Emulsifying behaviour of 11S globulin Vicia faba in mixtures with sulphated polysaccharides: comparison of thermal and high-pressure treatments

Food Hydrocolloids 13 (1999) 425–435 www.elsevier.com/locate/foodhyd

Emulsifying behaviour of 11S globulin Vicia faba in mixtures with sulphated polysaccharides: comparison of thermal and high-pressure treatments V.B. Galazka a,*, E. Dickinson a, D.A. Ledward b a

Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK Department of Food Science and Technology, University of Reading, Whiteknights, Reading RG6 6AP, UK

b

Received 8 March 1999; accepted 21 June 1999

Abstract The influence of heat (up to 808C for 2 min) and high-pressure (up to 250 MPa for 20 min) on the emulsifying properties of 11S globulin Vicia faba at pH 8.0 has been investigated for systems containing the sulphated polysaccharides i-carrageenan (i-CAR) and k-carrageenan (k-CAR). The emulsions (0.5 wt% 11S, 20 vol% n-tetradecane) made with heated or high-pressure treated 11S were found to give substantially larger droplets than those made with the native protein. Visual creaming behaviour has been monitored as a function of storage time. There was a consistent trend of decreasing emulsifying efficiency and emulsion stability with increase in treatment temperature or pressure. Addition of i-CAR or k-CAR (3:3–7:1 by weight) to the native protein at low ionic strength led to smaller droplets whose size decreased with increase in polysaccharide concentration and extent of high-pressure treatment (up to 200 MPa). Thermally treated biopolymer mixtures gave emulsions with droplets that did not significantly change with increase in temperature. In all cases, the presence of i-CAR led to a significant improvement in creaming stability. However, the presence of k-CAR in untreated and thermally treated (,758C) mixtures gave rapid serum separation probably due to depletion flocculation. Of the two polysaccharides studied, i-CAR gave the smallest droplets in fresh emulsions and the best stability with respect to visual creaming behaviour. The observations for 11S alone can be interpreted in terms of pressure or thermally induced unfolding of the protein, which results in a decrease in emulsion efficiency due to dissociation of subunits or protein aggregation. It appears that the strength of interaction of 11S with i- or k-CAR is dependent on the charge density on the polysaccharide. The presence of interacting polysaccharide in the heated and high-pressure processed samples seems to inhibit the formation of aggregates. High-pressure treatment of the mixed biopolymer solutions in the presence of sodium chloride (.0.01 M) destabilises the emulsion, and so the protective effect of polysaccharide is lost. q 1999 Elsevier Science Ltd. All rights reserved. Keywords: High-pressure treatment; Protein–polysaccharide interaction; 11S globulin Vicia faba; Polysaccharides; Carrageenan; Oil-in-water emulsions

1. Introduction Food emulsions commonly contain a mixture of proteins and polysaccharides, which both contribute to product texture and shelf life (Dickinson and Stainsby, 1982). Proteins are used as emulsifying agents because of their ability to form strong adsorbed layers at the oil–water interface, thus preventing droplet coalescence (Dickinson, Murray & Stainsby, 1988). On the contrary, polysaccharides of high molecular weights can modify the rheology of the disperse phase, thereby affecting flocculation and creaming velocity (Dickinson, 1988). Different factors, such as the ratio of protein to polysaccharide, the polysaccharide and protein charges and * Corresponding author. Tel.: 1 44-113-233-2970; fax: 1 44-113-2332982.

molecular weights, the ionic strength, the pH, and the temperature all can influence the formation and stability of protein–polysaccharide complexes (Dickinson, 1998; Schmitt, Sanchez, Desobry-Banon & Hardy, 1998). Under different conditions, one can expect macromolecular interactions to be specific or non-specific, strong or weak, attractive or repulsive. The interactions depend on the structure of the protein. For instance, studies have shown that the highly anionic polysaccharide, dextran sulphate (DS), forms interfacial electrostatic complex(es) with bovine serum albumin (BSA) in emulsions (Dickinson & Galazka, 1992) and foams (Galazka, Varley, Smith, Ledward & Dickinson, 1998; Izgi & Dickinson, 1995), but not in b-lactoglobulinstabilised emulsions (Dickinson & Galazka, 1991) and foams (Galazka, Varley, Smith, Ledward & Dickinson, 1998; Izgi & Dickinson, 1995) at neutral pH. Recent studies on the solution properties of BSA with

0268-005X/99/$ - see front matter q 1999 Elsevier Science Ltd. All rights reserved. PII: S0268-005 X( 99)00 028-4

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sulphated polysaccharides at pH # 7 and low ionic strength have shown that BSA forms complex(es) with DS (Galazka, Ledward, Sumner & Dickinson, 1997; Galazka, Sumner & Ledward, 1996a) as well as with i- or k-carrageenan (i- or k-CAR) (Galazka, Smith, Ledward & Dickinson, 1999a). Moreover, the ionic strength and pH were found to be important variables controlling the strength of complexation (Dickinson and Pawlowsky, 1998; Galazka et al., 1997, 1999a). A significant interaction occurs between BSA and DS at neutral pH and low ionic strength, whereas i- or k-CAR give a much weaker interaction that strengthens at pH 6.5. The application of high-pressure treatment was found to induce the formation of stronger complex(es) which appeared to protect the protein against –SH/–S–S– interchange during or after pressure treatment (Galazka et al., 1997, 1999a). Under similar experimental solution conditions and under pressurisation, another globular protein ovalbumin (OVA) formed electrostatic complex(es) with DS or i-CAR at pH # 7:0 and low ionic strength (Galazka, Smith, Ledward & Dickinson, 1999b). In both cases, it appears that the strength of complexation is related to the density of sulphate groups on the polysaccharide …DS . i-CAR . k-CAR†; and the complexation strengthens during high-pressure treatment at pH 6.5. It is also suggested that non-covalent protein–polysaccharide interactions are modified due to electrostatic interactions and pressure-induced changes in protein conformation (Galazka et al., 1999a). Recently it has been reported that bridging flocculation of BSA-stabilised oil-in-water emulsions can be induced at low ionic strength by the addition of i- or k-CAR (Dickinson & Pawlowsky, 1997, 1998) or DS (Dickinson & Pawlowsky, 1996). In agreement with the solution work (Galazka et al., 1999a), the nature of the flocculation and creaming behaviour seemed to be related to the charge density on the polysaccharide. Few comparisons have been made on the effects of highpressure and thermal treatment on mixtures of protein and polysaccharide. Okamoto, Kawamura and Kunugi (1990) reported that pressure-induced and thermal gelation mechanisms differed, presumably due to the fact that heatinduced denaturation involves the breaking down of non-covalent bonds (including hydrogen bonds) which are pressure resistant, whereas pressure denaturation involves predominantly the rupture of hydrophobic and ionic bonds. Recently we have demonstrated (Galazka et al., 1999b) that OVA heated alone at 808C for 10 min induces extensive precipitation, whereas solutions of the OVA 1 DS mixtures remained clear. It was therefore suggested that DS protects the protein against aggregation following thermal treatment by blocking direct hydrophobic interactions or by preventing sterically the protein physical entanglement during heating (Matsudomi, Tomonobu, Moriyoshi & Hasegawa, 1997). Interactions of polysaccharides with vegetable proteins are also of interest. The protein of leguminous plants,

such as soy, pea, and broad bean, may be used in the food industry for the formulation of new food products and as functional additives regulating the protein content in a given food. For instance, soy proteins are being widely used in the preparation of new forms of meat products, seasonings, dressings, and confectionery. The major storage protein in dicotyledonous seeds, 11S globulins, has a molecular weight in the region of 300–400 kDa (Derbyshire, Wright & Boulter, 1976). The 11S globulin of Vicia faba broad bean is homologous in physico-chemical properties and biological function to other 11S globulins from leguminous plants (soy, pea, etc.) (Derbyshire et al., 1976). In this paper we examine the effect of thermal treatment and high-pressure treatment on the emulsifying behaviour of 11S globulin of V. faba broad bean in the absence and presence of the food polysaccharides i- or k-CAR. In particular, we investigate: (i) the effect of heating or high-pressure treatment on the pure protein and biopolymer mixture before emulsification with (a) varying polysaccharide concentration and (b) variation of ionic strength; and (ii) the effect of high-pressure treatment on pure protein-stabilised and mixed protein–polysaccharide-stabilised emulsions after preparation. In order to compare the various emulsion systems, the emulsification conditions are kept constant, as are the concentrations of 11S (0.5 wt%) and oil (20 vol%). This low protein/oil ratio was selected to give a fairly stable coarse emulsion with the protein alone, thereby providing the opportunity for improved stability following high-pressure treatment and/or addition of polysaccharide.

2. Materials and methods 2.1. Materials The 11S globulin of V. faba of broad beans (legumin) was prepared at the Institute of Biochemical Physics of the Russian Academy of Sciences, Moscow, by a procedure described previously (Popello & Suchkow, 1988). The legumin sample contained 95% of the main protein with an 11S sedimentation coefficient; the remaining 5% was composed of an admixture with a 15S sedimentation coefficient, according to the sedimentation analysis. The 11S had a molecular weight of 345 kDa in phosphate buffer at pH 7.8 and ionic strength 0.1 M. Food grade k-CAR and i-CAR samples were kindly donated by Systems Bio Industries (Carentan, France). The k-CAR, made from Eucheuma cottoni, was in 60 mol% potassium form and 40 mol% sodium form with a small amount of contamination by i-CAR (,10%). Weight-average molecular weight was given by the suppliers as 720 kDa and the z-average hydrodynamic radius as 100 nm. The food grade i-CAR, manufactured from Eucheuma denticulatum, was almost in pure sodium form with a small amount of contamination by k-CAR (about 5%). The weight-average molecular weight

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during high-pressure treatment—and during compression and decompression—was maintained at 25 ^ 18C: Samples were used within 2 h of treatment.

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ANS Intensity (I )

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2.4. Spectrofluorimetry

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Surface hydrophobicity measurements were performed on solutions of 11S (0.1 wt%) or mixtures of 11S (0.1 wt%) plus i- or k-CAR (0.05 wt%) before and after thermal or high-pressure treatment using ANS ammonium salt as a fluorescent probe at pH 8 (Nakai and Li-Chan, 1988). 1 ml of 4 × 1025 M ANS was added to 1 ml of sample at room temperature. The intensity was then measured using a Perkin–Elmer LS50 spectrofluorimeter (excitation 350 nm, slit 2.5 nm; emission 470 nm, slit 2.5 nm), and the relative intensity measured at 470 nm. In another series of experiments, the concentration of ANS was kept constant (2.54 mM) and the concentration of native and pressure-treated 11S (200 MPa for 20 min) was varied (0.028–2.812 mM). Quoted values are the mean ^ SD from three determinations.

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Temperature (C) Fig. 1. Effect of thermal treatment on the relative fluorescence intensity I of 11S (0.1 wt%) solutions at pH 8.0, 0.02 M Tris–HCl buffer.

was given by the suppliers as 560 kDa with the z-average hydrodynamic radius as 80 nm. The k-CAR molecule carries ,25% ester sulphate groups by weight and i-CAR has about 32% (Enriquez & Flick, 1989). 1-Anilinonaphthalene-8-sulphonate (ANS) ammonium salt (316.4 Da) was purchased from SERVA (Feinbiochemica, Heidelberg, Germany). TRIZMAw (Tris) and research-grade n-tetradecane were obtained from Sigma Chemical Co. (St. Louis, MO). Sodium chloride was purchased from BDH Chemicals Ltd. (Poole, UK). Buffer solutions (20 mM Tris–HCl, pH 8.0) were prepared with double-distilled water. Carrageenan solutions (0.05–0.15 wt%) were prepared by dispersing the polysaccharide powder in pH 8.0 buffer and continuously stirring at 708C for 30 min. The resulting solution was then cooled to 258C. The 11S (0.1 or 0.5 wt%) was dissolved in buffer at the desired concentration and added to the polysaccharide solution. This solution was of low viscosity and close to Newtonian, with no sign of gelation. 2.2. Heat treatment Samples (5 or 20 ml) of solutions of 11S (0.1 or 0.5 wt%) and mixtures of 11S (0.1 or 0.5 wt%) plus i- or k-CAR (0.05 or 0.15 wt%) were placed in a water bath and heated to a range of temperatures (40–808C) for 2–10 min in glass test tubes or beakers. Samples were slowly cooled to room temperature and used within 2 h after treatment. 2.3. High-pressure treatment Solutions of 11S (0.1 or 0.5 wt%, 5 or 20 ml) and mixed biopolymer solutions (5 or 20 ml) or oil-in-water emulsions (5 ml) were hermetically sealed in Cryovac bags (CryovacW.R. Grace Ltd, London, UK). These were subjected to pressures up to 600 MPa for 20 min in a laboratory-scale high-pressure rig (Stansted Fluid Power Ltd, Stansted, UK). The rates of compression and decompression were controlled to avoid significant temperature changes arising from adiabatic heating/cooling. The sample temperature

2.5. Emulsion preparation and stability Oil-in-water emulsions (0.5 wt% protein or mixtures of 11S (0.5 wt%) plus i- or k-CAR (0.065–0.15 wt%) were prepared at room temperature with untreated, heated or pressurised samples using a small-scale ‘jet homogenizer’ (Burgaud, Dickinson & Nelson, 1990) operating at 400 bar. Sodium azide (0.02 wt%) was added to the samples before homogenisation to retard microbial growth. Immediately after emulsion preparation the droplet-size distribution was determined using a Malvern Mastersizer S2.01 laser light-scattering particle size analyser (presentation code 0405). The remaining emulsion sample of height 60 mm was stored in a glass tube at 258C. Creaming behaviour was followed by visually measuring the changes in thickness of the cream and serum layers [a distinct (semi-) transparent layer] at the bottom of the tube over a period of 7 days. 2.6. Variation of ionic strength The ionic strength dependence of the effect of high-pressure treatment on emulsion properties has been studied for the systems containing 11S plus i-CAR and 20 vol% n-tetradecane. Ionic strength at pH 8.0 was adjusted to 0.01, 0.02, 0.03, 0.04 and 0.1 M by the addition of an appropriate amount of NaCl to the aqueous phase prior to high-pressure treatment and emulsification. 3. Results and discussion 3.1. Thermal treatment We consider first the surface hydrophobicity of native and heat-treated 11S in the absence of i-CAR. Fig. 1 shows the

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Fig. 2. Influence of thermal treatment on the emulsifying efficiency of 11S (0.5 wt%, 20 vol% n-tetradecane) and mixtures of 11S (0.5 wt%) plus i- or k-carrageenan (3.3:1 by weight) at pH 8.0, 0.02 M Tris–HCl buffer. Oil-inwater emulsions were prepared by jet homogenisation at 400 bar and 258C. The average droplet diameter (d43) for freshly made emulsions is plotted against temperature: X, 11S alone; O, 11S 1 k-CAR; A, 11S 1 i-CAR:

ANS intensity I for 11S (0.1 wt%) as a function of temperature. The data indicate very little change in fluorescence intensity (arbitrary units) at temperatures # 608C, with a sharp fluorescence intensity response when the temperature is increased above 708C for 2 min. Native 11S has a relatively low fluorescence intensity (low surface hydrophobicity) whereas the sample heated at 808C for 2 min has a high value. It is noteworthy that, under similar experimental conditions the 7S globulin from broad bean (Galazka, unpublished) also exhibits an increase in ANS binding, but to a lesser degree. These appearances are in agreement

with other researchers (Kato, Osako, Matsudomi & Kobayashi, 1983) who have suggested that the increase in surface hydrophobicity for the 7S globulin from soybean was due to conformational changes of the protein, as big differences were observed at its denaturation temperature. The ANS binding data presented here for the 11S globulin suggest that under the conditions applied, a substantial part of the 11S globulin is present in the 7S form. This form has a lower denaturation temperature (,708C) (Kato et al., 1983) than the 11S globulin in broad bean which is about 858C (Belyakova, Semenova & Antipova, 1999). In follow-up work it would be interesting to check by differential scanning calorimetry what the heat denaturation temperature(s) is (are) of the broad bean protein samples to clarify if the 11S form is present in the 7S form. Studies carried out with other globular proteins show variable results; heating causes an increase in surface hydrophobicity for OVA (Galazka et al., 1999b, Hayakawa, Kajihara, Morikawa, Oda & Fujio, 1992; Hayakawa, Linko & Linko, 1996; Kato et al., 1983), but a decrease for BSA and b-lactoglobulin (Kato et al., 1983). The presence of i-CAR …11S : i-CAR weight ratio ˆ 2 : 1† in the untreated biopolymer mixtures (not shown) leads to no significant change in surface hydrophobicity …I ˆ 13:0†, and the surface hydrophobicity for the mixture heated at 808C for 2 min …I ˆ 50† is similar to the TT 11S alone. Heating the mixture to 808C for 10 min or 858C for 5 min gives a slight increase in surface hydrophobicity …I ˆ 52:0†: As with OVA in the absence or presence of DS (Galazka et al., 1999b), treatment of 11S alone at 808C for 3 min induces extensive precipitation, while the solution of 11S 1 i-CAR heated for 10 min at 808C remains clear. It can be speculated that the presence of i-CAR inhibits the formation of aggregates by limiting the number of accessible protein reactive sites (Schmitt et al., 1998). Moving now to the emulsion systems, we first discuss systems containing no polysaccharide. Under the homogenising conditions used here, we observe that the effect of heat treatment on 11S is to reduce its emulsifying capacity. Fig. 2 compares the average droplet size d43 as a function of temperature for freshly prepared 11S emulsions in the absence and presence of i- or k-CAR. The average droplet diameter d43 is defined by d43 ˆ

S ni di4 i

S ni di3

;

i

Fig. 3. Extent of serum separation of emulsions made with 11S (0.5 wt%, 20 vol% n-tetradecane) and mixtures of 11S (0.5 wt%) plus i- or k-carrageenan (3.3:1 by weight) at pH 8.0, 0.02 M Tris–HCl buffer, 258C. The thickness L of the serum layer (expressed as a percentage of total sample height) in a 7-day-old emulsion is plotted against temperature: X, 11S alone; O, 11S 1 k-CAR; A, 11S 1 i-CAR:

where ni is the number of droplets of diameter di : The average droplet size for the fresh n-tetradecane-in-water emulsion made with untreated 11S (0.5 wt%) at pH 8.0 is d43 ˆ 8:5 mm: The replacement of the native protein by a sample of 11S which had been heated (65, 70 or 758C for 2 min) gives emulsions with larger average droplets. There is a consistent trend towards larger droplets with increasing temperature. The moderate treatment regime of 658C for

V.B. Galazka et al. / Food Hydrocolloids 13 (1999) 425–435 60

ANS Intensity (I)

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Pressure (MPa) Fig. 4. Influence of high-pressure treatment on the relative fluorescence intensity I of 11S (0.1 wt%) and mixtures of 11S (0.1 wt%) plus i- or kcarrageenan (2:1 by weight) solutions at pH 8.0, 0.02 M Tris–HCl buffer: X, 11S alone; O, 11S 1 k-CAR; A, 11S 1 i-CAR:

2 min gives d43 ˆ 9:0 mm, whereas the treatment of 758C for 2 min gives d43 ˆ 24 mm: The addition of i-CAR …11S : i-CAR weight ratio ˆ 3:3 : 1† to the untreated protein leads to emulsions with smaller average droplets …d43 ˆ 4:8 ^ 0:6 mm†, and the average droplet diameter for the mixture heated at 808C for 2 min is d43 ˆ 5:7 mm: It is interesting to note that heating the biopolymer mixture to 808C for 6 min gives emulsions with no significant change in the average droplet size …d43 ˆ 5:7 mm†: Replacement of i-CAR with the less sulphated polysaccharide, k-CAR, gives emulsions with larger droplet diameters (untreated mixture d43 ˆ 6:3 mm; thermally treated mixture 808C for 2 min d43 ˆ 8:0 mm), and with a more severe treatment regime (808C for 6 min) there is a further increase in the droplet size …d43 ˆ 12 mm† (not shown). The creaming behaviour for emulsions prepared with 11S in the absence and presence of i- or k-CAR …11S : CAR weight ratio ˆ 3:3 : 1† is shown in Fig. 3. The Table 1 Influence of high pressure processing (200 MPa for 20 min) on the protein hydrophobicity (S0) of native and pressurised 11S (0.028–2.812 mM) and mixtures of 11S (0.028–2.812 mM) plus i- or k-CAR (1:0.5 by weight) in the presence of 2.54 mM ANS at pH 8.0, 20 mM Tris–HCl. Data are the averages of triplicate measurements with an error ^ 10% 11S:Polysaccharide ratio Treatment pressure (by weight) (MPa)

S0 (absorbance units/mol of ANS)

0 0 0.5 (k-CAR) 0.5 (k-CAR) 0.5 (i-CAR) 0.5 (i-CAR)

19.5 54.6 19.3 34.9 17.5 27.2

0 200 0 200 0 200

429

thickness, L, of the serum layer, expressed as a percentage of the total sample height (6 cm) (over a period of 7 days with emulsion samples not subject to agitation) is plotted as a function of temperature. Emulsions made with 11S alone generally gave rapid serum separation …L ˆ 29%†, with a sharp increase in L with samples subjected to thermal treatment above 708C for 2 min (treatment 758C: L ˆ 41:7%), which is consistent with the ANS and d43 data. The presence of i-CAR gave slower rates of serum separation as one would expect from the initial mean droplet size. We can see that the value of L for the untreated mixture is very similar to the value obtained for emulsions made with thermally treated mixtures at 808C for 2 min (untreated: L ˆ 9:5%; heated: L ˆ 6:6%). In the emulsion systems containing 11S 1 k-CAR; the creaming stability for the untreated mixture is noticeably worse …L ˆ 55%† than that with the 11S alone. And, for the emulsions made with 11S 1 k-CAR pre-treated at 65 or 708C for 2 min, the value of L is still higher than for the 11S heated alone. This rapid and extensive serum separation is probably indicative of depletion flocculation with the system containing a substantial concentration of free (unadsorbed) k-CAR (Cao, Dickinson & Wedlock, 1990). Similar appearances have been observed for emulsions containing b-lactoglobulin with dextran or DS (Dickinson and Galazka, 1991). Heating the mixture to 75 or 808C for 2 min leads to emulsions with much improved creaming behaviour …L ˆ 3:5%†: However, the value of L is twice that for the 11S 1 i-CAR emulsions under similar experimental conditions. This vast improvement in creaming behaviour can be attributed to the polysaccharide protecting the 11S against protein denaturation and aggregation. 3.2. High pressure treatment In the following series of experiments we first compare ANS binding to native and pressurised 11S in the absence of carrageenan. We see in Fig. 4 that the ANS intensity I for 11S (0.1 wt%, pH 8.0) in 20 mM Tris–HCl is plotted as function of pressure. The data show little change in intensity of fluorescence at pressures # 100 MPa, but an S-shaped fluorescence intensity response which increases rapidly at higher pressures ($150 MPa for 20 min). Addition of k-CAR …11S : polysaccharide weight ratio ˆ 2 : 1† to the untreated mixture leads to only a very small change in surface hydrophobicity (Native 11S: I ˆ 13:9; untreated 11S 1 k-CAR : I ˆ 12:3), and the fluorescence intensity for the pressurised (250 MPa for 20 min) mixture increases to give an I value ˆ 32. Replacement of k-CAR with the more highly anionic i-CAR leads to a further reduction in surface hydrophobicity for the untreated mixture …I ˆ 11:5†: High-pressure treatment at 250 MPa gives an I value ˆ 22.6. Protein surface hydrophobicity (So) data for these systems are presented in Table 1. These data have been determined in another series of experiments from those shown in Fig. 4.

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d 43 (µm)

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Pressure (MPa) Fig. 5. Effect of high-pressure treatment on the emulsifying efficiency of 11S (0.5 wt%, 20 vol% n-tetradecane) and mixtures of 11S (0.5 wt%) plus i- or k-carrageenan (3.3:1 by weight) at pH 8.0, 0.02 M Tris–HCl buffer. Oil-in-water emulsions were prepared by jet homogenisation at 400 bar and 258C. The average droplet diameter (d43) for freshly made emulsions is plotted against the applied pressure: X, 11S alone; O, 11S 1 k-CAR; A, 11S 1 i-CAR:

As with the heated samples (t758C), we see that the value for the pressure treated 11S (200 MPa for 20 min) is about three times that for the untreated sample. This increase in surface hydrophobicity was also observed with vicilin, the 7S storage globulin of peas (Pedrosa & Ferreira, 1994). The authors found that high-pressure treatment at 240 MPa caused dissociation and accumulation of assembly intermediates at pH 8.0, which markedly increased between pH 9.0 and 10.0. It was suggested that pH change may involve deprotonation of lysine residues which participate in salt bridges between the vicilin subunits. Preliminary gel 60

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L (%)

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Pressure (MPa) Fig. 6. Extent of serum separation of emulsions made with 11S (0.5 wt%, 20 vol% n-tetradecane) and mixtures of 11S (0.5 wt%) 1 i- or k-carrageenan (3.3:1 by weight) at pH 8.0, 0.02 M Tris–HCl buffer, 258C. The thickness L of the serum layer (expressed as a percentage of total sample height) in a 7-day-old emulsion is plotted against the applied pressure: X, 11S alone; O, 11S 1 k-CAR; A, 11S 1 i-CAR:

permeation data at pH 8.0 (Galazka, unpublished) suggest that there is subunit dissociation and some aggregation of 11S during or after high-pressure treatment (200 MPa for 20 min), presumably as a result of the weakening of hydrophobic interactions. It is well established (Galazka & Ledward, 1998) that many quaternary structures exhibit complex behaviour such as dissociation followed by aggregation of subunits (Schmid, Lu¨demann & Jaenicke, 1979) or precipitation after pressurisation (Morild, 1981). In some cases, it is suggested that subunit dissociation and localised unfolding can result in the exposure of buried groups within the molecule which are free to pair with newly exposed solvent groups. Therefore, any aggregation processes at subunit interfaces may be due to pressure-induced interactions (Masson, 1992). It is interesting to note that pressure treatment of OVA and b-lactoglobulin causes an increase in surface hydrophobicity (Hayakawa et al., 1992, 1996; Galazka et al., 1996a,b, 1999b) whereas this causes a decrease for BSA (Galazka et al., 1997, 1999a). Table 1 shows that the presence of k-CAR (1:0.5 by weight) in the untreated mixtures leads to no change in the S0 value (untreated 11S: S0 ˆ 19:5 AU=mol ANS; mixture: S0 ˆ 19:3 AU=mol ANS), and that the protein hydrophobicity S0 for the pressure-treated (200 MPa for 20 min) 11S 1 k-CAR (S0 ˆ 34:9 AU=mol ANS) is nearly 40% lower than for the 11S treated alone (So ˆ 54:6 AU=mol ANS). Replacement of k-CAR with i-CAR gives a small reduction in the S0 value in the untreated mixture (S0 ˆ 17:5 AU=mol ANS), and the S0 value for the pressurised mixture increases two-fold (S0 ˆ 27:2 AU=mol ANS). Due to the ANS not binding to the carrageenans alone, it is deduced that the reduction in surface hydrophobicity in the biopolymer mixtures is caused by the blocking of the hydrophobic binding sites on the surface of the protein by the bulky polysaccharide. We now turn to a comparison of emulsions made with native and high-pressure treated 11S without polysaccharide. For the freshly prepared emulsions, Fig. 5 shows that high-pressure treatment in each case leads to emulsions with bigger droplets, and that there is a general trend toward larger average droplet size with increasing applied pressure. A moderate treatment regime of 100 MPa for 20 min gives d43 ˆ 10:2 mm, and the most severe treatment of 250 MPa for 20 min gives d43 ˆ 22 mm: We found previously that pressure treatment of b-lactoglobulin and whey protein concentrate (WPC) both produced a substantial loss of emulsifying efficiency (Galazka, Ledward, Dickinson & Langley, 1995; Galazka, Dickinson & Ledward, 1996b). This loss of emulsifying efficiency (i.e. unable to make such small droplets) is thought to be due to protein aggregation induced by –SS– linkages, with polymerisation involving the free cysteine residue of the protein. In Fig. 5 we see that pressure treatment of 11S 1 k-CAR biopolymer mixtures …11S : k-CAR weight ratio ˆ 3:3 : 1† gives emulsions with smaller droplets, and that there is a general trend towards reduced droplet size with increasing

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Fig. 7. Effect of i- or k-carrageenan on the average droplet-size of freshly made emulsions (0.5 wt% 11S, 20 vol% n-tetradecane) at pH 8.0, 0.02 M Tris–HCl buffer, 258C. The average droplet size d43 is plotted against polysaccharide concentration C: O, 11S 1 k-CAR; A, 11S 1 i-CAR:

pressure (#200 MPa). At the higher treatment regimes (250 MPa), however, it appears that there is a slight increase again in the average droplet diameter (treatment at 200 MPa: d43 ˆ 3:1 ^ 0:1 mm; treatment at 250 MPa: d43 ˆ 3:9 ^ 0:2 mm). In the case of 11S 1 i-CAR we note the same general trend, only here the droplet sizes are rather smaller (treatment at 100 MPa: d43 ˆ 3:2 ^ 0:1 mm; treatment at 250 MPa: d43 ˆ 3:4 ^ 0:2 mm). Similar trends were observed for systems containing other 11S 1 carrageenan ratios (not shown). Serum separation data for 7-day-old emulsions made with 11S are presented in Fig. 6, where the thickness, L, of the serum layer is plotted as a function of pressure. We see that emulsions prepared with 11S alone give rapid serum separation …L ˆ 29%†, which gradually increases at pressures #150 MPa …L ˆ 36%†: Above 150 MPa there is a sharp 14 12

P (d)

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Particle Size (µm) Fig. 8. Effect of i-carrageenan on the droplet-size distribution P(d) of freshly made emulsions (0.5 wt% 11S, 20 vol% n-tetradecane) at pH 8.0, 0.02 M Tris–HCl buffer, 258C: X, 11S alone; – –, 0.065 wt% i-CAR; A, 0.15 wt% i-CAR.

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decrease in emulsion stability with respect to serum separation (treatment at 200 MPa for 20 min: L ˆ 49%). As with the heated emulsions, the addition of i-CAR gives much less serum separation (untreated: L ˆ 9:5%; treatment 200 MPa for 20 min: L ˆ 2%) with a trend of increased stability with increasing intensity of treatment. Emulsions prepared with k-CAR instead of i-CAR give poor stability for the untreated mixture L ˆ 55%, but the stability improves with increase in pressure. We note a big improvement even after a modest treatment of 100 MPa for 20 min …L ˆ 20%†: The rapid and extensive serum separation encountered with the untreated 11S 1 k-CAR emulsions is probably the result of depletion flocculation. Fig. 7 illustrates the influence of concentration of polysaccharide (i- or k-CAR) on the average droplet size for freshly made emulsions. Under these conditions (pH 8.0, 20 mM Tris–HCl), we notice a gradual decrease in d43 for unpressurised emulsions containing either i- or k-CAR with increasing polysaccharide content. It is also noteworthy that, for the corresponding protein:polysaccharide ratios, emulsions containing 11S 1 i-CAR have smaller droplets than those made with 11S 1 k-CAR: The corresponding droplet-size distributions (Fig. 8) for the emulsions containing 11S 1 i-CAR show that the original broad distribution for the 11S (modal value , 9.0 mm) emulsion is split into two peaks (at , 0.9 and 5.0 mm) on the addition of i-CAR …C ˆ 0:065 wt%†: Further addition of i-CAR …C ˆ 0:15 wt%† leads to a larger peak at , 0.9 mm and a smaller peak at , 5.0 mm. In the case of emulsions prepared with unpressurised 11S 1 k-CAR; we note qualitative similar behaviour, but to a much lesser extent. It is interesting to note that experiments carried out by Dickinson and Pawlowsky (1997) have shown that BSA 1 i-CAR emulsions become flocculated by bridging over a similar relative range of carrageenan concentrations. Droplet-size distributions for emulsions freshly made with pressure-treated (200 MPa) 11S and high-pressure treated mixtures of 11S 1 i-CAR are presented in Fig. 9. Here we see the broad distribution (modal value ,15.0 mm) for the 11S emulsion split into two similar sized peaks (at ,0.55 and 5.0 mm) on the addition of i-CAR …C ˆ 0:065 wt%†: Further addition of i-CAR …C ˆ 0:15 wt%† leads to a larger peak at ,0.55 mm with a corresponding tail at higher droplet diameters. Emulsions prepared with pressurised mixtures of 11S 1 k-CAR gives similar trends (not shown), but the effect of polysaccharide addition is much less apparent at the low k-CAR concentrations …C ˆ 0:1 wt%; d43 ˆ 7:7 mm†: Light microscopy (not shown) of the emulsions prepared with pressure treated (200 MPa) 11S alone show significant levels of droplet flocculation, whereas this is not the case with the untreated or high-pressure treated biopolymer mixtures. The effect of the concentration of polysaccharide (i- or k-CAR) on the serum layer thickness L of untreated and high-pressure treated (200 MPa) emulsions after 7 days of storage is shown in Fig. 10. We first compare emulsions

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16

25

14 12

20

P (d)

10 8

d 43 (µm)

6 4 2 0 0.01

0.1

1

10

15

10

100

Particle size (µm) Fig. 9. Effect of i-carrageenan on the droplet-size distribution P(d) of emulsions (0.5 wt% 11S, 20 vol% n-tetradecane) made with high-pressure treated (200 MPa for 20 min) 11S and mixtures of 11S 1 polysaccharide at pH 8.0, 0.02 M Tris–HCl buffer, 258C: X, 11S alone; – –, 0.065 wt% i-CAR; A, 0.15 wt% i-CAR.

prepared with untreated mixtures of 11S 1 i-CAR: With increasing polysaccharide content, there is a systematic reduction in L from a maximum at C ˆ 0 wt%, …L ˆ 29%† to a minimum at C ˆ 0:15 wt%, …L ˆ 8%†: In the case of pressure treated (200 MPa for 20 min) biopolymer mixtures, the presence of i-CAR greatly reduces L from a maximum C ˆ 0 wt%, …L ˆ 49%† to a minimum C ˆ 0:15 wt%, …L ˆ 2%†, thus greatly improving the creaming stability. In contrast, the replacement of i-CAR with k-CAR in the untreated mixture leads to a vast deterioration in creaming stability. At C ˆ 0:065 wt%, …L ˆ 55%† the extent of serum separation is twice that of 11S

Fig. 10. Effect of i- or k-carrageenan on the extent of creaming of emulsions (0.5 wt% 11S, 20 vol% n-tetradecane, pH 8.0, 0.02 M Tris–HCl buffer) stored for 7 days at 258C. The thickness L of the serum layer (expressed as a percentage of total sample height) is plotted against the polysaccharide concentration C: X, untreated 11S 1 k-CAR; × , treated (200 MPa) 11S 1 k-CAR; O, untreated 11S 1 i-CAR; A, treated (200 MPa) 11S 1 i-CAR:

5

0 0

10

20

30

40

I (mM) Fig. 11. Effect of ionic strength on droplet-size of freshly made 11S stabilised emulsions in the absence and presence of i-CAR (7:1 by weight) (0.5 wt% 11S, 20 vol% n-tetradecane) at pH 8.0, 0.02 M Tris–HCl buffer. The average diameter d43 is plotted against ionic strength I: X, native 11S; O, pressure-treated (200 MPa) 11S; B, untreated 11S 1 i-CAR; A, pressure-treated (200 MPa) 11S 1 i-CAR:

alone …L ˆ 29%† and further increments of k-CAR content slightly improve creaming stability …C ˆ 0:15 wt%; L ˆ 40%†, but it is still worse than with 11S alone. As with the emulsions made with heated (65 or 708C) biopolymer mixtures of 11S 1 k-CAR; the adverse influence of unadsorbed (non-complexed) polysaccharide on creaming stability is attributed to depletion flocculation (Cao et al., 1990). On the contrary, high-pressure treatment of 11S 1 k-CAR leads to a vast improvement in creaming behaviour (Fig. 10). At C ˆ 0:065 wt% the value of L is 18%, and further additions of polysaccharide gives less serum separation …C ˆ 0:15 wt%; L ˆ 6:8%†: In the presence of i-CAR, gel permeation data (Galazka, unpublished) suggest that there is no protein-protein aggregation in the high-pressure treated mixtures. Thus, the improvement in the emulsifying properties shown by pressure treated mixtures is probably due to the polysaccharide protecting the protein against pressure-induced aggregation. The influence of ionic strength on the droplet diameter (d43) for fresh emulsions made with native and pressuretreated 11S in the absence and presence of i-CAR (7:1 by weight) is shown in Fig. 11. We see that NaCl addition (up to 0.04 M) has little effect on d43 for emulsions prepared with native or pressure treated 11S or native 11S 1 i-CAR mixtures. However, high-pressure treatment of 11S 1 i-CAR in the presence of NaCl gives emulsions with larger droplets, which is enhanced by further addition of salt. The protective effect of the polysaccharide is then lost at the higher ionic strength. Fig. 12 shows the effect of ionic strength on the serum

V.B. Galazka et al. / Food Hydrocolloids 13 (1999) 425–435 60 50

L (%)

40 30 20 10 0 0

10

20

30

40

I (mM) Fig. 12. Effect of ionic strength on extent of creaming of 7-day-old 11S stabilised emulsions in the absence and presence of i-CAR (7:1 by weight) (0.5 wt% 11S, 20 vol% n-tetradecane) at pH 8.0, 0.02 M Tris–HCl buffer. The thickness L of the serum layer (expressed as percentage of total sample height) is plotted against ionic strength I: X, native 11S; O, pressure-treated (200 MPa) 11S; B, untreated 11S 1 i-CAR; A, pressure-treated (200 MPa) 11S 1 i-CAR:

layer thickness L after 7 days of storage for the same set of emulsions. The data indicate little change in serum separation for emulsions made from the native and pressure treated 11S alone. However, at ionic strengths above 0.03 M there is a rapid increase in serum separation for emulsions made with native and high-pressure processed 11S 1 i-CAR: The presence of NaCl in concentrations greater than 0.05 M (not shown) leads to precipitation and phase separation of the biopolymer mixture. The exact salt content causing phase separation depends on the nature of the protein–polysaccharide interaction, the molecular weight of the polysaccharide and the relative concentrations of the two biopolymers (Dickinson and Semenova, 1992). In this study, we have mainly concentrated on systems in

Fig. 13. Influence of high-pressure processing on emulsions (0.5 wt% protein, 20 vol% n-tetradecane, pH 8.0, 0.02 M Tris–HCl buffer, 258C) made with native 11S and mixtures of 11S 1 i-CAR (7:1 by weight). The average droplet size d43 is plotted against applied pressure: X, 11S; A, 11S 1 i-CAR:

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which the protein and polysaccharide were subjected to high-pressure treatment before homogenisation. Now we turn briefly to see how the state of aggregation of droplets is affected by high-pressure after emulsification. The influence of pressure treatment on the average droplet diameter of fresh emulsions made with native 11S in the absence and presence of i-CAR (7:1 by weight), and then subjected to high-pressure treatment, is presented in Fig. 13. It is seen that pressurisation at 200 MPa for 20 min leads to a slight increase in the average droplet size from d43 ˆ 8:5 mm (no treatment) to d43 ˆ 10:4 mm for the 11S alone. In the presence of i-CAR, high-pressure treatment (200 MPa) also leads to larger droplets from d43 ˆ 6:4 mm (no treatment) to d43 ˆ 8:6 mm: There was no change in the overall droplet-size distribution, and no real evidence for droplet flocculation. Overall, the average droplet size was found to increase slightly with increasing applied pressure. Creaming data for the 7-day-old emulsions (not shown) also show increases in serum layer formation after high-pressure processing. As with previous studies on b-lactoglobulin (Galazka, Dickinson & Ledward, 1996b; Dickinson and James, 1998) and WPC (Galazka et al., 1995), it appears that the effect on d43 and creaming behaviour is less when high-pressure treatment is applied after emulsion formation. These results may be explained in terms that during homogenisation a protein probably becomes partially unfolded at the interface, and subsequent pressure treatment causes no further conformational change. Finally, to provide a reference point for this study, it is of interest to compare an emulsion prepared with a commercially efficient protein emulsifier such as sodium caseinate with the most stable emulsion made here with the 11S globulin. Fig. 14 shows droplet-size distributions of fresh n-tetradecane-in-water emulsions made with untreated sodium caseinate (0.5 wt% protein, 20 vol% oil), and high-pressure treated (200 MPa) 11S 1 i-CAR (weight ratio ˆ 3:3 : 1†: The average droplet diameter for the milk protein emulsion is d43 ˆ 1:0 mm and the d43 value for the plant protein 1 carrageenan emulsion is 3.2 mm. The creaming behaviour (not shown) for the 7-day-old emulsions are very similar with the extent of serum separation being L ˆ 6:0% for the milk protein emulsion and L ˆ 2:0% for the mixed system. Hence, by using optimum mixtures with carrageenan and applying high-pressure processing, we can improve the emulsifying properties of 11S globulin with behaviour approaching that of sodium caseinate. Based on experimental data reported in previous work with BSA 1 DS or BSA 1 carrageenans (Galazka et al., 1997, 1999a,b), we assume that high-pressure treatment causes dissociation of reversible electrostatic protein–polysaccharide complex(es). It is thought that protein becomes denatured and exposes more charged groups during the holding of pressure, and during pressure release attractive electrostatic protein–polysaccharide interactions are restored more strongly than ever. Recomplexation of the

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strengthens during thermal or high-pressure treatment. It seems highly likely that these interactions protect the protein against loss of functionality from aggregation during or after treatment (heating or pressurisation).

16 14 12

P (d)

10

Acknowledgements

8 6 4 2 0 0.01

0.1

1

10

100

Particle size (µm) Fig. 14. Comparison of droplet-size distributions of freshly prepared emulsions made with (a) native sodium caseinate (0.5 wt%) (X); and (b) pressure-treated (200 MPa) 11S (0.5 wt%) 1 i-CAR (3.3:1 by weight) (A) at pH 8.0, 0.02 M Tris–HCl.

polysaccharide with the unfolded protein occurs on decompression, which then protects the denatured protein against protein–protein aggregation.

4. Summarising remarks In this article, we have demonstrated that emulsions prepared with thermal or high-pressure treated 11S have poorer emulsifying and stabilising ability with respect to initial droplet size and creaming behaviour, when compared with the native protein. This is probably mainly due to enhanced dissociation of subunits and/or aggregation through disulphide bridging. Moderate thermal treatment appears to have a far greater effect than high-pressure treatment in terms of the associated changes in droplet-size and serum separation. This is in agreement with Dickinson and James (1998) who have reported that high-pressure processing destabilises b-lactoglobulin emulsion systems to a much lesser extent than heating, with high-pressure treatment considered to be a gentler processing operation in comparison to thermal processing. Emulsions prepared with untreated or thermally treated (,758C) biopolymer mixtures of 11S 1 k-CAR have been found to have smaller droplets but more extensive serum separation compared with the native protein. However, emulsions made with i-CAR have significantly better emulsifying efficiency and creaming stability. In all cases, highpressure treatment of 11S 1 i- or k-CAR mixtures leads to a remarkable improvement in emulsifying efficiency and emulsion stability. As in previous studies with BSA or OVA combined with sulphated polysaccharides at pH # 7:0 (Galazka et al., 1997, 1999ab), we have shown that the strength of the protein–polysaccharide interaction is dependent on the charge density of sulphate groups on the polysaccharide …i-CAR . k-CAR† and that the extent of interaction

We thank Dr M.G. Semenova for supplying the protein sample. We acknowledge funding from the Biotechnology and Biological Sciences Research Council, and thank the BBSRC Institute of Food Research (Reading) for use of the luminescence spectrometer for making the surface hydrophobicity measurements.

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