsions containing 1 .O and 1.5% tryptic fractions was higher than that of the commercial mayonnaise (Table 1). This phenomenon could be related to one or more of the following interlinked factors: a thicker adsorbed layer, stronger interactions between globules, smaller globules, stronger water immobilization by adsorbed peptides. No significant differences were found between the MP and TH fractions, nor between those derived from WPC and H-WPC.

As aforementioned, the loss tangent defines the relative viscous characteristic of the material. A perfectly elastic gel will have zero G” and tan 6. Emulsions containing 1.0 or 1.5% tryptic fractions had tan fi values lower than 0.3 and were the most stable. These values were close to those measured on the commercial mayonnaise (0.12) which was stable over 6 months. For chymotryptic digest emulsions, the tan 6 values were larger than 0.5 and samples were rather liquid.

DISCUSSION

Emulsions with a high oil volume fraction, such as salad dressing-type mayonnaise, need maximal stability against coalescence. Indeed, when increasing

Peptide fractions based salad dressing 655

the volume fraction until the close-packing value, fat globules come closer and closer. The membrane surrounding fat globules, therefore, must resist coalescence, otherwise rapid destabilization occurs, causing oil separation (Coia & Stauffer, 1987). In these emulsions, the proteins and peptides making fat globule membranes are close and may create a semi-continuous flocculated structure stabilizing the whole system. In such a system, destabilization is likely to occur by exclusion of the aqueous phase at the emulsion bottom according to a process similar to syneresis (Dickinson, 1987).

The occurrence of very unstable WPC-based emulsions showing three phases suggests that whey proteins are unable to form resistant membranes against coalescence at the concentrations studied. Another likely explanation is that whey proteins have insufficient emulsifying properties to reduce effectively the fat globule size, which increases creaming and coalescence ability. The compact globular structure of whey proteins prevents their effective adsorption and, consequently, higher concentrations would be necessary to obtain better emulsifying properties (Yamauchi et al., 1980). The orange colour of these emulsions suggests further that fat globules are larger in the unstable emulsions than in the more stable ones, which were colourless. Other known examples of this phenomenon are homogenization of milk or cream cheese which produces whiter products (Walstra & Jenness, 1984; Sanchez et al., 1994). The low viscosity of unstable emulsions causes extensive air bubble incorporation during manufacturing. The presence of air in emulsions strongly impairs their stability during mixing (Dolby, 1957). A thermal modification, increasing the accessi- bility of hydrophobic areas, may improve the stabilizing properties as shown in this study. However, the stabilities at 4 or 25°C of these emulsions containing 0.5 or 1.0% heat-treated whey protein concentrate did not exceed 1 month. A higher protein concentration, 1.5%, was required to improve the stability.

Enzymatic hydrolysis has been studied often in order to modify the functional properties of proteins. Generally, emulsifying capacity is improved while stability is lowered (Kuehler & Stine, 1974; Chobert et al., 1988). The peptidic fractions from tryptic hydrolysis gave more stable emulsions than their chymotryptic equivalents, and at lower concentrations. Jost & Monti (1982) also pointed out the need for adding more chymotryptic peptides (5 vs 1% for tryptic peptides) in order to stabilize an emulsion (4 = 0.20) against coalescence. The dynamic viscoelastic properties of these emulsions suggest the occurrence of strong peptideepeptide and peptideepolypeptide interactions stabilizing the flocculated network. The tryptic digests are characterized by a higher content of high molecular weight peptides and more intact hydrophobic areas than the chymotryptic ones (Turgeon & Gauthier, 1990). We suggest that these structural features possibly led to thicker adsorption layer (and thus more resisting film), stronger water immobilization and a more structured and stable network.

Storage at 4’C improved the stability of most emulsions, probably by increasing the viscosity of systems, as a consequence of stronger solvent-protein interactions, but also by favouring proteinprotein interactions between adjacent globules to the detriment of protein-protein interactions in the film of individual globule. The H-WPC-based emulsions were stabilized greatly by cooling. It is likely that, at 4’C, fat globule membrane structuring was faster than emulsion destabilization. Rearrangements of the fat globule membrane have been

656 S. L. Turgeon et al

hypothesized by Britten & Giroux (1991) in order to explain the good storage stability at 4°C (after several months) of oil emulsified by heat-treated whey protein isolate, in comparison with the centrifugal stability at 25°C of the same fresh emulsion. Rheological measurements could not show this phenomenon since they were carried out 24 h after manufacturing at room temperature. Rivas & Sherman (1983) obtained an increase in viscoelastic parameters of protein- based emulsion after 3 and 7 days of storage. These results indicate that protein inter-linking continues during this period and dominates the mechanisms retarding coalescence (Sherman, 1968). This phenomenon also could explain the higher stability of the emulsion based on MP from the chymotryptic hydrolysis of H-WPC (1.5% w/w) in spite of a very low viscosity, low dynamic moduli and high tan 6. The evolution of the viscosity during storage should be studied in order to understand the destabilization process of this emulsion.

Many rheological parameters can be related to stability. It is of practical interest to predict the storage stability of emulsions from instrumental measurements carried on freshly prepared samples. An example is high viscosity, which improves storage stability. This observation is related generally to a critical value of tan 6 z 0.5, thus to the development of a network sufficiently strong to delay creaming and coalescence. However, this is mainly true at higher protein and peptide concentrations. The optimum concentration of each emulsifier in a given formulation has to be known in order to attain the desired stability.

CONCLUSIONS

This study of the stabilizing properties of peptidic fractions of whey proteins in a complex formulation shows the need to work with a real food to reach specific conclusions. It was shown in a model emulsion that H-WPC hydrolysed by trypsin had lower emulsifying capacity than both WPC and H-WPC (Turgeon et al., 199la); in the present study it was one of the best stabilizers. Our study demonstrated clearly that the most important parameter influencing the salad dressing emulsifying stability was the incorporation level of proteins and peptidic fractions. At a high level, dynamic testing enables satisfactory prediction of the storage stability of most emulsions, although accounting for possible rearrange- ments of fat globule membranes during storage may be difficult.

ACKNOWLEDGEMENTS

This research was supported by the Fonds pour la formation de Chercheurs et 1’Aide a la Recherche (FCAR) and le Centre Qutbecois de Valorisation de la Biomasse (CQVB).

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Pep tide f&c tions based sulud dressing 651

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