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Effect of sugars on the rheological properties of acid caseinate-stabilized emulsion gels
Food Hydrocolloids 16 (2002) 321±331
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Effect of sugars on the rheological properties of acid caseinate-stabilized emulsion gels Eric Dickinson*, Lara Matia Merino Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK Received 3 May 2001; revised 13 September 2001; accepted 24 September 2001
Abstract Rheological properties of sweetened acid-induced caseinate-stabilized emulsion gels (30% v/v n-tetradecane) and the corresponding acidinduced caseinate gels have been investigated. Emulsions were typically prepared with sodium caseinate (1.4% w/v) as emulsi®er and with added sucrose (0±66% w/v) or a mixture of sucrose 1 glucose (76% w/v) present in the aqueous phase. The development of a threedimensional network, following slow acidi®cation on addition of glucono-d-lactone granules (0.3 g GDL/g protein), was followed through the resulting increase in the small-deformation shear moduli (G 0 and G 00 ) at 258C. Sugar concentrations greater than 60% w/v led to larger emulsion droplets. Added sugar was found to reduce the gelation time and increase substantially the elastic modulus of these emulsion gels and protein gels, especially at high sugar/protein ratio. Sugar was found also to affect the large-deformation rheology, promoting strainweakening behavior and a shorter linear regime. Emulsion gels of high sugar content showed the typically expected behavior on changing oil volume fraction or protein content. Overall, the ®ndings are consistent with an enhancement in the strength of the protein±protein interactions in the presence of sugars. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Sugar; Acid-induced gelation; Emulsion gel; Sodium caseinate; Rheology
1. Introduction Milk protein-stabilized oil-in-water emulsions can be converted into emulsion gels by various methods such as heating (Dickinson & Chen, 1999), high-pressure treatment (Dickinson & James, 1998), and enzymic cross-linking (Dickinson & Yamamoto, 1996). All of these methods are effective for whey protein-coated emulsion droplets, and pH reduction may be used in the case of casein(ate)-based emulsions (Chen, Dickinson, & Edwards, 1999). The formation of acid-induced milk protein emulsion gels is brought about by the aggregation of the casein component (molecules, submicelles or micelles) on approaching its isoelectric point. Thus, upon acidi®cation, casein-coated oil droplets stick together, leading to formation of a particulate network structure, which can be characterized by its rheological properties. The properties of acid milk gels have been extensively studied (Horne, 1999, 2001; Lucey & Singh, 1998; van Vliet, Roefs, Zoon, & Walstra, 1989). However, most common fermented dairy products (e.g. yoghurt-style * Corresponding author. Tel.: 144-1132-332956; fax: 144-1132332982. E-mail address: [email protected] (E. Dickinson).
desserts) formed by the mechanism of acid-induced casein aggregation are far from being simple model systems since they involve the presence of other interacting species (e.g. whey proteins, lactose, emulsi®ers, polysaccharides, added sugars, etc.). This paper focuses speci®cally on the effect of added sugar on the rheology of acid-induced emulsion gels in model systems containing sodium caseinate, sucrose and emulsi®ed hydrocarbon oil as the three main functional components. The protection against globular protein unfolding that is conferred by high sugar/protein ratios is a well-known phenomenon with consequences for heat stability (Boye, Alli, & Ismail, 1996; Jou & Harper, 1996) and also for stability against pressure-induced denaturation (Dumay, Kalichevsky, & Cheftel, 1994). The phenomenon has been interpreted as follows. First, the direct contact between protein and water is considered thermodynamically unfavourable in the presence of sugars (Arakawa & Timasheff, 1982; Lee & Timasheff, 1981), and this can be correlated directly with an enhancement of hydrophobic interactions (Phillips, Whitehead, & Kinsella, 1994). Secondly, a diminished water activity in the presence of concentrated carbohydrates is considered to make water±protein interactions less effective (Barone, Del Vecchio, Giancola, & Notaro, 1992). Thirdly, direct sugar±protein interactions through
0268-005X/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0268- 005 X( 01) 00 105-9
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hydrogen bonding may lead to a change in protein surface hydrophilicity (Antipova & Semenova, 1995, 1997; Antipova, Semenova, & Belyakova, 1999). Novel aspects of the in¯uence of sugars on the gelation of globular proteins (mainly whey protein) have recently been studied. In terms of gelation rate, the differing effects of low sucrose concentration (0±10% w/w) or high sucrose concentration (10±30% w/w) on the `cold' gelation of heat-denatured whey protein isolate has been noted by Bryant & McClements (2000) and Kulmyrzaev, Cancelliere, and McClements (2000b). An increase in the extent of droplet ¯occulation after heating whey protein-stabilized emulsions in the presence of 10 wt% sucrose has also been reported (Kulmyrzaev, Bryant, & McClements, 2000a). In terms of the fracture properties of gels, the effect of sucrose (Boye, Alli, Ramaswamy, & Raghavan, 1997) and ribose or lactose (Rich & Foegeding, 2000) on the large-deformation properties of whey protein gels has been investigated. The kinetics of heat coagulation of concentrated milk proteins at high sucrose contents have also been studied by Pauletti, Castelao, and Seguro (1996), as well as the in¯uence of reducing sugars and sucrose on texture of thermally processed soy protein isolate-glucono-d-lactone gels (Peng, Ismail, & Easa, 2000), where `Maillard crosslinks' are considered to contribute to the hardness of the gels. Against this background, the in¯uence of sugars on the acid-induced gelation of caseins still needs to be properly characterized. 2. Materials and methods 2.1. Materials Spray-dried sodium caseinate (.82 wt% dry protein, ,6 wt% moisture, ,6 wt% fat and ash) was supplied by DMV international (Veghel, Netherlands). The hydrocarbon oil n-tetradecane (.99%), the acidulant glucono-d-lactone (GDL) (.99%) and d-(1)-glucose (.99.5%) were purchased from the Sigma Chemical Co. (St Louis, MO). Sucrose (.99.5%, derived from sugar beet) was supplied by British Sugar plc. All solutions were prepared using doubledistilled water. In what follows, the term `sugar' refers to sucrose or a mixture of sucrose 1 glucose. 2.2. Solution and emulsion preparation Sodium caseinate (2% w/v in emulsion gels; 3% w/v in protein gels) and sugar solutions were prepared overnight by gentle stirring at room temperature until dissolution was complete. The sugar concentration ranged from 0 to 76% w/v, using sucrose in all cases except for a mixture of 70% sucrose 1 30% glucose (dry weight) which was used to produce the highest total sugar concentration (76% w/v and 60% w/w). The sugar 1 protein solution constituted the aqueous phase of the oil-in-water emulsions (30% v/v n-tetradecane), made using a small-scale high-pressure jet
homogenizer operating at 300 bar. The content of the `base emulsion' was 30% v/v oil and 1.4% w/v sodium caseinate. This latter protein content was assumed suf®cient to emulsify the oil to a surface coverage of 2±3 mg/m 2 without introducing a signi®cant amount of non-adsorbed caseinate (Dickinson & Golding, 1997). Different contents of oil (5±25% v/v) and protein (0.46±2.0% w/v) were derived from the `base emulsion' by diluting appropriately with sucrose or sucrose/caseinate solutions. 2.3. Characterization of emulsions Emulsion droplet-size distributions were measured using a Malvern Mastersizer MS2000 static laser light-scattering analyzer. Mean droplet size was characterized by two average diameters, d32 and d43, de®ned by X 3 X 2 X 4 X 3 di = ni di ; d43 ni di = ni di ; d32 i
i
i
i
where ni is the number of droplets of diameter di. 2.4. Preparation of acid gels and rheological measurements Protein-stabilized emulsion gels and protein gels were investigated in situ by dynamic oscillatory rheometry using a controlled stress Bohlin CS50 rheometer (Bohlin Instruments, Cirencester, UK). Fresh emulsion and solution samples (equilibrated at 258C) were acidi®ed by adding GDL granules (0.3 g GDL/g protein) to 20 ml of emulsion or solution, respectively. This volume was chosen in order to minimize the errors encountered when measuring small quantities of GDL. Thorough mixing, for 10 min, was required to ensure uniform GDL distribution and hence the formation of a homogeneous gel. High sugar content samples were subjected to de-gassing by applying high-intensity ultrasound after initial mixing. Whilst still fresh, 12 ml of emulsion were introduced into the measurement cell of the rheometer using the coaxial cylinder (C25) geometry known as a `cup and bob' (27.5 mm o.d., 25 mm i.d.). A thin layer of low viscosity silicone oil was spread over the surface of the emulsion to prevent evaporation. Three consecutive oscillation tests were performed at 258C: (i) A test at single frequency (1 Hz) to study the development of viscoelasticity over time (storage modulus G 0 and loss modulus G 00 ) monitored in the linear regime at a shear strain of 0.005 (i.e. 0.5%) for 8 h. The pH of a portion of the acidi®ed sample stored in parallel to the portion in the rheometer was measured using a pH meter (Model HI8519N) from Hanna Instruments (Portugal). (ii) A frequency sweep test, where the timescale of the applied deformation increased stepwise from 0.001 to 10 Hz over ca. 2 h at a target strain of 0.005. (iii) An amplitude sweep at ®xed frequency (1 Hz), where the stress amplitude was increased stepwise from 0.025 to 1200 Pa over an average period of 2 min, in order to study the properties of gels at large deformation.
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Fig. 1. Effect of sugar concentration in the aqueous phase on the average droplet diameter of oil-in-water emulsions (30% v/v) prepared with sodium caseinate (1.4% w/v): (W) d32; (X) d43. Error bars are based on standard deviations for sets of ®ve measurements at each concentration.
3. Results and discussion 3.1. Particle-size distribution of the emulsions Fig. 1 shows the average particle size of sodium caseinate-stabilized oil-in-water emulsions (30% v/v) with increasing sugar concentration in the aqueous phase. Droplet diameters ranged between 0.1 and 10 mm, this span becoming broader (0.1±15 mm) at the highest sugar (sucrose 1 glucose) concentration (76% w/v). Emulsions having similar average diameters (d32 0:45 ^ 0:04 mm and d43 0:77 ^ 0:1 mm) were formed at sugar concentrations #60% w/v. The effectiveness of sodium caseinate as an emulsi®er seemed not to be affected by the presence of sugars, at least up to the mentioned concentration, since 21 there was no change in the speci®c surface area ( / d32 ) over that concentration range. Further incorporation of sugars produced a substantial increase in d43 and a moderate increase in d32. As expected, the quantity d43 showed a greater sensitivity to the presence of sugars and homogenization conditions, as re¯ected in the higher standard deviation of this parameter for $50% w/v concentration. The larger mean droplet diameter(s) for the emulsions made at high sugar content implies a poorer ef®ciency of emulsi®cation. This may be due to less effective droplet disruption in the homogenizer, probably with some larger droplets produced by recoalescence during/after emulsi®cation, as well as some kind of bridging ¯occulation Ð as was detected by conventional light microscopy. The in¯uence of sugars can be interpreted in terms of the in¯uence on protein aggregation, as well as on the effect of the viscosity of the bulk phase in reducing the ef®ciency of droplet disruption by turbulent ¯ow in the homogenizer (Dickinson, 1994). At 218C, the aqueous phase viscosity virtually doubles from 66 to 76% w/v sugar, and the mean diameters change from d43 1:26 ^ 0:24 mm and d32 0:56 ^ 0:08 mm to d43 2:49 ^ 0:33 mm and d32 0:91 ^ 0:11 mm over the same concentration range. The presence of sugar dissolved in the aqueous phase in¯uences optical properties as well as rheological ones.
Our concentrated emulsion samples showed a gradual transformation from turbid to nearly clear with increasing sugar content. As a consequence, the resulting acid-induced emulsion gels appeared translucent, in contrast to the normal `milky' appearance of emulsion gels not containing sugar. The physical basis for this is, of course, that adding sugar to the aqueous phase of a hydrocarbon oil-in-water emulsion lowers considerably the relative refractive index at the oil±water interface (Weiss & Liao, 2000). 3.2. Determination of the gelation time on lowering of the pH While a high GDL concentration reduces the gelation time, it can also cause over-acidi®cation (the pH being lowered to a value beyond the isoelectric point), which is detected rheologically as a maximum in the storage modulus beyond the critical gelation time (Horne, 2001). Consequently, in this study, different GDL/protein ratios were tested in order to attempt to establish the conditions for relatively fast gelation without causing any over-acidi®cation in the presence of sucrose (50% w/v in the aqueous phase). Fig. 2 shows the time development of the storage modulus G 0 at different GDL/protein ratios. Gelation time was de®ned as the time of sharp increase in the shear modulus G 0 from the baseline, this being signaled in practice by the detection of a modulus above the instrument noise level (ca. 1 Pa). According to this criterion, the measured gelation time decreased from 3.5 to 0.5 h when going from 0.15 g GDL/g caseinate to 0.8 g GDL/g caseinate. For all GDL concentrations studied, the plots of G 0 and G 00 (the latter not shown) reached almost plateau values of ca. 3500 and 750 Pa, respectively. Although there was still some slow increase in the shear moduli after this time, for the purposes of our analysis of the effect of added sugars we assume the establishment after several hours of essentially complete network formation and `®nal' gel strength, with the ®nal pH value being in the range 3.5±4 after 18 h for the data shown in Fig. 2. Based on the preceding preliminary results, it was
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Fig. 2. Time development of the storage modulus G 0 of acid-induced caseinate emulsion gels (1.4% w/v caseinate, 35% w/v sucrose, 30% v/v n-tetradecane) made at different GDL/protein (w/w) ratios: (e) 0.15; (W) 0.2; (A) 0.3; (X) 0.8.
thereafter decided to use a constant ratio of 0.3 g GDL/g caseinate (which gives a gelation time tgel < 1 h), and to investigate all the samples over a period of 8 h at single oscillation frequency. During this period, suf®cient time was available for the development of the ®nal viscoelasticity of these gels. Fig. 3 shows the gradual lowering of pH with time in these systems in the presence of sucrose (35% w/v) and its correlation with the development of the rheological parameters at the chosen GDL concentration. Gelation onset, as de®ned above, was found to occur at pH < 5:6: It can be inferred that the main body of the threedimensional gel network has already been formed after 3 h (between pH 5.6 and 4.6), with the ®nal pH value (ca. 4.0) reached after 17 h. Previously, Chen et al. (1999) found no rheological evidence for a three-dimensional network in acid-induced sodium caseinate emulsion gels when the pH value was outside the range 3.2±5.8. It appears from our observations that the presence of sucrose keeps the pH signi®cantly higher at extended times following GDL addition. To con®rm that this behavior was indeed a characteristic feature of the casein/sucrose system, the rate of pH lowering over the same 8 h period was also followed for pure protein gels (3% w/v sodium caseinate, no oil) without and with sucrose (66% w/v). Similar plots to Fig. 3 (data not shown) were obtained. While the ®rst 30 min of observation always gave the same rapid drop of pH from 6.9±7.0 to 5.8±5.9, two parallel curves were obtained after this time, the pH values of systems containing sucrose always lying ca. 0.2 units above those of the sugar-free systems. The implication of these observations, with gel points detected at pH < 5:6 and < 5.4 for caseinate gels with and without sucrose, respectively Ð and gelation always being faster in the former cases Ð will be discussed in Section 3.6. 3.3. Effect of sugar on gel strength 1 and gelation kinetics Fig. 4 shows the experimental dependence of storage 1 For clarity, in our terminology in this paper, the expression `gel strength' (or stiffness) refers to the small deformation elastic property (modulus); it is not a fracture property.
modulus G 0 (after 8 h) on sugar content for emulsion gels in the form of a log±log plot. Gels with ®nal shear moduli of up to ,5 kPa were formed. A rather weak dependence on sugar content was found up to 50% w/v, whereas at the higher sugar concentrations there was a greater rate of increase in gel strength. The system with the highest sugar concentration, achieved with a mixture of sucrose 1 glucose (to avoid crystallization of pure sucrose) behaved slightly differently: the modulus did not increase so strongly with increasing sugar concentration (76% w/v) following the stronger dependency observed for the pure sucrose systems (50±66% w/v). Fig. 5 shows the gel point (de®ned as above) of the caseinate-stabilized emulsion gels as a function of sugar concentration in the range 0±76% w/v (as measured in the aqueous phase of the emulsions). Whereas gelation time tgel was reduced from ca. 65 to 60 min on adding 10% sucrose, the increasing incorporation of sucrose up to 50% in the aqueous phase did not have such a substantial in¯uence in reducing tgel (down to 55 min at 50% sucrose). Above this concentration, however, a greater increase in gelling rate was found, with tgel falling from 55 to 39 min on increasing the sugar content from 50 to 76% w/v. This mirrors the large increase in gel strength at .50% w/v sugar (Fig. 4). These results indicate that sugar addition is more effective in enhancing the gelling properties of these protein-based emulsion systems at either low sugar content (0±10%) or high sugar content (50±76%). Horne (1995, 1996, 1999) has demonstrated that, under certain conditions, kinetic gelation pro®les for casein particle gel formation exhibit a scaling behavior that can be described by just two parameters: the gelation time tgel, and the gel strength at in®nite time (or, in practice, a ®xed fraction of this after a fairly long time). The ®rst parameter describes the dynamics of gel development, whereas the second speci®es a characteristic static rheological attribute. Fig. 6 was obtained by analyzing our kinetic data using this simple scaling model, with each pro®le G 0 (t) replotted as normalized elastic modulus (G 0 /G 0 max) against reduced time (t/tgel), where G 0 max refers to the value of G 0 after 8 h and tgel is the gelation time for the speci®c sugar concentration. A
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325
Fig. 3. Time development of the viscoelastic parameters, G 0 and G 00 , and their correlation with the lowering of pH for acid-induced sodium caseinate-stabilized emulsion gels containing sucrose (1.4% w/v caseinate, 35% w/v sucrose, 30% v/v oil, 0.3 g GDL/g protein) at 258C and 1 Hz: (e) pH; (X) G 0 ; (W) G 00 .
sequence of partially overlapping parallel curves of similar shape, ordered consistently according to ultimate gel strength, were obtained from the set of individual gelation pro®les as shown in Fig. 6(a). Consistent with the model of Horne (1995, 1996) devised for rennet milk gels, the individual pro®les corresponding to different sugar contents reduce to a kind of `master curve' within a certain acceptable tolerance. However, a more pronounced deviation from the rest of the set of curves is evident at the highest sugar concentration, 76% w/v, corresponding to the system containing the mixture of sucrose 1 glucose. To compare with systems not containing oil droplets, equivalent pure protein gels were formed by acidifying suf®cient sodium caseinate in solution to produce networks of notionally similar maximum gel strength (i.e. 2 £ 102 ±5 £ 103 Pa; as compared with 2 £ 103 ±5 £ 103 Pa for the emulsion gels). Following rheologically (at 1 Hz) the gelation of a solution of 3.0% w/v caseinate, it was noted that the resulting acid-induced protein gel started to lose strength beyond a certain peak G 0 value at around t=tgel < 3 (see Fig. 6(b)). With incorporation of sucrose at 50% w/v, however, this phenomenon of the loss of gel strength at longer times was seen to disappear, and a single monotonic increase was recorded (as in Fig. 6(a)). A reduction in the elastic modulus of an acid-induced casein gel following
initial gel rheology build-up is normally attributed to `overacidi®cation', which causes enhancement of repulsive electrostatic interactions between caseinate particles of net positive charge at pH values below the isoelectric point. Disappearance of this overacidi®cation modulus overshoot phenomenon in the sugar-containing systems would be consistent with the reduced extent of lowering of pH in the presence of sugars (see Section 3.2). But probably a more plausible alternative (and maybe additional) explanation is that there is substantial strengthening of the protein± protein interactions in the presence of sugars. That is, one may envisage that, at this low caseinate concentration (3.0% w/v) in the absence of oil droplets and sugars, the relatively weak initial network (,200 Pa) rapidly rearranges its structure, resulting in partial breakage of some of the load-bearing protein±protein bonds already formed. The fact that sugar addition contributes to eliminating the modulus `overshoot' is therefore consistent with most of the network-forming protein±protein interactions becoming stronger. Oscillatory sweep tests were performed to check the frequency dependence of the small-deformation rheology. Plotting log G 0 vs. log frequency gave essentially straight lines with positive slopes varying from 0.16 to 0.18 (data not shown) for all emulsion gels at different
Fig. 4. Effect of sugar concentration in the aqueous phase on the `®nal' storage modulus G 0 (after 8 h) for acid-induced sodium caseinate stabilized emulsion gels (1.4% w/v caseinate, 30% v/v n-tetradecane) at 258C and 1 Hz.
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Fig. 5. Effect of sugar concentration in the aqueous phase on the gelation time of acid-induced emulsion gels (30% v/v oil and 1.4% w/v sodium caseinate) at 258C and 1 Hz. Error bars re¯ect the fact that rheological measurements were taken every 2.5 min.
sugar concentrations. Values from 0.13 to 0.16 have been reported for acid skim milk gels of different fat content by Lucey, Munro, and Singh (1998). This con®rms that the rheological properties depend on the timescale of the applied deformation. The value of tan d G 00 =G 0 was found to decrease with increasing frequency for all the gels, suggesting that, as expected, the systems become more elastic in character at higher
frequencies (shorter timescales). For systems behaving like this, it can be reasonably assumed (Bryant & McClements, 2000) that microstructural rearrangements are able to relax within the timescale of the applied stress at low frequencies, and so the system appears less rigid. Conversely, at higher frequencies, there is insuf®cient time for such relaxation processes to take place; hence the system appears more rigid.
Fig. 6. Effect of sugar concentration on the time development of the storage modulus for (a) acid-induced emulsion gels (30% v/v oil, 1.4% w/v sodium caseinate) and (b) acid-induced protein gels (3% w/v sodium caseinate). The normalized elastic modulus G 0 /G 0 max is plotted against reduced time t/tgel, where G 0 max refers to the value of G 0 after 8 h and tgel is the gelation time for each speci®c sugar concentration: (W) 0% w/v; (e) 10% w/v; O, 30% w/v; (A) 50% w/v; ( £ ) 60% w/v; (X) 66% w/v; (B) 76% w/v. The sugar concentrations refer to the aqueous phase in the case of emulsion gels and overall concentrations in the case of protein gels.
E. Dickinson, L. Matia Merino / Food Hydrocolloids 16 (2002) 321±331
Fig. 7. Large-deformation behavior of (a) acid-induced emulsion gels (30% v/v oil, 1.4% w/v sodium caseinate) and (b) acid-induced protein gels (3% w/v sodium caseinate). The reduced complex modulus G p/G0p at 1 Hz is plotted against the strain for various sugar concentrations in the aqueous phase: (a) (W) 0% w/v; (e) 15% w/v; (O) 30% w/v; (A) 55% w/v; ( £ ) 60% w/v; (±) 63% w/v; (X) 66% w/v; (B) 76% w/v; (b) (W) 0.0% w/v; (e) 10% w/v; O, 30% w/v; (A) 50% w/v; £ , 60% w/v; (X) 66% w/v.
3.4. Large-deformation behavior Large-deformation rheology was explored by investigating the change in complex shear modulus G p in oscillatory mode as a function of the shear strain amplitude. To facilitate comparison between different systems, data were normalized with respect to the limiting low-strain modulus G0p. By gradually increasing stepwise the stress amplitude from very low values, the strain obtained in the sample is initially increased proportionally. This is the so-called linear viscoelastic range. Deviations from linearity occur when the gel is deformed to a strain at which some of the weak (physical) bonds of the aggregated network structure are destroyed. In general, particle gels have a much shorter linear region than crosslinked polymer gels. Fig. 7(a) shows that strain-weakening behavior is exhibited for all our emulsion gels. Samples with high sugar concentrations (30±76% w/v) showed deviations from linear behavior at strain values in the range 1±2% (corresponding to shear stresses of 50±80 Pa), whereas the linear regime was longer at lower sugar concentrations (15% w/v), and it extended to a strain of ,7%
327
(corresponding to a shear stress of ,130 Pa) for the emulsion sample without added sugar. Whereas the value of G p fell drastically with increasing strain in samples of high sugar content, suggesting extensive bond disruption during straining, the decrease in G p with strain was less pronounced at low sugar content. Fig. 7(b) shows the change in large-deformation behavior for 3.0% w/v caseinate gels with increasing incorporation of sugars in the system. It should ®rst be noted that the effect of sugar incorporation increases the small-deformation complex modulus G0p by more than an order of magnitude. So, for example, at a shear stress of ca. 20 Pa, the sample without sugar undergoes a shear deformation of ,10%, whereas samples at high sugar concentration are deformed by only ,0.3%. There is some evidence in Fig. 7(b) of a slight strain-hardening in the non-linear regime. This could be considered to resemble more closely typical polymer gel rheological behavior, i.e. strain hardening, a long linear region, and a large rupture strain (Dickinson & Chen, 1999). By incorporating more sugar into the system, the behavior changes to more like that of a typical particle gel with shorter breakable bonds (,1.5% strain before fracture at 66% w/v sucrose). The phase angle in the linear region reveals a greater degree of elastic gel character in the absence of sugar d < 138 compared to that at high sugar content d < 188: In agreement with previous studies of large-deformation behavior of particle gels (Yanez, Laarz, & Bergstrom, 1999), our results indicate that stronger gels are more brittle: that is, the maximum strain the gel can take before it `breaks' decreases with the sugar content. 3.5. Effects of oil volume fraction and protein content at constant sucrose content The in¯uence of oil volume fraction was studied systematically by diluting a sucrose-rich base emulsion (30% v/v oil, 1.4% w/v caseinate, 46.2% w/v sucrose) (i.e. 66% w/v in aqueous phase) with aqueous phase down to 5.0% oil, thereby keeping the mean emulsion droplet size d32 0:57 mm exactly constant throughout. A content of 46.2% w/v sucrose and a protein/oil ratio of 0.046 g caseinate/ml oil were maintained in all the diluted emulsions. The effect of protein concentration, varying from 2.0% w/v down to 0.46% w/v, was also studied at constant oil volume fraction (10% v/v) and sucrose concentration (46.2% w/v). Tables 1 and 2 show the characteristics of the emulsion gels in terms of overall composition, gel strength after 8 h (G 0 8 h), and the time to detect visible syneresis during storage after gelation. Using the data in Table 1 to plot the logarithm of the storage modulus against the logarithm of the oil volume fraction f , a strong linear dependence is found with a scaling exponent of ca. 4. (The actual linear ®t is log G 08 h 3:82 log f 2 2:018; with R2 0:9985: The general rule of ®lled gels is followed here: the higher the volume fraction of incorporated active ®ller particles, the greater the enhancement of the gel strength. It is important
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E. Dickinson, L. Matia Merino / Food Hydrocolloids 16 (2002) 321±331
Table 1 Storage modulus G 0 after 8 h and time of visible indication of syneresis (based on observations up to one week) for emulsion gels of different oil volume fraction f but constant sucrose content (46.2% w/v) and protein/oil ratio (0.046 g/ml)
Table 2 Storage modulus G 0 after 8 h and time of visible indication of syneresis (based on observations up to one week) for emulsion gels of different protein content but constant sucrose content (46.2% w/v) and oil volume fraction (0.1)
f
Protein content (% w/v)
G 08 h (Pa)
Syneresis?
Protein content (% w/v)
G 08 h (Pa)
Syneresis?
0.3 0.25 0.2 0.15 0.1 0.05
1.40 1.16 0.93 0.70 0.46 0.23
4:4 £ 103 1:7 £ 103 9 £ 102 3 £ 102 5 £ 101 , 1 £ 101
No No No No After 20 h After 10 h
0.6 0.8 1.2 1.6 2.0
9 £ 101 1:7 £ 102 4:6 £ 102 9:7 £ 102 1:6 £ 103
After 24 h After 36 h No No No
to note that a reduction in the number of oil droplets in the network also implies here a reduction in the total amount of protein present, since we are dealing with a base emulsion system in which nearly all the protein is coating the oil droplets (little unadsorbed protein). Previous studies in our laboratory (Dickinson & Chen, 1999) have found that there is a very substantial effect of the oil volume fraction on the viscoelasticity of heat-set protein emulsion gels. In this latter case, however, the nature of the network was rather different, with non-adsorbed aggregated whey protein contributing substantially to the rheology, and whey protein-coated emulsion droplets present as active ®llers. The effect of varying the total protein content, whilst maintaining the same number of droplets, corresponds to changing the ratio of unadsorbed to adsorbed caseinate in the emulsion gels (Table 2). The plot of logarithm of storage modulus against protein concentration Cp (not shown) is ®tted by the relationship: log G 08 h 2:434Cp0:429 R2 0:998: The weak emulsion gels containing sugars G 08 h , 200 Pa were observed to exhibit syneresis, with distinct vertical separation into a clear aqueous phase at the bottom and a turbid gel network above. Emulsion gel compositions associated with this phenomenon are detailed in Tables 1 and 2. This information roughly identi®es the system compositions promoting formation of particle gel networks that can maintain microstructural stability for an extended period of time. Beyond this stability regime, although there is enough aggregating material actually present to form the
gel, subsequent rearrangements of transient clusters via breaking and reforming of insuf®ciently strong protein± protein interactions lead, ®rst, to local (micro)-phase separation, and then to macroscopic syneresis. Fig. 8 shows that the gelation time is systematically reduced with increasing oil volume fraction. A qualitatively similar type of plot can be drawn for increasing protein content (not shown). The biopolymer gel literature indicates that gelation time depends on sample conditions, the polymer concentration being the most important factor (Clark, 1991). The gelling time generally increases as particle concentration decreases, which is in agreement with the trend exhibited here. Fig. 9 shows the time-dependent reduced storage modulus for different oil volume fractions. In accordance with the scaling analysis methodology of Horne (1996), all the data sets are seen to follow roughly the same sigmoidal curve in the covered oil volume fraction range (0.1±0.3), as do the equivalent plots for changing protein concentration (0.46±2.0% w/v) at constant oil content (not shown). The fact that there is no signi®cant change in kinetics upon diluting the emulsions in the presence of sugar seems to suggest that there is a similar aggregate growth following collapse of the caseinate protection layer upon acidi®cation. For the ®nal gelled emulsions (after 8±12 h), the phase angle remained essentially constant 14:5 ^ 0:58 throughout the linear region upon changing the oil volume fraction, but it varied slightly with protein content (from 14.0 to 16.88). This latter behavior shows that the viscous/elastic ratio changes on
Fig. 8. Effect of oil volume fraction f on gelation time tgel of acid-induced emulsion gels at high sugar concentration (46.2% w/v sucrose in whole system). Base emulsions (30% v/v oil, 1.4% w/v sodium caseinate) were diluted to obtain the different oil volume fractions (see text).
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329
Fig. 9. Kinetics of gelation for acid-induced emulsion gels at high sugar concentration (46.2% w/v) at different oil volume fractions: (W) 0.1; (e) 0.15; ( £ ) 0.2; (O) 0.25; (X) 0.3. The normalized elastic modulus G 0 /G 0 max is plotted against reduced time t/tgel, where G 0 max refers to the value of G 0 after 8 h and tgel is the gelation time.
manipulating the amount of non-adsorbed protein. This is presumably due to a transformation from a rheology dominated by a network of protein-coated oil droplets to one in which the aggregated interdroplet protein contributes signi®cantly to the network. Finally, Fig. 10 shows the large-deformation rheological behavior for the sets of emulsion gels with the compositions recorded in Table 1. At the lowest oil and protein contents, we see that the rheology is relatively insensitive to strain, and that there is even some evidence of a slight strain-hardening
behavior in the range 20±40%. A shortening of the linear region is seen to be promoted on increasing either (a) the oil droplet concentration or (b) the protein concentration. 3.6. Interpretation in terms of protein interactions The general effect of sugars on protein functionality is typically discussed in terms of the in¯uence on the protein± protein interactions. What we ®nd here is that the rigidity of an acid-induced caseinate-stabilized emulsion gel (or pure
Fig. 10. Large deformation behavior of acid-induced emulsion gels at high sugar content (46.2% w/v in whole system). The reduced complex modulus G p/G0p at 1 Hz is plotted against the strain. (a) Effect of changing oil volume fraction: (W) 0.1; (O) 0.15; (e) 0.2; (X) 0.25; ( £ ) 0.3. (b) Effect of changing protein content (% w/v): (B) 0.46; (W) 0.6; (O) 0.8; (e) 1.2; (X) 1.6; ( £ ) 2.0.
330
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caseinate gel) can be substantially enhanced by the presence of sugars in the aqueous phase, especially at concentrations above 50% w/v. Moreover, the gelation kinetics is affected over the whole range of studied sugar concentrations (0±76% w/v) leading to a reduction in gelation time compared with the sugar-free system. The presence of sugar also changes qualitatively the evolving structural and rheological development by inhibiting the reduction in elastic modulus at longer times. All of these facts are broadly consistent with the strength of the casein±casein molecular interactions being enhanced by the presence of added sugars. There is considerable independent evidence in the literature supporting the role of sugar as an enhancer of protein±protein interactions and protein aggregation, e.g. (i) an inferred increased attraction between unfolded whey protein molecules in the presence of sucrose (Kulmyrzaev et al., 2000a,b), (ii) a stronger attractive electrostatic interaction of casein molecules caused by the addition of sugars (Mora-Gutierrez, Kumosinski, & Farrell, 1997), and (iii) a corresponding strengthening of the protein±protein attractive interactions in caseinate (Antipova et al., 1999). As noted by van Vliet et al. (1989), the most important interaction forces in casein gels are probably the hydrophobic and the unlike (^) electrostatic interactions. One could reasonably expect that, the faster the lowering of the pH, the faster will be the gelation Ð on the basis that pH change is the dominant factor inducing the aggregation, with electrostatic casein±casein repulsive interactions being gradually overwhelmed by combined hydrophobic and electrostatic attractive interactions. However, the fact that the pH in systems containing sugars at the gelation point (ca. 5.6) was consistently higher than in the equivalent sugar-free system (ca. 5.4), and that it stayed 0.2 units higher at post-gelation times, could be interpreted as implying that the attractive hydrophobic bonds are more enhanced by the presence of sugars than the unlike (^) electrostatic interactions. This view is supported, for instance, by the work of Phillips et al. (1994) who have attributed the main effect of sugars to the promotion of hydrophobic interactions through the modi®cation of water structure surrounding the proteins. Based on the measured gel points of our caseinatestabilized emulsions, it would seem that sensitivity to sugar addition is rather greater at the extreme ranges of low and high sugar concentrations. The substantial dependence of the strength of protein±sugar interactions on the actual sugar concentration itself is implied by much published work in this ®eld (see, for instance, Antipova & Semenova, 1997). Abbasi & Dickinson (2001) have recently reported the existence of critical ranges of sugar concentration for producing pressure-induced gels from dispersions of skim milk powder at micellar casein concentrations down to ,2% w/v. This latter work has illustrated the importance of minimum sugar±protein ratios for promoting milk protein gelation, and has also identi®ed the existence of high sugar concentrations (.50 wt%) at which pressure-induced
gelation of casein micellar systems is completely inhibited. At these high sugar concentrations, the sugar appears to `protect' the casein micelles against pressure-induced dissociation by modifying the interactions of the casein molecules with colloidal calcium phosphate and with other casein molecules. This dual role of added sugars, in promoting or delaying protein gelation, depending on the sugar concentration, has also been noted by Kulmyrzaev et al. (2000b) for the case of cold gelation of heat-denatured whey proteins in the presence of sucrose. Our results suggest that the mechanism underlying the gelation kinetics is dependent not only on the ®nal gel strength but also on the initial aggregation period (i.e. the gelation time). Bryan & McClements (2000) have explained the varying effects of sugar concentration on the kinetics in terms of whether or not sugars retard or enhance the gelation by, respectively, reducing or increasing the collision frequency. In our casein-stabilized emulsions, the presence of sugars could increase the effective number of interacting particles and the strength of interparticle interactions, thus leading to a greater degree of `interconnectivity' of the gel microstructure. The signi®cant deviation in the characteristics of the aggregation process for the case of the system containing sucrose 1 glucose (76% w/v), compared with the pure sucrose systems, could be due either to some different speci®c effect of the glucose on the protein interactions or, more likely, the predominant kinetic effect of the high viscosity over the thermodynamic effects on the protein± protein interactions. Factors affecting the large-deformation rheology of gels include the number of bonds per cross section of the strand, the strength of each bond (van Vliet, Luyten, & Walstra, 1991), and the tortuosity of the gel network (Bremer, Bijsterbosch, Schrijvers, van Vliet, & Walstra, 1990). We can speculate that, by incorporating sugars into emulsion gels or protein gels, the strength of bonds between caseinate-coated droplets and caseinate particles might be increased, making connections between structural units less ¯exible and of shorter range, and thereby leading to a shorter linear viscoelastic range. Assuming that the sugars in interaction with the protein can be regarded as being an active part of the network, the larger rigidity of the resulting (emulsion) gels can be reasonably attributable to the increased number of structurally important bonds. In the presence of sugars, the combination of just 10% v/v oil and 0.8% w/v protein has been found to be suf®cient to initiate network formation. Nevertheless, syneresis experienced by such acid-induced gels of low protein and oil contents would seem to suggest that the droplet±droplet interactions are not strong enough to maintain the network structure following rearrangements on storage. Acknowledgements We gratefully acknowledge receipt by L.M.M. of an
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EPSRC Research Studentship and ®nancial support from Nestle Product Technology Centre (York, UK). References Abbasi, S., & Dickinson, E. (2001). In¯uence of sugars on high-pressure induced gelation of skim milk dispersions. Food Hydrocolloids, 15, 315±319. Antipova, A. S., & Semenova, M. G. (1995). Effect of sucrose on the thermodynamic incompatibility of different biopolymers. Carbohydrate Polymers, 28, 359±365. Antipova, A. S., & Semenova, M. G. (1997). Effect of neutral carbohydrate structure in the set glucose/sucrose/maltodextrin/dextran on protein surface activity at the air/water interface. Food Hydrocolloids, 11, 71±77. Antipova, A. S., Semenova, M. G., & Belyakova, L. E. (1999). Effect of sucrose on the thermodynamic properties of ovalbumin and sodium caseinate in bulk solution and at air±water interface. Colloids and Surfaces B, 12, 261±270. Arakawa, T., & Timasheff, S. N. (1982). Stabilization of protein structure by sugars. Biochemistry, 21, 6536±6544. Barone, G., Del Vecchio, P., Giancola, C., & Notaro, G. (1992). Conformational stability of proteins and peptide±peptide interactions in the presence of carbohydrates. Thermochimica Acta, 199, 189±196. Boye, J. I., Alli, I., & Ismail, A. A. (1996). Interactions involved in the gelation of bovine serum albumin. Journal of Agricultural and Food Chemistry, 44, 996±1004. Boye, J. I., Alli, I., Ramaswamy, H., & Raghavan, V. G. S. (1997). Interactive effects of factors affecting gelation of whey proteins. Journal of Food Science, 62, 56±65. Bremer, L. G. B., Bijsterbosch, B. H., Schrijvers, R., van Vliet, T., & Walstra, P. (1990). On the fractal nature of the structure of acid casein gels. Colloids and Surfaces, 51, 159±170. Bryant, C. M., & McClements, D. J. (2000). In¯uence of sucrose on NaCl-induced gelation of heat denatured whey protein solutions. Food Research International, 33, 649±653. Chen, J., Dickinson, E., & Edwards, M. (1999). Rheology of acid-induced sodium caseinate stabilized emulsion gels. Journal of Texture Studies, 30, 377±396. Clark, A. H. (1991). Structural and mechanical properties of biopolymer gels. In E. Dickinson, Food polymers, gels and colloids (pp. 322±338). Cambridge: Royal Society of Chemistry. Dickinson, E. (1994). Emulsions and droplet-size control. In D. J. Wedlock, Controlled particle, droplet and bubble formation (pp. 191±216). Oxford: Butterworth-Heinemann. Dickinson, E., & Chen, J. (1999). Heat-set whey protein emulsion gels: role of active and inactive ®ller particles. Journal of Dispersion Science and Technology, 20, 197±213. Dickinson, E., & Golding, M. (1997). Depletion ¯occulation of emulsions containing unadsorbed sodium caseinate. Food Hydrocolloids, 11, 13±18. Dickinson, E., & James, J. D. (1998). Rheology and ¯occulation of high-pressure-treated b-lactoglobulin-stabilized emulsions: Comparison with thermal treatment. Journal of Agricultural and Food Chemistry, 46, 2565±2571. Dickinson, E., & Yamamoto, Y. (1996). Rheology of milk protein gels and protein-stabilized emulsion gels cross-linked with transglutaminase. Journal of Agricultural and Food Chemistry, 44, 1371±1377.
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Dumay, E. M., Kalichevsky, M. T., & Cheftel, J. C. (1994). High-pressure unfolding and aggregation of b-lactoglobulin and the baroprotective effects of sucrose. Journal of Agricultural and Food Chemistry, 42, 1861±1868. Horne, D. S. (1995). Scaling behaviour of shear moduli during the formation of rennet milk gels. In E. Dickinson & D. Lorient, Food macromolecules and colloids (pp. 456±461). Cambridge: Royal Society of Chemistry. Horne, D. S. (1996). Aspects of scaling behaviour in the kinetics of particle gel formation. Journal de Chime Physique et de Physico Chimie Biologique, 93, 977±986. Horne, D. S. (1999). Formation and structure of acidi®ed milk gels. International Dairy Journal, 9, 261±268. Horne, D. S. (2001). Factors in¯uencing acid-induced gelation of skim milk. In E. Dickinson & R. Miller, Food colloids: fundamentals of formulation (pp. 345±351). Cambridge: Royal Society of Chemistry. Jou, K. D., & Harper, W. J. (1996). Effect of di-saccaharides on the thermal properties of whey proteins determined by differential scanning calorimetry. Milchwissenschaft, 51, 509±512. Kulmyrzaev, A., Bryant, C., & McClements, D. J. (2000a). In¯uence of sucrose on the thermal denaturation, gelation, and emulsion stabilization of whey proteins. Journal of Agricultural and Food Chemistry, 48, 1593±1597. Kulmyrzaev, A., Cancelliere, C., & McClements, D. J. (2000b). In¯uence of sucrose on cold gelation of heat-denatured whey protein isolate. Journal of the Science of Food and Agriculture, 80, 1314±1318. Lee, J. C., & Timasheff, S. N. (1981). The stabilization of proteins by sucrose. Journal of Biological Chemistry, 256, 7193±7201. Lucey, J. A., & Singh, H. (1998). Formation and physical properties of acid milk gels: a review. Food Research International, 30, 529±542. Lucey, J. A., Munro, P. A., & Singh, H. (1998). Rheological properties and microstructure of acid milk gels as affected by fat content and heat treatment. Journal of Food Science, 63, 660±664. Mora-Gutierrez, A., Kumosinski, T. F., & Farrell Jr., H. M. (1997). Oxygen-17 nuclear magnetic resonance studies of bovine and caprine hydration and activity in deuterated sugar solutions. Journal of Agricultural and Food Chemistry, 45, 4545±4553. Pauletti, M. S., Castelao, E. L., & Seguro, E. (1996). Kinetics of heat coagulation of concentrated milk proteins at high sucrose contents. Journal of Food Science, 61, 1207±1210. Peng, L. K., Ismail, N., & Easa, A. M. (2000). Effects of reducing sugars on texture of thermally processed soy protein isolate-glucono-delta-lactone gels. Journal of Food Science and Technology Ð Mysore, 37, 188±190. Phillips, L. G., Whitehead, D. M., & Kinsella, J. (1994). Structure±function properties of food proteins, London: Academic Press. Rich, L. M., & Foegeding, E. A. (2000). Effects of sugars on whey protein isolate gelation. Journal of Agricultural and Food Chemistry, 48, 5046±5052. van Vliet, T., Roefs, S. P. M. F., Zoon, P., & Walstra, P. (1989). Rheological properties of casein gels. Journal of Dairy Research, 56, 529±534. van Vliet, T., Luyten, H., & Walstra, P. (1991). Fracture and yielding of gels. In E. Dickinson, Food polymers, gels and colloids (pp. 392±405). Cambridge: Royal Society of Chemistry. Weiss, J., & Liao, W. (2000). Addition of sugars in¯uences colour of oil-inwater emulsions. Journal of Agricultural and Food Chemistry, 48, 5053±5060. Yanez, J. A., Laarz, E., & Bergstrom, L. (1999). Viscoelastic properties of particle gels. Journal of Colloid and Interface Science, 209, 162±172.
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Effect of sugars on the rheological properties of acid caseinate-stabilized emulsion gels Eric Dickinson*, Lara Matia Merino Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK Received 3 May 2001; revised 13 September 2001; accepted 24 September 2001
Abstract Rheological properties of sweetened acid-induced caseinate-stabilized emulsion gels (30% v/v n-tetradecane) and the corresponding acidinduced caseinate gels have been investigated. Emulsions were typically prepared with sodium caseinate (1.4% w/v) as emulsi®er and with added sucrose (0±66% w/v) or a mixture of sucrose 1 glucose (76% w/v) present in the aqueous phase. The development of a threedimensional network, following slow acidi®cation on addition of glucono-d-lactone granules (0.3 g GDL/g protein), was followed through the resulting increase in the small-deformation shear moduli (G 0 and G 00 ) at 258C. Sugar concentrations greater than 60% w/v led to larger emulsion droplets. Added sugar was found to reduce the gelation time and increase substantially the elastic modulus of these emulsion gels and protein gels, especially at high sugar/protein ratio. Sugar was found also to affect the large-deformation rheology, promoting strainweakening behavior and a shorter linear regime. Emulsion gels of high sugar content showed the typically expected behavior on changing oil volume fraction or protein content. Overall, the ®ndings are consistent with an enhancement in the strength of the protein±protein interactions in the presence of sugars. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Sugar; Acid-induced gelation; Emulsion gel; Sodium caseinate; Rheology
1. Introduction Milk protein-stabilized oil-in-water emulsions can be converted into emulsion gels by various methods such as heating (Dickinson & Chen, 1999), high-pressure treatment (Dickinson & James, 1998), and enzymic cross-linking (Dickinson & Yamamoto, 1996). All of these methods are effective for whey protein-coated emulsion droplets, and pH reduction may be used in the case of casein(ate)-based emulsions (Chen, Dickinson, & Edwards, 1999). The formation of acid-induced milk protein emulsion gels is brought about by the aggregation of the casein component (molecules, submicelles or micelles) on approaching its isoelectric point. Thus, upon acidi®cation, casein-coated oil droplets stick together, leading to formation of a particulate network structure, which can be characterized by its rheological properties. The properties of acid milk gels have been extensively studied (Horne, 1999, 2001; Lucey & Singh, 1998; van Vliet, Roefs, Zoon, & Walstra, 1989). However, most common fermented dairy products (e.g. yoghurt-style * Corresponding author. Tel.: 144-1132-332956; fax: 144-1132332982. E-mail address: [email protected] (E. Dickinson).
desserts) formed by the mechanism of acid-induced casein aggregation are far from being simple model systems since they involve the presence of other interacting species (e.g. whey proteins, lactose, emulsi®ers, polysaccharides, added sugars, etc.). This paper focuses speci®cally on the effect of added sugar on the rheology of acid-induced emulsion gels in model systems containing sodium caseinate, sucrose and emulsi®ed hydrocarbon oil as the three main functional components. The protection against globular protein unfolding that is conferred by high sugar/protein ratios is a well-known phenomenon with consequences for heat stability (Boye, Alli, & Ismail, 1996; Jou & Harper, 1996) and also for stability against pressure-induced denaturation (Dumay, Kalichevsky, & Cheftel, 1994). The phenomenon has been interpreted as follows. First, the direct contact between protein and water is considered thermodynamically unfavourable in the presence of sugars (Arakawa & Timasheff, 1982; Lee & Timasheff, 1981), and this can be correlated directly with an enhancement of hydrophobic interactions (Phillips, Whitehead, & Kinsella, 1994). Secondly, a diminished water activity in the presence of concentrated carbohydrates is considered to make water±protein interactions less effective (Barone, Del Vecchio, Giancola, & Notaro, 1992). Thirdly, direct sugar±protein interactions through
0268-005X/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0268- 005 X( 01) 00 105-9
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hydrogen bonding may lead to a change in protein surface hydrophilicity (Antipova & Semenova, 1995, 1997; Antipova, Semenova, & Belyakova, 1999). Novel aspects of the in¯uence of sugars on the gelation of globular proteins (mainly whey protein) have recently been studied. In terms of gelation rate, the differing effects of low sucrose concentration (0±10% w/w) or high sucrose concentration (10±30% w/w) on the `cold' gelation of heat-denatured whey protein isolate has been noted by Bryant & McClements (2000) and Kulmyrzaev, Cancelliere, and McClements (2000b). An increase in the extent of droplet ¯occulation after heating whey protein-stabilized emulsions in the presence of 10 wt% sucrose has also been reported (Kulmyrzaev, Bryant, & McClements, 2000a). In terms of the fracture properties of gels, the effect of sucrose (Boye, Alli, Ramaswamy, & Raghavan, 1997) and ribose or lactose (Rich & Foegeding, 2000) on the large-deformation properties of whey protein gels has been investigated. The kinetics of heat coagulation of concentrated milk proteins at high sucrose contents have also been studied by Pauletti, Castelao, and Seguro (1996), as well as the in¯uence of reducing sugars and sucrose on texture of thermally processed soy protein isolate-glucono-d-lactone gels (Peng, Ismail, & Easa, 2000), where `Maillard crosslinks' are considered to contribute to the hardness of the gels. Against this background, the in¯uence of sugars on the acid-induced gelation of caseins still needs to be properly characterized. 2. Materials and methods 2.1. Materials Spray-dried sodium caseinate (.82 wt% dry protein, ,6 wt% moisture, ,6 wt% fat and ash) was supplied by DMV international (Veghel, Netherlands). The hydrocarbon oil n-tetradecane (.99%), the acidulant glucono-d-lactone (GDL) (.99%) and d-(1)-glucose (.99.5%) were purchased from the Sigma Chemical Co. (St Louis, MO). Sucrose (.99.5%, derived from sugar beet) was supplied by British Sugar plc. All solutions were prepared using doubledistilled water. In what follows, the term `sugar' refers to sucrose or a mixture of sucrose 1 glucose. 2.2. Solution and emulsion preparation Sodium caseinate (2% w/v in emulsion gels; 3% w/v in protein gels) and sugar solutions were prepared overnight by gentle stirring at room temperature until dissolution was complete. The sugar concentration ranged from 0 to 76% w/v, using sucrose in all cases except for a mixture of 70% sucrose 1 30% glucose (dry weight) which was used to produce the highest total sugar concentration (76% w/v and 60% w/w). The sugar 1 protein solution constituted the aqueous phase of the oil-in-water emulsions (30% v/v n-tetradecane), made using a small-scale high-pressure jet
homogenizer operating at 300 bar. The content of the `base emulsion' was 30% v/v oil and 1.4% w/v sodium caseinate. This latter protein content was assumed suf®cient to emulsify the oil to a surface coverage of 2±3 mg/m 2 without introducing a signi®cant amount of non-adsorbed caseinate (Dickinson & Golding, 1997). Different contents of oil (5±25% v/v) and protein (0.46±2.0% w/v) were derived from the `base emulsion' by diluting appropriately with sucrose or sucrose/caseinate solutions. 2.3. Characterization of emulsions Emulsion droplet-size distributions were measured using a Malvern Mastersizer MS2000 static laser light-scattering analyzer. Mean droplet size was characterized by two average diameters, d32 and d43, de®ned by X 3 X 2 X 4 X 3 di = ni di ; d43 ni di = ni di ; d32 i
i
i
i
where ni is the number of droplets of diameter di. 2.4. Preparation of acid gels and rheological measurements Protein-stabilized emulsion gels and protein gels were investigated in situ by dynamic oscillatory rheometry using a controlled stress Bohlin CS50 rheometer (Bohlin Instruments, Cirencester, UK). Fresh emulsion and solution samples (equilibrated at 258C) were acidi®ed by adding GDL granules (0.3 g GDL/g protein) to 20 ml of emulsion or solution, respectively. This volume was chosen in order to minimize the errors encountered when measuring small quantities of GDL. Thorough mixing, for 10 min, was required to ensure uniform GDL distribution and hence the formation of a homogeneous gel. High sugar content samples were subjected to de-gassing by applying high-intensity ultrasound after initial mixing. Whilst still fresh, 12 ml of emulsion were introduced into the measurement cell of the rheometer using the coaxial cylinder (C25) geometry known as a `cup and bob' (27.5 mm o.d., 25 mm i.d.). A thin layer of low viscosity silicone oil was spread over the surface of the emulsion to prevent evaporation. Three consecutive oscillation tests were performed at 258C: (i) A test at single frequency (1 Hz) to study the development of viscoelasticity over time (storage modulus G 0 and loss modulus G 00 ) monitored in the linear regime at a shear strain of 0.005 (i.e. 0.5%) for 8 h. The pH of a portion of the acidi®ed sample stored in parallel to the portion in the rheometer was measured using a pH meter (Model HI8519N) from Hanna Instruments (Portugal). (ii) A frequency sweep test, where the timescale of the applied deformation increased stepwise from 0.001 to 10 Hz over ca. 2 h at a target strain of 0.005. (iii) An amplitude sweep at ®xed frequency (1 Hz), where the stress amplitude was increased stepwise from 0.025 to 1200 Pa over an average period of 2 min, in order to study the properties of gels at large deformation.
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Fig. 1. Effect of sugar concentration in the aqueous phase on the average droplet diameter of oil-in-water emulsions (30% v/v) prepared with sodium caseinate (1.4% w/v): (W) d32; (X) d43. Error bars are based on standard deviations for sets of ®ve measurements at each concentration.
3. Results and discussion 3.1. Particle-size distribution of the emulsions Fig. 1 shows the average particle size of sodium caseinate-stabilized oil-in-water emulsions (30% v/v) with increasing sugar concentration in the aqueous phase. Droplet diameters ranged between 0.1 and 10 mm, this span becoming broader (0.1±15 mm) at the highest sugar (sucrose 1 glucose) concentration (76% w/v). Emulsions having similar average diameters (d32 0:45 ^ 0:04 mm and d43 0:77 ^ 0:1 mm) were formed at sugar concentrations #60% w/v. The effectiveness of sodium caseinate as an emulsi®er seemed not to be affected by the presence of sugars, at least up to the mentioned concentration, since 21 there was no change in the speci®c surface area ( / d32 ) over that concentration range. Further incorporation of sugars produced a substantial increase in d43 and a moderate increase in d32. As expected, the quantity d43 showed a greater sensitivity to the presence of sugars and homogenization conditions, as re¯ected in the higher standard deviation of this parameter for $50% w/v concentration. The larger mean droplet diameter(s) for the emulsions made at high sugar content implies a poorer ef®ciency of emulsi®cation. This may be due to less effective droplet disruption in the homogenizer, probably with some larger droplets produced by recoalescence during/after emulsi®cation, as well as some kind of bridging ¯occulation Ð as was detected by conventional light microscopy. The in¯uence of sugars can be interpreted in terms of the in¯uence on protein aggregation, as well as on the effect of the viscosity of the bulk phase in reducing the ef®ciency of droplet disruption by turbulent ¯ow in the homogenizer (Dickinson, 1994). At 218C, the aqueous phase viscosity virtually doubles from 66 to 76% w/v sugar, and the mean diameters change from d43 1:26 ^ 0:24 mm and d32 0:56 ^ 0:08 mm to d43 2:49 ^ 0:33 mm and d32 0:91 ^ 0:11 mm over the same concentration range. The presence of sugar dissolved in the aqueous phase in¯uences optical properties as well as rheological ones.
Our concentrated emulsion samples showed a gradual transformation from turbid to nearly clear with increasing sugar content. As a consequence, the resulting acid-induced emulsion gels appeared translucent, in contrast to the normal `milky' appearance of emulsion gels not containing sugar. The physical basis for this is, of course, that adding sugar to the aqueous phase of a hydrocarbon oil-in-water emulsion lowers considerably the relative refractive index at the oil±water interface (Weiss & Liao, 2000). 3.2. Determination of the gelation time on lowering of the pH While a high GDL concentration reduces the gelation time, it can also cause over-acidi®cation (the pH being lowered to a value beyond the isoelectric point), which is detected rheologically as a maximum in the storage modulus beyond the critical gelation time (Horne, 2001). Consequently, in this study, different GDL/protein ratios were tested in order to attempt to establish the conditions for relatively fast gelation without causing any over-acidi®cation in the presence of sucrose (50% w/v in the aqueous phase). Fig. 2 shows the time development of the storage modulus G 0 at different GDL/protein ratios. Gelation time was de®ned as the time of sharp increase in the shear modulus G 0 from the baseline, this being signaled in practice by the detection of a modulus above the instrument noise level (ca. 1 Pa). According to this criterion, the measured gelation time decreased from 3.5 to 0.5 h when going from 0.15 g GDL/g caseinate to 0.8 g GDL/g caseinate. For all GDL concentrations studied, the plots of G 0 and G 00 (the latter not shown) reached almost plateau values of ca. 3500 and 750 Pa, respectively. Although there was still some slow increase in the shear moduli after this time, for the purposes of our analysis of the effect of added sugars we assume the establishment after several hours of essentially complete network formation and `®nal' gel strength, with the ®nal pH value being in the range 3.5±4 after 18 h for the data shown in Fig. 2. Based on the preceding preliminary results, it was
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Fig. 2. Time development of the storage modulus G 0 of acid-induced caseinate emulsion gels (1.4% w/v caseinate, 35% w/v sucrose, 30% v/v n-tetradecane) made at different GDL/protein (w/w) ratios: (e) 0.15; (W) 0.2; (A) 0.3; (X) 0.8.
thereafter decided to use a constant ratio of 0.3 g GDL/g caseinate (which gives a gelation time tgel < 1 h), and to investigate all the samples over a period of 8 h at single oscillation frequency. During this period, suf®cient time was available for the development of the ®nal viscoelasticity of these gels. Fig. 3 shows the gradual lowering of pH with time in these systems in the presence of sucrose (35% w/v) and its correlation with the development of the rheological parameters at the chosen GDL concentration. Gelation onset, as de®ned above, was found to occur at pH < 5:6: It can be inferred that the main body of the threedimensional gel network has already been formed after 3 h (between pH 5.6 and 4.6), with the ®nal pH value (ca. 4.0) reached after 17 h. Previously, Chen et al. (1999) found no rheological evidence for a three-dimensional network in acid-induced sodium caseinate emulsion gels when the pH value was outside the range 3.2±5.8. It appears from our observations that the presence of sucrose keeps the pH signi®cantly higher at extended times following GDL addition. To con®rm that this behavior was indeed a characteristic feature of the casein/sucrose system, the rate of pH lowering over the same 8 h period was also followed for pure protein gels (3% w/v sodium caseinate, no oil) without and with sucrose (66% w/v). Similar plots to Fig. 3 (data not shown) were obtained. While the ®rst 30 min of observation always gave the same rapid drop of pH from 6.9±7.0 to 5.8±5.9, two parallel curves were obtained after this time, the pH values of systems containing sucrose always lying ca. 0.2 units above those of the sugar-free systems. The implication of these observations, with gel points detected at pH < 5:6 and < 5.4 for caseinate gels with and without sucrose, respectively Ð and gelation always being faster in the former cases Ð will be discussed in Section 3.6. 3.3. Effect of sugar on gel strength 1 and gelation kinetics Fig. 4 shows the experimental dependence of storage 1 For clarity, in our terminology in this paper, the expression `gel strength' (or stiffness) refers to the small deformation elastic property (modulus); it is not a fracture property.
modulus G 0 (after 8 h) on sugar content for emulsion gels in the form of a log±log plot. Gels with ®nal shear moduli of up to ,5 kPa were formed. A rather weak dependence on sugar content was found up to 50% w/v, whereas at the higher sugar concentrations there was a greater rate of increase in gel strength. The system with the highest sugar concentration, achieved with a mixture of sucrose 1 glucose (to avoid crystallization of pure sucrose) behaved slightly differently: the modulus did not increase so strongly with increasing sugar concentration (76% w/v) following the stronger dependency observed for the pure sucrose systems (50±66% w/v). Fig. 5 shows the gel point (de®ned as above) of the caseinate-stabilized emulsion gels as a function of sugar concentration in the range 0±76% w/v (as measured in the aqueous phase of the emulsions). Whereas gelation time tgel was reduced from ca. 65 to 60 min on adding 10% sucrose, the increasing incorporation of sucrose up to 50% in the aqueous phase did not have such a substantial in¯uence in reducing tgel (down to 55 min at 50% sucrose). Above this concentration, however, a greater increase in gelling rate was found, with tgel falling from 55 to 39 min on increasing the sugar content from 50 to 76% w/v. This mirrors the large increase in gel strength at .50% w/v sugar (Fig. 4). These results indicate that sugar addition is more effective in enhancing the gelling properties of these protein-based emulsion systems at either low sugar content (0±10%) or high sugar content (50±76%). Horne (1995, 1996, 1999) has demonstrated that, under certain conditions, kinetic gelation pro®les for casein particle gel formation exhibit a scaling behavior that can be described by just two parameters: the gelation time tgel, and the gel strength at in®nite time (or, in practice, a ®xed fraction of this after a fairly long time). The ®rst parameter describes the dynamics of gel development, whereas the second speci®es a characteristic static rheological attribute. Fig. 6 was obtained by analyzing our kinetic data using this simple scaling model, with each pro®le G 0 (t) replotted as normalized elastic modulus (G 0 /G 0 max) against reduced time (t/tgel), where G 0 max refers to the value of G 0 after 8 h and tgel is the gelation time for the speci®c sugar concentration. A
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325
Fig. 3. Time development of the viscoelastic parameters, G 0 and G 00 , and their correlation with the lowering of pH for acid-induced sodium caseinate-stabilized emulsion gels containing sucrose (1.4% w/v caseinate, 35% w/v sucrose, 30% v/v oil, 0.3 g GDL/g protein) at 258C and 1 Hz: (e) pH; (X) G 0 ; (W) G 00 .
sequence of partially overlapping parallel curves of similar shape, ordered consistently according to ultimate gel strength, were obtained from the set of individual gelation pro®les as shown in Fig. 6(a). Consistent with the model of Horne (1995, 1996) devised for rennet milk gels, the individual pro®les corresponding to different sugar contents reduce to a kind of `master curve' within a certain acceptable tolerance. However, a more pronounced deviation from the rest of the set of curves is evident at the highest sugar concentration, 76% w/v, corresponding to the system containing the mixture of sucrose 1 glucose. To compare with systems not containing oil droplets, equivalent pure protein gels were formed by acidifying suf®cient sodium caseinate in solution to produce networks of notionally similar maximum gel strength (i.e. 2 £ 102 ±5 £ 103 Pa; as compared with 2 £ 103 ±5 £ 103 Pa for the emulsion gels). Following rheologically (at 1 Hz) the gelation of a solution of 3.0% w/v caseinate, it was noted that the resulting acid-induced protein gel started to lose strength beyond a certain peak G 0 value at around t=tgel < 3 (see Fig. 6(b)). With incorporation of sucrose at 50% w/v, however, this phenomenon of the loss of gel strength at longer times was seen to disappear, and a single monotonic increase was recorded (as in Fig. 6(a)). A reduction in the elastic modulus of an acid-induced casein gel following
initial gel rheology build-up is normally attributed to `overacidi®cation', which causes enhancement of repulsive electrostatic interactions between caseinate particles of net positive charge at pH values below the isoelectric point. Disappearance of this overacidi®cation modulus overshoot phenomenon in the sugar-containing systems would be consistent with the reduced extent of lowering of pH in the presence of sugars (see Section 3.2). But probably a more plausible alternative (and maybe additional) explanation is that there is substantial strengthening of the protein± protein interactions in the presence of sugars. That is, one may envisage that, at this low caseinate concentration (3.0% w/v) in the absence of oil droplets and sugars, the relatively weak initial network (,200 Pa) rapidly rearranges its structure, resulting in partial breakage of some of the load-bearing protein±protein bonds already formed. The fact that sugar addition contributes to eliminating the modulus `overshoot' is therefore consistent with most of the network-forming protein±protein interactions becoming stronger. Oscillatory sweep tests were performed to check the frequency dependence of the small-deformation rheology. Plotting log G 0 vs. log frequency gave essentially straight lines with positive slopes varying from 0.16 to 0.18 (data not shown) for all emulsion gels at different
Fig. 4. Effect of sugar concentration in the aqueous phase on the `®nal' storage modulus G 0 (after 8 h) for acid-induced sodium caseinate stabilized emulsion gels (1.4% w/v caseinate, 30% v/v n-tetradecane) at 258C and 1 Hz.
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Fig. 5. Effect of sugar concentration in the aqueous phase on the gelation time of acid-induced emulsion gels (30% v/v oil and 1.4% w/v sodium caseinate) at 258C and 1 Hz. Error bars re¯ect the fact that rheological measurements were taken every 2.5 min.
sugar concentrations. Values from 0.13 to 0.16 have been reported for acid skim milk gels of different fat content by Lucey, Munro, and Singh (1998). This con®rms that the rheological properties depend on the timescale of the applied deformation. The value of tan d G 00 =G 0 was found to decrease with increasing frequency for all the gels, suggesting that, as expected, the systems become more elastic in character at higher
frequencies (shorter timescales). For systems behaving like this, it can be reasonably assumed (Bryant & McClements, 2000) that microstructural rearrangements are able to relax within the timescale of the applied stress at low frequencies, and so the system appears less rigid. Conversely, at higher frequencies, there is insuf®cient time for such relaxation processes to take place; hence the system appears more rigid.
Fig. 6. Effect of sugar concentration on the time development of the storage modulus for (a) acid-induced emulsion gels (30% v/v oil, 1.4% w/v sodium caseinate) and (b) acid-induced protein gels (3% w/v sodium caseinate). The normalized elastic modulus G 0 /G 0 max is plotted against reduced time t/tgel, where G 0 max refers to the value of G 0 after 8 h and tgel is the gelation time for each speci®c sugar concentration: (W) 0% w/v; (e) 10% w/v; O, 30% w/v; (A) 50% w/v; ( £ ) 60% w/v; (X) 66% w/v; (B) 76% w/v. The sugar concentrations refer to the aqueous phase in the case of emulsion gels and overall concentrations in the case of protein gels.
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Fig. 7. Large-deformation behavior of (a) acid-induced emulsion gels (30% v/v oil, 1.4% w/v sodium caseinate) and (b) acid-induced protein gels (3% w/v sodium caseinate). The reduced complex modulus G p/G0p at 1 Hz is plotted against the strain for various sugar concentrations in the aqueous phase: (a) (W) 0% w/v; (e) 15% w/v; (O) 30% w/v; (A) 55% w/v; ( £ ) 60% w/v; (±) 63% w/v; (X) 66% w/v; (B) 76% w/v; (b) (W) 0.0% w/v; (e) 10% w/v; O, 30% w/v; (A) 50% w/v; £ , 60% w/v; (X) 66% w/v.
3.4. Large-deformation behavior Large-deformation rheology was explored by investigating the change in complex shear modulus G p in oscillatory mode as a function of the shear strain amplitude. To facilitate comparison between different systems, data were normalized with respect to the limiting low-strain modulus G0p. By gradually increasing stepwise the stress amplitude from very low values, the strain obtained in the sample is initially increased proportionally. This is the so-called linear viscoelastic range. Deviations from linearity occur when the gel is deformed to a strain at which some of the weak (physical) bonds of the aggregated network structure are destroyed. In general, particle gels have a much shorter linear region than crosslinked polymer gels. Fig. 7(a) shows that strain-weakening behavior is exhibited for all our emulsion gels. Samples with high sugar concentrations (30±76% w/v) showed deviations from linear behavior at strain values in the range 1±2% (corresponding to shear stresses of 50±80 Pa), whereas the linear regime was longer at lower sugar concentrations (15% w/v), and it extended to a strain of ,7%
327
(corresponding to a shear stress of ,130 Pa) for the emulsion sample without added sugar. Whereas the value of G p fell drastically with increasing strain in samples of high sugar content, suggesting extensive bond disruption during straining, the decrease in G p with strain was less pronounced at low sugar content. Fig. 7(b) shows the change in large-deformation behavior for 3.0% w/v caseinate gels with increasing incorporation of sugars in the system. It should ®rst be noted that the effect of sugar incorporation increases the small-deformation complex modulus G0p by more than an order of magnitude. So, for example, at a shear stress of ca. 20 Pa, the sample without sugar undergoes a shear deformation of ,10%, whereas samples at high sugar concentration are deformed by only ,0.3%. There is some evidence in Fig. 7(b) of a slight strain-hardening in the non-linear regime. This could be considered to resemble more closely typical polymer gel rheological behavior, i.e. strain hardening, a long linear region, and a large rupture strain (Dickinson & Chen, 1999). By incorporating more sugar into the system, the behavior changes to more like that of a typical particle gel with shorter breakable bonds (,1.5% strain before fracture at 66% w/v sucrose). The phase angle in the linear region reveals a greater degree of elastic gel character in the absence of sugar d < 138 compared to that at high sugar content d < 188: In agreement with previous studies of large-deformation behavior of particle gels (Yanez, Laarz, & Bergstrom, 1999), our results indicate that stronger gels are more brittle: that is, the maximum strain the gel can take before it `breaks' decreases with the sugar content. 3.5. Effects of oil volume fraction and protein content at constant sucrose content The in¯uence of oil volume fraction was studied systematically by diluting a sucrose-rich base emulsion (30% v/v oil, 1.4% w/v caseinate, 46.2% w/v sucrose) (i.e. 66% w/v in aqueous phase) with aqueous phase down to 5.0% oil, thereby keeping the mean emulsion droplet size d32 0:57 mm exactly constant throughout. A content of 46.2% w/v sucrose and a protein/oil ratio of 0.046 g caseinate/ml oil were maintained in all the diluted emulsions. The effect of protein concentration, varying from 2.0% w/v down to 0.46% w/v, was also studied at constant oil volume fraction (10% v/v) and sucrose concentration (46.2% w/v). Tables 1 and 2 show the characteristics of the emulsion gels in terms of overall composition, gel strength after 8 h (G 0 8 h), and the time to detect visible syneresis during storage after gelation. Using the data in Table 1 to plot the logarithm of the storage modulus against the logarithm of the oil volume fraction f , a strong linear dependence is found with a scaling exponent of ca. 4. (The actual linear ®t is log G 08 h 3:82 log f 2 2:018; with R2 0:9985: The general rule of ®lled gels is followed here: the higher the volume fraction of incorporated active ®ller particles, the greater the enhancement of the gel strength. It is important
328
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Table 1 Storage modulus G 0 after 8 h and time of visible indication of syneresis (based on observations up to one week) for emulsion gels of different oil volume fraction f but constant sucrose content (46.2% w/v) and protein/oil ratio (0.046 g/ml)
Table 2 Storage modulus G 0 after 8 h and time of visible indication of syneresis (based on observations up to one week) for emulsion gels of different protein content but constant sucrose content (46.2% w/v) and oil volume fraction (0.1)
f
Protein content (% w/v)
G 08 h (Pa)
Syneresis?
Protein content (% w/v)
G 08 h (Pa)
Syneresis?
0.3 0.25 0.2 0.15 0.1 0.05
1.40 1.16 0.93 0.70 0.46 0.23
4:4 £ 103 1:7 £ 103 9 £ 102 3 £ 102 5 £ 101 , 1 £ 101
No No No No After 20 h After 10 h
0.6 0.8 1.2 1.6 2.0
9 £ 101 1:7 £ 102 4:6 £ 102 9:7 £ 102 1:6 £ 103
After 24 h After 36 h No No No
to note that a reduction in the number of oil droplets in the network also implies here a reduction in the total amount of protein present, since we are dealing with a base emulsion system in which nearly all the protein is coating the oil droplets (little unadsorbed protein). Previous studies in our laboratory (Dickinson & Chen, 1999) have found that there is a very substantial effect of the oil volume fraction on the viscoelasticity of heat-set protein emulsion gels. In this latter case, however, the nature of the network was rather different, with non-adsorbed aggregated whey protein contributing substantially to the rheology, and whey protein-coated emulsion droplets present as active ®llers. The effect of varying the total protein content, whilst maintaining the same number of droplets, corresponds to changing the ratio of unadsorbed to adsorbed caseinate in the emulsion gels (Table 2). The plot of logarithm of storage modulus against protein concentration Cp (not shown) is ®tted by the relationship: log G 08 h 2:434Cp0:429 R2 0:998: The weak emulsion gels containing sugars G 08 h , 200 Pa were observed to exhibit syneresis, with distinct vertical separation into a clear aqueous phase at the bottom and a turbid gel network above. Emulsion gel compositions associated with this phenomenon are detailed in Tables 1 and 2. This information roughly identi®es the system compositions promoting formation of particle gel networks that can maintain microstructural stability for an extended period of time. Beyond this stability regime, although there is enough aggregating material actually present to form the
gel, subsequent rearrangements of transient clusters via breaking and reforming of insuf®ciently strong protein± protein interactions lead, ®rst, to local (micro)-phase separation, and then to macroscopic syneresis. Fig. 8 shows that the gelation time is systematically reduced with increasing oil volume fraction. A qualitatively similar type of plot can be drawn for increasing protein content (not shown). The biopolymer gel literature indicates that gelation time depends on sample conditions, the polymer concentration being the most important factor (Clark, 1991). The gelling time generally increases as particle concentration decreases, which is in agreement with the trend exhibited here. Fig. 9 shows the time-dependent reduced storage modulus for different oil volume fractions. In accordance with the scaling analysis methodology of Horne (1996), all the data sets are seen to follow roughly the same sigmoidal curve in the covered oil volume fraction range (0.1±0.3), as do the equivalent plots for changing protein concentration (0.46±2.0% w/v) at constant oil content (not shown). The fact that there is no signi®cant change in kinetics upon diluting the emulsions in the presence of sugar seems to suggest that there is a similar aggregate growth following collapse of the caseinate protection layer upon acidi®cation. For the ®nal gelled emulsions (after 8±12 h), the phase angle remained essentially constant 14:5 ^ 0:58 throughout the linear region upon changing the oil volume fraction, but it varied slightly with protein content (from 14.0 to 16.88). This latter behavior shows that the viscous/elastic ratio changes on
Fig. 8. Effect of oil volume fraction f on gelation time tgel of acid-induced emulsion gels at high sugar concentration (46.2% w/v sucrose in whole system). Base emulsions (30% v/v oil, 1.4% w/v sodium caseinate) were diluted to obtain the different oil volume fractions (see text).
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329
Fig. 9. Kinetics of gelation for acid-induced emulsion gels at high sugar concentration (46.2% w/v) at different oil volume fractions: (W) 0.1; (e) 0.15; ( £ ) 0.2; (O) 0.25; (X) 0.3. The normalized elastic modulus G 0 /G 0 max is plotted against reduced time t/tgel, where G 0 max refers to the value of G 0 after 8 h and tgel is the gelation time.
manipulating the amount of non-adsorbed protein. This is presumably due to a transformation from a rheology dominated by a network of protein-coated oil droplets to one in which the aggregated interdroplet protein contributes signi®cantly to the network. Finally, Fig. 10 shows the large-deformation rheological behavior for the sets of emulsion gels with the compositions recorded in Table 1. At the lowest oil and protein contents, we see that the rheology is relatively insensitive to strain, and that there is even some evidence of a slight strain-hardening
behavior in the range 20±40%. A shortening of the linear region is seen to be promoted on increasing either (a) the oil droplet concentration or (b) the protein concentration. 3.6. Interpretation in terms of protein interactions The general effect of sugars on protein functionality is typically discussed in terms of the in¯uence on the protein± protein interactions. What we ®nd here is that the rigidity of an acid-induced caseinate-stabilized emulsion gel (or pure
Fig. 10. Large deformation behavior of acid-induced emulsion gels at high sugar content (46.2% w/v in whole system). The reduced complex modulus G p/G0p at 1 Hz is plotted against the strain. (a) Effect of changing oil volume fraction: (W) 0.1; (O) 0.15; (e) 0.2; (X) 0.25; ( £ ) 0.3. (b) Effect of changing protein content (% w/v): (B) 0.46; (W) 0.6; (O) 0.8; (e) 1.2; (X) 1.6; ( £ ) 2.0.
330
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caseinate gel) can be substantially enhanced by the presence of sugars in the aqueous phase, especially at concentrations above 50% w/v. Moreover, the gelation kinetics is affected over the whole range of studied sugar concentrations (0±76% w/v) leading to a reduction in gelation time compared with the sugar-free system. The presence of sugar also changes qualitatively the evolving structural and rheological development by inhibiting the reduction in elastic modulus at longer times. All of these facts are broadly consistent with the strength of the casein±casein molecular interactions being enhanced by the presence of added sugars. There is considerable independent evidence in the literature supporting the role of sugar as an enhancer of protein±protein interactions and protein aggregation, e.g. (i) an inferred increased attraction between unfolded whey protein molecules in the presence of sucrose (Kulmyrzaev et al., 2000a,b), (ii) a stronger attractive electrostatic interaction of casein molecules caused by the addition of sugars (Mora-Gutierrez, Kumosinski, & Farrell, 1997), and (iii) a corresponding strengthening of the protein±protein attractive interactions in caseinate (Antipova et al., 1999). As noted by van Vliet et al. (1989), the most important interaction forces in casein gels are probably the hydrophobic and the unlike (^) electrostatic interactions. One could reasonably expect that, the faster the lowering of the pH, the faster will be the gelation Ð on the basis that pH change is the dominant factor inducing the aggregation, with electrostatic casein±casein repulsive interactions being gradually overwhelmed by combined hydrophobic and electrostatic attractive interactions. However, the fact that the pH in systems containing sugars at the gelation point (ca. 5.6) was consistently higher than in the equivalent sugar-free system (ca. 5.4), and that it stayed 0.2 units higher at post-gelation times, could be interpreted as implying that the attractive hydrophobic bonds are more enhanced by the presence of sugars than the unlike (^) electrostatic interactions. This view is supported, for instance, by the work of Phillips et al. (1994) who have attributed the main effect of sugars to the promotion of hydrophobic interactions through the modi®cation of water structure surrounding the proteins. Based on the measured gel points of our caseinatestabilized emulsions, it would seem that sensitivity to sugar addition is rather greater at the extreme ranges of low and high sugar concentrations. The substantial dependence of the strength of protein±sugar interactions on the actual sugar concentration itself is implied by much published work in this ®eld (see, for instance, Antipova & Semenova, 1997). Abbasi & Dickinson (2001) have recently reported the existence of critical ranges of sugar concentration for producing pressure-induced gels from dispersions of skim milk powder at micellar casein concentrations down to ,2% w/v. This latter work has illustrated the importance of minimum sugar±protein ratios for promoting milk protein gelation, and has also identi®ed the existence of high sugar concentrations (.50 wt%) at which pressure-induced
gelation of casein micellar systems is completely inhibited. At these high sugar concentrations, the sugar appears to `protect' the casein micelles against pressure-induced dissociation by modifying the interactions of the casein molecules with colloidal calcium phosphate and with other casein molecules. This dual role of added sugars, in promoting or delaying protein gelation, depending on the sugar concentration, has also been noted by Kulmyrzaev et al. (2000b) for the case of cold gelation of heat-denatured whey proteins in the presence of sucrose. Our results suggest that the mechanism underlying the gelation kinetics is dependent not only on the ®nal gel strength but also on the initial aggregation period (i.e. the gelation time). Bryan & McClements (2000) have explained the varying effects of sugar concentration on the kinetics in terms of whether or not sugars retard or enhance the gelation by, respectively, reducing or increasing the collision frequency. In our casein-stabilized emulsions, the presence of sugars could increase the effective number of interacting particles and the strength of interparticle interactions, thus leading to a greater degree of `interconnectivity' of the gel microstructure. The signi®cant deviation in the characteristics of the aggregation process for the case of the system containing sucrose 1 glucose (76% w/v), compared with the pure sucrose systems, could be due either to some different speci®c effect of the glucose on the protein interactions or, more likely, the predominant kinetic effect of the high viscosity over the thermodynamic effects on the protein± protein interactions. Factors affecting the large-deformation rheology of gels include the number of bonds per cross section of the strand, the strength of each bond (van Vliet, Luyten, & Walstra, 1991), and the tortuosity of the gel network (Bremer, Bijsterbosch, Schrijvers, van Vliet, & Walstra, 1990). We can speculate that, by incorporating sugars into emulsion gels or protein gels, the strength of bonds between caseinate-coated droplets and caseinate particles might be increased, making connections between structural units less ¯exible and of shorter range, and thereby leading to a shorter linear viscoelastic range. Assuming that the sugars in interaction with the protein can be regarded as being an active part of the network, the larger rigidity of the resulting (emulsion) gels can be reasonably attributable to the increased number of structurally important bonds. In the presence of sugars, the combination of just 10% v/v oil and 0.8% w/v protein has been found to be suf®cient to initiate network formation. Nevertheless, syneresis experienced by such acid-induced gels of low protein and oil contents would seem to suggest that the droplet±droplet interactions are not strong enough to maintain the network structure following rearrangements on storage. Acknowledgements We gratefully acknowledge receipt by L.M.M. of an
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EPSRC Research Studentship and ®nancial support from Nestle Product Technology Centre (York, UK). References Abbasi, S., & Dickinson, E. (2001). In¯uence of sugars on high-pressure induced gelation of skim milk dispersions. Food Hydrocolloids, 15, 315±319. Antipova, A. S., & Semenova, M. G. (1995). Effect of sucrose on the thermodynamic incompatibility of different biopolymers. Carbohydrate Polymers, 28, 359±365. Antipova, A. S., & Semenova, M. G. (1997). Effect of neutral carbohydrate structure in the set glucose/sucrose/maltodextrin/dextran on protein surface activity at the air/water interface. Food Hydrocolloids, 11, 71±77. Antipova, A. S., Semenova, M. G., & Belyakova, L. E. (1999). Effect of sucrose on the thermodynamic properties of ovalbumin and sodium caseinate in bulk solution and at air±water interface. Colloids and Surfaces B, 12, 261±270. Arakawa, T., & Timasheff, S. N. (1982). Stabilization of protein structure by sugars. Biochemistry, 21, 6536±6544. Barone, G., Del Vecchio, P., Giancola, C., & Notaro, G. (1992). Conformational stability of proteins and peptide±peptide interactions in the presence of carbohydrates. Thermochimica Acta, 199, 189±196. Boye, J. I., Alli, I., & Ismail, A. A. (1996). Interactions involved in the gelation of bovine serum albumin. Journal of Agricultural and Food Chemistry, 44, 996±1004. Boye, J. I., Alli, I., Ramaswamy, H., & Raghavan, V. G. S. (1997). Interactive effects of factors affecting gelation of whey proteins. Journal of Food Science, 62, 56±65. Bremer, L. G. B., Bijsterbosch, B. H., Schrijvers, R., van Vliet, T., & Walstra, P. (1990). On the fractal nature of the structure of acid casein gels. Colloids and Surfaces, 51, 159±170. Bryant, C. M., & McClements, D. J. (2000). In¯uence of sucrose on NaCl-induced gelation of heat denatured whey protein solutions. Food Research International, 33, 649±653. Chen, J., Dickinson, E., & Edwards, M. (1999). Rheology of acid-induced sodium caseinate stabilized emulsion gels. Journal of Texture Studies, 30, 377±396. Clark, A. H. (1991). Structural and mechanical properties of biopolymer gels. In E. Dickinson, Food polymers, gels and colloids (pp. 322±338). Cambridge: Royal Society of Chemistry. Dickinson, E. (1994). Emulsions and droplet-size control. In D. J. Wedlock, Controlled particle, droplet and bubble formation (pp. 191±216). Oxford: Butterworth-Heinemann. Dickinson, E., & Chen, J. (1999). Heat-set whey protein emulsion gels: role of active and inactive ®ller particles. Journal of Dispersion Science and Technology, 20, 197±213. Dickinson, E., & Golding, M. (1997). Depletion ¯occulation of emulsions containing unadsorbed sodium caseinate. Food Hydrocolloids, 11, 13±18. Dickinson, E., & James, J. D. (1998). Rheology and ¯occulation of high-pressure-treated b-lactoglobulin-stabilized emulsions: Comparison with thermal treatment. Journal of Agricultural and Food Chemistry, 46, 2565±2571. Dickinson, E., & Yamamoto, Y. (1996). Rheology of milk protein gels and protein-stabilized emulsion gels cross-linked with transglutaminase. Journal of Agricultural and Food Chemistry, 44, 1371±1377.
331
Dumay, E. M., Kalichevsky, M. T., & Cheftel, J. C. (1994). High-pressure unfolding and aggregation of b-lactoglobulin and the baroprotective effects of sucrose. Journal of Agricultural and Food Chemistry, 42, 1861±1868. Horne, D. S. (1995). Scaling behaviour of shear moduli during the formation of rennet milk gels. In E. Dickinson & D. Lorient, Food macromolecules and colloids (pp. 456±461). Cambridge: Royal Society of Chemistry. Horne, D. S. (1996). Aspects of scaling behaviour in the kinetics of particle gel formation. Journal de Chime Physique et de Physico Chimie Biologique, 93, 977±986. Horne, D. S. (1999). Formation and structure of acidi®ed milk gels. International Dairy Journal, 9, 261±268. Horne, D. S. (2001). Factors in¯uencing acid-induced gelation of skim milk. In E. Dickinson & R. Miller, Food colloids: fundamentals of formulation (pp. 345±351). Cambridge: Royal Society of Chemistry. Jou, K. D., & Harper, W. J. (1996). Effect of di-saccaharides on the thermal properties of whey proteins determined by differential scanning calorimetry. Milchwissenschaft, 51, 509±512. Kulmyrzaev, A., Bryant, C., & McClements, D. J. (2000a). In¯uence of sucrose on the thermal denaturation, gelation, and emulsion stabilization of whey proteins. Journal of Agricultural and Food Chemistry, 48, 1593±1597. Kulmyrzaev, A., Cancelliere, C., & McClements, D. J. (2000b). In¯uence of sucrose on cold gelation of heat-denatured whey protein isolate. Journal of the Science of Food and Agriculture, 80, 1314±1318. Lee, J. C., & Timasheff, S. N. (1981). The stabilization of proteins by sucrose. Journal of Biological Chemistry, 256, 7193±7201. Lucey, J. A., & Singh, H. (1998). Formation and physical properties of acid milk gels: a review. Food Research International, 30, 529±542. Lucey, J. A., Munro, P. A., & Singh, H. (1998). Rheological properties and microstructure of acid milk gels as affected by fat content and heat treatment. Journal of Food Science, 63, 660±664. Mora-Gutierrez, A., Kumosinski, T. F., & Farrell Jr., H. M. (1997). Oxygen-17 nuclear magnetic resonance studies of bovine and caprine hydration and activity in deuterated sugar solutions. Journal of Agricultural and Food Chemistry, 45, 4545±4553. Pauletti, M. S., Castelao, E. L., & Seguro, E. (1996). Kinetics of heat coagulation of concentrated milk proteins at high sucrose contents. Journal of Food Science, 61, 1207±1210. Peng, L. K., Ismail, N., & Easa, A. M. (2000). Effects of reducing sugars on texture of thermally processed soy protein isolate-glucono-delta-lactone gels. Journal of Food Science and Technology Ð Mysore, 37, 188±190. Phillips, L. G., Whitehead, D. M., & Kinsella, J. (1994). Structure±function properties of food proteins, London: Academic Press. Rich, L. M., & Foegeding, E. A. (2000). Effects of sugars on whey protein isolate gelation. Journal of Agricultural and Food Chemistry, 48, 5046±5052. van Vliet, T., Roefs, S. P. M. F., Zoon, P., & Walstra, P. (1989). Rheological properties of casein gels. Journal of Dairy Research, 56, 529±534. van Vliet, T., Luyten, H., & Walstra, P. (1991). Fracture and yielding of gels. In E. Dickinson, Food polymers, gels and colloids (pp. 392±405). Cambridge: Royal Society of Chemistry. Weiss, J., & Liao, W. (2000). Addition of sugars in¯uences colour of oil-inwater emulsions. Journal of Agricultural and Food Chemistry, 48, 5053±5060. Yanez, J. A., Laarz, E., & Bergstrom, L. (1999). Viscoelastic properties of particle gels. Journal of Colloid and Interface Science, 209, 162±172.