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Defining the conditions for heat-induced gelation of a caseinate-stabilized emulsion
Colloids and Surfaces B: Biointerfaces 29 (2003) 89 /97 www.elsevier.com/locate/colsurfb
Defining the conditions for heat-induced gelation of a caseinate-stabilized emulsion Eric Dickinson *, Caroline Eliot Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, United Kingdom Received 13 March 2002; received in revised form 9 July 2002; accepted 13 August 2002
Abstract Depending on pH and calcium ion content, a concentrated emulsion (45 vol.% oil) stabilized by sodium caseinate (8.1 wt.% in aqueous phase) as sole emulsifying agent can be made to transform from a liquid-like emulsion at 0/25 8C to a solid-like emulsion gel by heating to a temperature in the range 30 /45 8C. The gelation process can be described as thermoreversible when characterized in terms of viscometry measurements made at a reasonably high applied shear stress (40 Pa). It has been established that reversible heat-induced emulsion gelation does not occur for calcium ion concentrations below 10 mM in the aqueous phase or pH values outside the range 5.4 B/pH 0/6.4. # 2003 Published by Elsevier Science B.V. Keywords: Emulsion gel; Thermoreversible gelation; Caseinate; Calcium ions; Hydrophobic interaction; Flocculation
1. Introduction Amongst biopolymers, the phenomenon of thermoreversible gelation on heating is relatively uncommon [1]. Probably the most widely recognized examples are solutions of hydrophobically modified cellulose derivatives (e.g. methylcellulose) [2 /5] which can be made to gel on heating above approximately 40 8C. Of course, concentrated solutions of many food proteins (e.g. bovine serum albumin, b-lactoglobulin) will form gels following globular protein denaturation on heating above approximately 70 8C [6], but in these cases the molecular changes and the thermal * Corresponding author. Fax: /44-113-3432-2982. E-mail address: [email protected] (E. Dickinson).
gelation are irreversible, with the modulus even increasing substantially on subsequent cooling. We reported recently [7] that a concentrated oilin-water emulsion based on sodium caseinate as sole emulsifying agent, with a controlled amount of additional ionic calcium present, could exhibit gelation when heated through just a few degrees over the temperature range 35/45 8C. Below 30 8C the emulsion had a free-flowing liquid-like consistency, but on heating to 45/50 8C at / 2 8C min 1 it was converted into a viscoelastic gel that could support its own weight. Gelation of this caseinate-based emulsion on heating is in contrast to the thermoreversible gelation on cooling, over roughly the same temperature range, for gelatin solutions or for emulsions based on sodium dodecyl sulfate and gelatin [8].
0927-7765/03/$ - see front matter # 2003 Published by Elsevier Science B.V. doi:10.1016/S0927-7765(02)00146-7
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The amount of additional ionic calcium required to enable a caseinate-stabilized emulsion system to gel on heating is dependent on the casein composition [9] and the pH [7]. The onset of gelation is attributed to a temperature-induced flocculation of casein-coated oil droplets, presumably as a result of strengthening hydrophobic interactions between protein molecules adsorbed on the different droplets [10]. The presence of calcium ions is assumed to affect the delicate balance of electrostatic and hydrophobic forces controlling the change from net repulsive to net attractive interdroplet interactions. We have found [7] that this gelation exhibits some of the characteristics of thermoreversibility, in that the protein-stabilized emulsion droplets could be made to deflocculate slowly on cooling, with the return to the original low-viscosity state greatly accelerated by stirring. This type of potentially reversible flocculation/ gelation behaviour is not expected to be unique to casein-containing systems. An oil-in-water emulsion stabilized by a synthetic-copolymer has also recently, been reported [11,12] as exhibiting heatinduced thermoreversible gelation. In this paper we examine the phenomenon in more detail, with particular attention given to monitoring of pH and precise control of the conditions for preparing the protein solutions prior to emulsion preparation. Our objective is to construct a ‘state diagram’ showing the ranges of pH and calcium ion concentration over which this potential emulsion gel thermoreversibility can be observed.
2. Materials and methods Sodium caseinate (90.0% protein, 5.0% moisture, 800 ppm calcium ions) from DMV (Veghel, Netherlands) was supplied by Unilever Research Colworth (Bedford, UK) in January 2001. Acidulant glucono-d-lactone (GDL) (99%), n -tetradecane (99%), dihydrated calcium chloride (CaCl2 ×/ 2H2O) (99%) and standard buffer solutions for pH meter calibration were obtained from Sigma Chemicals (St. Louis, MO). Powder chemicals were stored in hermetically sealed small containers placed in separate desiccators.
Aqueous solutions of sodium caseinate (8.1 wt.%) were prepared with stirring for approximately 17 h at room temperature, followed by 30 min at 70 8C, and approximately 2 h at room temperature. The pH was adjusted by addition of an appropriate amount of GDL followed by stirring for 16 h at room temperature. The pH was measured using a Hanna Instruments 8519 pH meter, calibrated using pH 4 and 7 reference solutions (9/0.01). The ionic calcium level was carefully adjusted (0 /30 mM) by dropwise addition of 1 M CaCl2 solution at 5 8C with vigorous stirring. It was found that overall experimental repeatability was enhanced by making large batches of acidified and calcium-adjusted protein solutions and freezing them until required for emulsion preparation. This solution freezing procedure had no effect on emulsion droplet-size distributions or emulsion rheology. During defrosting, solutions were stirred gently at ambient temperature prior to further use. Oil-in-water emulsions (5 wt.% protein, 45 vol.% oil) were prepared by jet homogenization [13] of n tetradecane and 8.1 wt.% sodium caseinate solution in the volume ratio 45:55. To ensure that the temperature of any freshly prepared emulsion was below 30 8C, the cylinder blocks of the homogenizer were stored in a refrigerator prior to use. Droplet-size distributions of emulsions were determined using a Malvern Mastersizer 2000 with an optical absorption parameter of 0.005. At moderately high concentrations of CaCl2 and/or moderately low pH values, the emulsion droplets were aggregated. Gentle stirring whilst diluting some of these samples with distilled water broke down some of the weak flocs that could have been misinterpreted as larger droplets in the size distribution. Stronger flocculation was apparent from the bimodal shape of the particle-size distribution, which reverted to a monomodal distribution on addition of excess nonionic surfactant (Tween 20). Small-deformation oscillatory shear rheology was carried out using a Bohlin CVO or Bohlin CS50 rheometer with a C14 concentric cylinder measuring cell (inner diameter 14 mm, sample volume 2 ml). As previously [7], the maximum strain was set at 0.5%, and storage and loss moduli (G ? and G ƒ) were recorded at a constant frequency
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of 0.1 Hz, with temperature increased from 25 to 45 8C in increments of 5 8C. Prior to these measurements the emulsions were subjected to a pre-shearing treatment at a constant stress of 40 Pa at 25 8C for 1 h. Large-deformation steady-state shear rheology was carried out on the same device with another portion of the same sample which had been stored in the refrigerator. The viscosity was recorded at 40 Pa applied stress every 5 8C over a 25 8C 0/ T 8C 0/25 8C cycle, where T is the ‘final’ temperature set in the small-deformation study. Several further cycles were then applied, adjusting the ‘final’ temperature by 1 8C, after having continuously sheared the sample at 40 Pa for 1 h. This enabled accurate reproducible determination of the ‘gelation temperature’, i.e. the temperature at which the apparent viscosity increased by several orders of magnitude. For some emulsion compositions, yet another portion of the same sample stored in the refrigerator was subjected to temperature cycles with decreasing shear stresses applied at the temperature of interest, again with continuous shearing at 40 Pa for 1 h between cycles.
3. Results and discussion The concentration of ionic calcium required to induce flocculation of the sodium caseinate-stabilized emulsion (45 vol.% oil, 8.1 wt.% protein in aqueous phase) is reduced as the pH is lowered towards the isoelectric point of the protein. So, based on particle-size distributions from lightscattering, 10 mM CaCl2 in the aqueous phase produces no discernible aggregation at neutral pH (Fig. 1) but significant aggregation at pH 5.6 (of the emulsion) (Fig. 2). Increasing the added ionic calcium content shifts the bimodal distribution for flocculated emulsions towards larger particle sizes, as reported previously [14]. (This shift could be completely reversed by addition of excess surfactant, demonstrating that the changes were indeed caused by droplet flocculation and not by droplet coalescence.) Hence, we observe that calcium ions and hydrogen ions have synergistic effects on the destabilization of the emulsion with respect to
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aggregation, as detected by the Malvern Mastersizer. Temperature-dependent small-deformation shear rheology data are presented in Fig. 3 for the set of emulsions made with sodium caseinate initially at pH 7 (no addition of GDL) with 0 /30 mM CaCl2 added prior to emulsification. The complex shear modulus G */(G ?2/G ƒ2)1/2 at 0.1 Hz is plotted as a function of increasing temperature over the range 25 /45 8C. In the absence of added ionic calcium, the emulsion shows a steady decrease in complex modulus on heating up to 45 8C, corresponding to predominantly viscous character with phase angle d /tan 1(G ƒ/G ?) / 708, and certainly no evidence of any heat-induced gelation. Fig. 3 shows that the incorporation of 20 mM CaCl2 in the aqueous phase leads to an emulsion of substantially lower complex modulus, suggesting a less flocculated structure. This can be interpreted in terms of a reduction in the extent of depletion flocculation by unadsorbed caseinate in the presence of added calcium ions [15] arising as a result of the changes in caseinate self-assembly and sub-micelle structure caused by calcium binding to the protein [16]. It should be noted that addition of CaCl2 itself leads to a lowering of the pH of the sodium caseinate solution (down to pH 6.5) and of the emulsion (down to pH 6.4). Hence, the results in Fig. 3 relate to systems of both changing pH and changing ionic calcium content, although the latter is clearly much more important at these relatively high pH values. On increasing the CaCl2 content in the aqueous phase by just another 5/25 mM, the modulus of the resulting emulsion (pH 6.3) was found to increase strongly with temperature over range 30 /45 8C. This trend becomes even more pronounced for the emulsion containing 30 mM CaCl2 (pH 6.2), with the value of G * increasing strongly from 25 to 30 8C, and by more than three orders of magnitude over the 30/45 8C interval. Fig. 4 shows equivalent temperature-dependent complex modulus data for emulsions (pH 6.1) made with CaCl2 added to sodium caseinate solutions adjusted to pH 6.0 with 0.30 wt.% GDL. For the emulsions containing 20 mM CaCl2 (solution pH 5.7, emulsion pH 5.8) and 25
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Fig. 1. Particle-size distribution of emulsions (45 vol.% oil) made with 8.1 wt.% sodium caseinate solution with various concentrations of CaCl2 added prior to emulsification: */ */, 0 mM; . . .. . ., 20 mM; / / /, 25 mM. Differences between samples reflect differences in the extent of flocculation of the droplets.
mM CaCl2 (solution pH 5.6, emulsion pH 5.7), the value of G * changes little from 25 to 30 8C, by more than an order of magnitude from 30 to 35 8C, and by at least another order of magnitude from 35 to 45 8C. At 30 mM CaCl2 (solution pH 5.6, emulsion pH 5.7), the G * values are consistently higher and they smoothly increase with increasing temperature. There is no unique way to define the (emulsion) gel state or the gelation point. Often the condition G ?/G ƒ(dB/ 458) is adopted [17] at some arbitrary frequency. However, for many of the ‘gelling’ emulsion systems studied here, it was observed that the viscous component developed with temperature almost as strongly as the elastic component, and so there was no sharp G ?/G ƒ cross-over at some characteristic gel point. Hence, we prefer
here to adopt a more pragmatic definition related to the ability of the sample to support its own weight for a reasonable time ( /10 s) when inverted in a test tube. A convenient pragmatic definition based on the temperature-dependent small-deformation rheology measurements is that the system forms a heat-induced emulsion gel if G * goes above 10 Pa in the temperature range 30 / 45 8C. According to this definition, the gel points of the samples with 20, 25 and 30 mM CaCl2 contents in the aqueous phase in Fig. 4 are approximately 40, 35 and 30 8C, respectively. The very substantial increase in shear modulus on elevation of temperature, as indicated in Fig. 4, can be attributed to an increase in droplet /droplet attraction leading to strong droplet aggregation. On lowering the temperature again back to 25 8C,
Fig. 2. Particle-size distribution of emulsions (45 vol.% oil) made with 8.1 wt.% sodium caseinate solution adjusted to pH 5.6 with 0.5 wt.% GDL with various concentrations of CaCl2 added prior to emulsification: */ */ (thick line), 0 mM; / / /, 10 mM; . . .. . ., 15 mM; */ */ (thin line), 20 mM. Differences between samples reflect differences in the extent of flocculation of the droplets.
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Fig. 3. Temperature-dependent complex modulus G * at 0.1 Hz of sodium caseinate-stabilized emulsions (45 vol.% oil, 8.1 wt.% protein in aqueous phase) heated from 25 to 45 8C as a function of added CaCl2 concentration: I, 0 mM; ', 20 mM; k, 25 mM; ", 30 mM.
there was generally found to be a gradual reduction in storage and loss moduli, with the values falling back to their pre-heating values over periods from hours to days. Repeating the heating/cooling cycle led to the same reversible behaviour, suggesting that the inferred droplet aggregation/disruption process does not lead to
any irreversible change in the properties of the system (e.g. any significant change in the structure/ composition of the adsorbed layer). Thermoreversibility over a short experimental time-scale in these systems can, however, be achieved by using an applied shear field to disrupt the aggregates formed on heating.
Fig. 4. As Fig. 3, except that 8.1 wt.% sodium caseinate solution adjusted to pH 6.0 with 0.3 wt.% GDL prior to addition of CaCl2: ', 20 mM; k, 25 mM; ", 30 mM.
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So let us now consider the large-deformation rheology behaviour. In order to be able to summarize the gelling properties of the caseinatestabilized emulsion systems as a function of both GDL concentration (or pH) and calcium ion content, it was decided to categorize the emulsions into three classes: (I) liquid-like emulsions that remain liquid when heated up to 45 8C, (II) liquidlike emulsions that become emulsion gels on heating to 30/45 8C, and (III) emulsions that are already ‘solid-like’ at ambient temperature (on emerging from the homogenizer) or form emulsion gels (G * /10 Pa) below 30 8C. Examples of the three kinds of behaviour are illustrated in Fig. 5. The apparent viscosity, determined at a controlled stress of 40 Pa, is plotted against temperature. The category I emulsion retains a low temperatureinsensitive viscosity (B/0.1 Pa s) on heating from 25 to 45 8C. Also the G * remains below 10 Pa over this temperature range (e.g. see Fig. 3). This type of system does not form a heat-induced emulsion gel. The category II emulsion exhibits a large increase in viscosity from a low value ( B/0.1 Pa s) at low temperature ( 0/25 8C) to a value at least an order of magnitude bigger over a 5 8C
Fig. 5. Three categories of emulsion large-deformation rheological behaviour distinguished according to changes in apparent viscosity as a function of heating (solid lines) and back-cooling (dashed lines): j, category I; \, category II (potentially reversible emulsion gel); m, category III.
heating increment in the range 30/45 8C, the viscosity reducing substantially again on cooling back down to 25 8C. The category II system also satisfies the small-deformation definition of the gel state mentioned above (G * /0.1 at 25 8C, G * / 10 Pa on heating in the range 30/45 8C). This is the type of emulsion system that can be considered to possess potentially reversible heat-induced gelation characteristics. The category III emulsion typically has G * /10 Pa even below 30 8C. Its apparent viscosity increases by several orders of magnitude on heating above 30 8C, without returning to low values on cooling back to ambient temperature whilst shearing at 40 Pa. That is, the category III emulsion is already in the gel state even before heating above 30 8C. On the basis of these criteria, we can summarize our temperature-dependent rheological experiments for sodium caseinate emulsions of different composition with respect to the added GDL and CaCl2 concentrations in terms of the state diagram in Fig. 6. Three different symbols distinguish (I) emulsions that do not gel on heating, (II) emul-
Fig. 6. Diagram showing state of sodium caseinate-stabilized emulsions (45 vol.% oil, 8.1 wt.% protein in aqueous phase) with respect to heat-induced gelation as a function of added GDL concentration and added CaCl2 concentration in aqueous phase: j, category I; \, category II (potentially reversible emulsion gel); m, category III; /, solution exhibits visible gelation before emulsification.
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sions that do form gels on heating with some potentially reversible character, and (III) emulsions that are already gelled before heating and become very much thicker after heating. Also indicated in Fig. 6 are compositions for which the protein solution was already gelled before emulsification (no emulsions were prepared in these cases). Fig. 7 shows an alternative representation of the state diagram in terms of a plot of emulsion pH versus CaCl2 concentration in the aqueous phase. As discussed above, the measured pH is dependent not only on the GDL concentration but also the CaCl2 concentration. We can see from Fig. 7 that there are clear boundaries in composition space defining the limits of potentially reversible heatinduced gelation (category II behaviour). The triangular region of heat-induced emulsion gel behaviour is centred on a composition with pH :/5.8 and [CaCl2] :/25 mM. According to the criteria adopted, no category II systems can be formed at calcium ion concentrations below 10
Fig. 7. State diagram of sodium caseinate-stabilized emulsions (45 vol.% oil, 8.1 wt.% protein in aqueous phase) as a function of final emulsion pH and added CaCl2 concentration in aqueous phase: j, category I; \, category II (potentially reversible emulsion gel); m, category III. The boundary lines are drawn to guide the eye.
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mM or pH values outside the range 5.4 B/pH 0/ 6.4. Some of the changes in aggregation behaviour with pH and CaCl2 concentration indicated in Fig. 7 may be attributable to differing adsorbed layer compositions arising from the different sensitivities of the casein monomers (especially as1 and b) towards self-association in the presence of calcium ions [9,10]. This could explain, for instance, the ‘unexpected’ liquid-like character, at ambient temperatures, of the emulsions made at low pH, whereas the equivalent systems appear solid-like at neutral pH. It should be noted, however, that the ‘thermoreversibility’ referred to here, in the context of the category II emulsions, relates to the large-strain apparent viscosity behaviour and not to the smallstrain complex modulus behaviour. What this means is that the flocculated network of proteincoated droplets formed on heating is not spontaneously disrupted on cooling under the influence of Brownian motion. Rather the dissociation of aggregated droplets requires the application of external hydrodynamic forces (as during the viscosity measurement) in order to break down the emulsion gel network and to convert the system back to the fully dispersed liquid-like emulsion on cooling. As the state diagram in Fig. 7 is based largely on steady-state viscometry, its boundaries are undoubtedly influenced by the conditions (i.e. stress) set in the viscosity measurements. Were the viscosities to have been measured at lower stresses, some category II emulsions would have been allocated to category III. Conversely, if an even higher stress had been used, some category III emulsions might have been put in category II. However, the general form of the state diagram should not be affected by such considerations */ nor the position of the low pH boundary. Presumably as a result of its proximity to the casein’s isoelectric point, the location of the border between category II and category III at pH :/5.4 was found to be uninfluenced by the viscometry flow conditions. The sensitivity of the potentially reversible character to the stress applied during viscosity measurement is illustrated in Fig. 8 for the case of
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Fig. 8. Sensitivity of apparent viscosity versus temperature plot to the shear stress applied during viscosity measurement. Four consecutive heating/cooling cycles over the range 25 /45 8C were carried out with different applied stresses: m, 1st cycle (10 Pa); 2, 2nd cycle (20 Pa); ', 3rd cycle (30 Pa); \, 4th cycle (40 Pa). Solid and dotted lines refer to heating and cooling, respectively.
an emulsion to which 0.30 wt.% GDL and 20 mM CaCl2 were added prior to emulsification. Four temperature cycles between 25 and 45 8C were consecutively carried out on this same sample, with a 40 Pa shear stress applied for 1 h before each cycle. In this case, category II behaviour was indicated by a jump in apparent viscosity by at least an order of magnitude on heating from 40 to 45 8C. Fig. 8 shows that the measured viscosities on heating are relatively insensitive to the applied stress in the range 10/40 Pa. However, on cooling back to 25 8C during the 1st cycle, the viscosity measured at stress 10 Pa remains consistently constant at around the high value induced by the heating to 45 8C. So, it would appear that a stress of 10 Pa is insufficient to disrupt the droplet / droplet interactions leading to the heat-induced emulsion gel network. On the other hand, during the 2nd cycle involving a 20 Pa applied stress, there was observed to be a substantial reduction in viscosity on cooling, with the viscosity returning almost to its original value at 25 8C. The stresses
of 30 and 40 Pa applied during the 3rd and 4th cycles, respectively, were sufficient to allow complete thermoreversibility. Finally, we note that the likely molecular origin of this heat-induced emulsion gelation behaviour in terms of casein /casein interactions in bulk solution and at the surface of emulsion droplets has been discussed previously [7]. Hydrophobic interaction provides the main molecular driving force for the self-association of the caseins and hence, the aggregation of casein-coated droplets, but this can be moderated by changes in electrostatic interactions caused by pH-sensitive hydrogen ion equilibria involving the ionizable aminoacid side-chains and by specific calcium-ion binding to the phosphoserine residues [10,18]. This calcium binding is promoted by increase in temperature [19]. Moreover, it is well established that hydrophobic interactions become stronger with increasing temperature over the temperature range explored in this study. In the case of sodium caseinate, it would appear that some calcium ion binding combined with pH reduction is required to provide the optimum balance of intermolecular forces conducive to heat-induced reversible gelation.
Acknowledgements C.E. acknowledges receipt of a BBSRC Research Studentship in collaboration with Unilever Research (Colworth House).
References [1] J.-M. Guenet, Thermoreversible Gelation of Polymers and Biopolymers, Academic Press, New York, 1992. [2] A. Haque, E.R. Morris, Carbohydr. Polym. 22 (1993) 161. [3] K. Kobayashi, C. Huang, T.P. Lodge, Macromolecules 32 (1999) 7070. [4] J. Desbrieres, M. Hirrien, S.B. Ross-Murphy, Polymer 41 (2000) 2451. [5] L. Li, P.M. Thangamathesvaran, C.Y. Yue, K.C. Tam, X. Hu, Y.C. Lam, Langmuir 17 (2001) 8062. [6] L.G. Phillips, D.M. Whitehead, J. Kinsella, Structure / Function Properties of Food Proteins (Chapter 9), Academic Press, San Diego, 1994.
E. Dickinson, C. Eliot / Colloids and Surfaces B: Biointerfaces 29 (2003) 89 /97 [7] E. Dickinson, H. Casanova, Food Hydrocoll. 13 (1999) 285. [8] M. Lechtenfeld, W. Borchard, Phys. Chem. Chem. Phys. 1 (1999) 3129. [9] H. Casanova, E. Dickinson, J. Coll. Interface Sci. 207 (1998) 82. [10] D.S. Horne, Int. Dairy J. 8 (1998) 171. [11] A. Koh, B.R. Saunders, Chem. Commun. 24 (2000) 2461. [12] A.Y.C. Koh, C. Prestidge, I. Ametov, B.R. Saunders, Phys. Chem. Chem. Phys. 4 (2002) 96.
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[13] I. Burgaud, E. Dickinson, P.V. Nelson, Int. J. Food Sci. Technol. 25 (1990) 39. [14] E. Dickinson, E. Davies, Coll. Surf. B 12 (1999) 203. [15] E. Dickinson, M. Golding, Coll. Surf. A 144 (1998) 167. [16] E. Dickinson, M.G. Semenova, L.E. Belyakova, A.S. Antipova, M.M. In’in, E.N. Tsapkina, C. Ritzoulis, J. Coll. Interface Sci. 239 (2001) 87. [17] S.B. Ross-Murphy, Polym. Gels Networks 2 (1994) 229. [18] D.S. Horne, J. Biol. Macromol. 5 (1983) 296. [19] D.G. Dalgleish, T.G. Parker, J. Dairy Res. 47 (1980) 113.
Defining the conditions for heat-induced gelation of a caseinate-stabilized emulsion Eric Dickinson *, Caroline Eliot Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, United Kingdom Received 13 March 2002; received in revised form 9 July 2002; accepted 13 August 2002
Abstract Depending on pH and calcium ion content, a concentrated emulsion (45 vol.% oil) stabilized by sodium caseinate (8.1 wt.% in aqueous phase) as sole emulsifying agent can be made to transform from a liquid-like emulsion at 0/25 8C to a solid-like emulsion gel by heating to a temperature in the range 30 /45 8C. The gelation process can be described as thermoreversible when characterized in terms of viscometry measurements made at a reasonably high applied shear stress (40 Pa). It has been established that reversible heat-induced emulsion gelation does not occur for calcium ion concentrations below 10 mM in the aqueous phase or pH values outside the range 5.4 B/pH 0/6.4. # 2003 Published by Elsevier Science B.V. Keywords: Emulsion gel; Thermoreversible gelation; Caseinate; Calcium ions; Hydrophobic interaction; Flocculation
1. Introduction Amongst biopolymers, the phenomenon of thermoreversible gelation on heating is relatively uncommon [1]. Probably the most widely recognized examples are solutions of hydrophobically modified cellulose derivatives (e.g. methylcellulose) [2 /5] which can be made to gel on heating above approximately 40 8C. Of course, concentrated solutions of many food proteins (e.g. bovine serum albumin, b-lactoglobulin) will form gels following globular protein denaturation on heating above approximately 70 8C [6], but in these cases the molecular changes and the thermal * Corresponding author. Fax: /44-113-3432-2982. E-mail address: [email protected] (E. Dickinson).
gelation are irreversible, with the modulus even increasing substantially on subsequent cooling. We reported recently [7] that a concentrated oilin-water emulsion based on sodium caseinate as sole emulsifying agent, with a controlled amount of additional ionic calcium present, could exhibit gelation when heated through just a few degrees over the temperature range 35/45 8C. Below 30 8C the emulsion had a free-flowing liquid-like consistency, but on heating to 45/50 8C at / 2 8C min 1 it was converted into a viscoelastic gel that could support its own weight. Gelation of this caseinate-based emulsion on heating is in contrast to the thermoreversible gelation on cooling, over roughly the same temperature range, for gelatin solutions or for emulsions based on sodium dodecyl sulfate and gelatin [8].
0927-7765/03/$ - see front matter # 2003 Published by Elsevier Science B.V. doi:10.1016/S0927-7765(02)00146-7
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The amount of additional ionic calcium required to enable a caseinate-stabilized emulsion system to gel on heating is dependent on the casein composition [9] and the pH [7]. The onset of gelation is attributed to a temperature-induced flocculation of casein-coated oil droplets, presumably as a result of strengthening hydrophobic interactions between protein molecules adsorbed on the different droplets [10]. The presence of calcium ions is assumed to affect the delicate balance of electrostatic and hydrophobic forces controlling the change from net repulsive to net attractive interdroplet interactions. We have found [7] that this gelation exhibits some of the characteristics of thermoreversibility, in that the protein-stabilized emulsion droplets could be made to deflocculate slowly on cooling, with the return to the original low-viscosity state greatly accelerated by stirring. This type of potentially reversible flocculation/ gelation behaviour is not expected to be unique to casein-containing systems. An oil-in-water emulsion stabilized by a synthetic-copolymer has also recently, been reported [11,12] as exhibiting heatinduced thermoreversible gelation. In this paper we examine the phenomenon in more detail, with particular attention given to monitoring of pH and precise control of the conditions for preparing the protein solutions prior to emulsion preparation. Our objective is to construct a ‘state diagram’ showing the ranges of pH and calcium ion concentration over which this potential emulsion gel thermoreversibility can be observed.
2. Materials and methods Sodium caseinate (90.0% protein, 5.0% moisture, 800 ppm calcium ions) from DMV (Veghel, Netherlands) was supplied by Unilever Research Colworth (Bedford, UK) in January 2001. Acidulant glucono-d-lactone (GDL) (99%), n -tetradecane (99%), dihydrated calcium chloride (CaCl2 ×/ 2H2O) (99%) and standard buffer solutions for pH meter calibration were obtained from Sigma Chemicals (St. Louis, MO). Powder chemicals were stored in hermetically sealed small containers placed in separate desiccators.
Aqueous solutions of sodium caseinate (8.1 wt.%) were prepared with stirring for approximately 17 h at room temperature, followed by 30 min at 70 8C, and approximately 2 h at room temperature. The pH was adjusted by addition of an appropriate amount of GDL followed by stirring for 16 h at room temperature. The pH was measured using a Hanna Instruments 8519 pH meter, calibrated using pH 4 and 7 reference solutions (9/0.01). The ionic calcium level was carefully adjusted (0 /30 mM) by dropwise addition of 1 M CaCl2 solution at 5 8C with vigorous stirring. It was found that overall experimental repeatability was enhanced by making large batches of acidified and calcium-adjusted protein solutions and freezing them until required for emulsion preparation. This solution freezing procedure had no effect on emulsion droplet-size distributions or emulsion rheology. During defrosting, solutions were stirred gently at ambient temperature prior to further use. Oil-in-water emulsions (5 wt.% protein, 45 vol.% oil) were prepared by jet homogenization [13] of n tetradecane and 8.1 wt.% sodium caseinate solution in the volume ratio 45:55. To ensure that the temperature of any freshly prepared emulsion was below 30 8C, the cylinder blocks of the homogenizer were stored in a refrigerator prior to use. Droplet-size distributions of emulsions were determined using a Malvern Mastersizer 2000 with an optical absorption parameter of 0.005. At moderately high concentrations of CaCl2 and/or moderately low pH values, the emulsion droplets were aggregated. Gentle stirring whilst diluting some of these samples with distilled water broke down some of the weak flocs that could have been misinterpreted as larger droplets in the size distribution. Stronger flocculation was apparent from the bimodal shape of the particle-size distribution, which reverted to a monomodal distribution on addition of excess nonionic surfactant (Tween 20). Small-deformation oscillatory shear rheology was carried out using a Bohlin CVO or Bohlin CS50 rheometer with a C14 concentric cylinder measuring cell (inner diameter 14 mm, sample volume 2 ml). As previously [7], the maximum strain was set at 0.5%, and storage and loss moduli (G ? and G ƒ) were recorded at a constant frequency
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of 0.1 Hz, with temperature increased from 25 to 45 8C in increments of 5 8C. Prior to these measurements the emulsions were subjected to a pre-shearing treatment at a constant stress of 40 Pa at 25 8C for 1 h. Large-deformation steady-state shear rheology was carried out on the same device with another portion of the same sample which had been stored in the refrigerator. The viscosity was recorded at 40 Pa applied stress every 5 8C over a 25 8C 0/ T 8C 0/25 8C cycle, where T is the ‘final’ temperature set in the small-deformation study. Several further cycles were then applied, adjusting the ‘final’ temperature by 1 8C, after having continuously sheared the sample at 40 Pa for 1 h. This enabled accurate reproducible determination of the ‘gelation temperature’, i.e. the temperature at which the apparent viscosity increased by several orders of magnitude. For some emulsion compositions, yet another portion of the same sample stored in the refrigerator was subjected to temperature cycles with decreasing shear stresses applied at the temperature of interest, again with continuous shearing at 40 Pa for 1 h between cycles.
3. Results and discussion The concentration of ionic calcium required to induce flocculation of the sodium caseinate-stabilized emulsion (45 vol.% oil, 8.1 wt.% protein in aqueous phase) is reduced as the pH is lowered towards the isoelectric point of the protein. So, based on particle-size distributions from lightscattering, 10 mM CaCl2 in the aqueous phase produces no discernible aggregation at neutral pH (Fig. 1) but significant aggregation at pH 5.6 (of the emulsion) (Fig. 2). Increasing the added ionic calcium content shifts the bimodal distribution for flocculated emulsions towards larger particle sizes, as reported previously [14]. (This shift could be completely reversed by addition of excess surfactant, demonstrating that the changes were indeed caused by droplet flocculation and not by droplet coalescence.) Hence, we observe that calcium ions and hydrogen ions have synergistic effects on the destabilization of the emulsion with respect to
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aggregation, as detected by the Malvern Mastersizer. Temperature-dependent small-deformation shear rheology data are presented in Fig. 3 for the set of emulsions made with sodium caseinate initially at pH 7 (no addition of GDL) with 0 /30 mM CaCl2 added prior to emulsification. The complex shear modulus G */(G ?2/G ƒ2)1/2 at 0.1 Hz is plotted as a function of increasing temperature over the range 25 /45 8C. In the absence of added ionic calcium, the emulsion shows a steady decrease in complex modulus on heating up to 45 8C, corresponding to predominantly viscous character with phase angle d /tan 1(G ƒ/G ?) / 708, and certainly no evidence of any heat-induced gelation. Fig. 3 shows that the incorporation of 20 mM CaCl2 in the aqueous phase leads to an emulsion of substantially lower complex modulus, suggesting a less flocculated structure. This can be interpreted in terms of a reduction in the extent of depletion flocculation by unadsorbed caseinate in the presence of added calcium ions [15] arising as a result of the changes in caseinate self-assembly and sub-micelle structure caused by calcium binding to the protein [16]. It should be noted that addition of CaCl2 itself leads to a lowering of the pH of the sodium caseinate solution (down to pH 6.5) and of the emulsion (down to pH 6.4). Hence, the results in Fig. 3 relate to systems of both changing pH and changing ionic calcium content, although the latter is clearly much more important at these relatively high pH values. On increasing the CaCl2 content in the aqueous phase by just another 5/25 mM, the modulus of the resulting emulsion (pH 6.3) was found to increase strongly with temperature over range 30 /45 8C. This trend becomes even more pronounced for the emulsion containing 30 mM CaCl2 (pH 6.2), with the value of G * increasing strongly from 25 to 30 8C, and by more than three orders of magnitude over the 30/45 8C interval. Fig. 4 shows equivalent temperature-dependent complex modulus data for emulsions (pH 6.1) made with CaCl2 added to sodium caseinate solutions adjusted to pH 6.0 with 0.30 wt.% GDL. For the emulsions containing 20 mM CaCl2 (solution pH 5.7, emulsion pH 5.8) and 25
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Fig. 1. Particle-size distribution of emulsions (45 vol.% oil) made with 8.1 wt.% sodium caseinate solution with various concentrations of CaCl2 added prior to emulsification: */ */, 0 mM; . . .. . ., 20 mM; / / /, 25 mM. Differences between samples reflect differences in the extent of flocculation of the droplets.
mM CaCl2 (solution pH 5.6, emulsion pH 5.7), the value of G * changes little from 25 to 30 8C, by more than an order of magnitude from 30 to 35 8C, and by at least another order of magnitude from 35 to 45 8C. At 30 mM CaCl2 (solution pH 5.6, emulsion pH 5.7), the G * values are consistently higher and they smoothly increase with increasing temperature. There is no unique way to define the (emulsion) gel state or the gelation point. Often the condition G ?/G ƒ(dB/ 458) is adopted [17] at some arbitrary frequency. However, for many of the ‘gelling’ emulsion systems studied here, it was observed that the viscous component developed with temperature almost as strongly as the elastic component, and so there was no sharp G ?/G ƒ cross-over at some characteristic gel point. Hence, we prefer
here to adopt a more pragmatic definition related to the ability of the sample to support its own weight for a reasonable time ( /10 s) when inverted in a test tube. A convenient pragmatic definition based on the temperature-dependent small-deformation rheology measurements is that the system forms a heat-induced emulsion gel if G * goes above 10 Pa in the temperature range 30 / 45 8C. According to this definition, the gel points of the samples with 20, 25 and 30 mM CaCl2 contents in the aqueous phase in Fig. 4 are approximately 40, 35 and 30 8C, respectively. The very substantial increase in shear modulus on elevation of temperature, as indicated in Fig. 4, can be attributed to an increase in droplet /droplet attraction leading to strong droplet aggregation. On lowering the temperature again back to 25 8C,
Fig. 2. Particle-size distribution of emulsions (45 vol.% oil) made with 8.1 wt.% sodium caseinate solution adjusted to pH 5.6 with 0.5 wt.% GDL with various concentrations of CaCl2 added prior to emulsification: */ */ (thick line), 0 mM; / / /, 10 mM; . . .. . ., 15 mM; */ */ (thin line), 20 mM. Differences between samples reflect differences in the extent of flocculation of the droplets.
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Fig. 3. Temperature-dependent complex modulus G * at 0.1 Hz of sodium caseinate-stabilized emulsions (45 vol.% oil, 8.1 wt.% protein in aqueous phase) heated from 25 to 45 8C as a function of added CaCl2 concentration: I, 0 mM; ', 20 mM; k, 25 mM; ", 30 mM.
there was generally found to be a gradual reduction in storage and loss moduli, with the values falling back to their pre-heating values over periods from hours to days. Repeating the heating/cooling cycle led to the same reversible behaviour, suggesting that the inferred droplet aggregation/disruption process does not lead to
any irreversible change in the properties of the system (e.g. any significant change in the structure/ composition of the adsorbed layer). Thermoreversibility over a short experimental time-scale in these systems can, however, be achieved by using an applied shear field to disrupt the aggregates formed on heating.
Fig. 4. As Fig. 3, except that 8.1 wt.% sodium caseinate solution adjusted to pH 6.0 with 0.3 wt.% GDL prior to addition of CaCl2: ', 20 mM; k, 25 mM; ", 30 mM.
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So let us now consider the large-deformation rheology behaviour. In order to be able to summarize the gelling properties of the caseinatestabilized emulsion systems as a function of both GDL concentration (or pH) and calcium ion content, it was decided to categorize the emulsions into three classes: (I) liquid-like emulsions that remain liquid when heated up to 45 8C, (II) liquidlike emulsions that become emulsion gels on heating to 30/45 8C, and (III) emulsions that are already ‘solid-like’ at ambient temperature (on emerging from the homogenizer) or form emulsion gels (G * /10 Pa) below 30 8C. Examples of the three kinds of behaviour are illustrated in Fig. 5. The apparent viscosity, determined at a controlled stress of 40 Pa, is plotted against temperature. The category I emulsion retains a low temperatureinsensitive viscosity (B/0.1 Pa s) on heating from 25 to 45 8C. Also the G * remains below 10 Pa over this temperature range (e.g. see Fig. 3). This type of system does not form a heat-induced emulsion gel. The category II emulsion exhibits a large increase in viscosity from a low value ( B/0.1 Pa s) at low temperature ( 0/25 8C) to a value at least an order of magnitude bigger over a 5 8C
Fig. 5. Three categories of emulsion large-deformation rheological behaviour distinguished according to changes in apparent viscosity as a function of heating (solid lines) and back-cooling (dashed lines): j, category I; \, category II (potentially reversible emulsion gel); m, category III.
heating increment in the range 30/45 8C, the viscosity reducing substantially again on cooling back down to 25 8C. The category II system also satisfies the small-deformation definition of the gel state mentioned above (G * /0.1 at 25 8C, G * / 10 Pa on heating in the range 30/45 8C). This is the type of emulsion system that can be considered to possess potentially reversible heat-induced gelation characteristics. The category III emulsion typically has G * /10 Pa even below 30 8C. Its apparent viscosity increases by several orders of magnitude on heating above 30 8C, without returning to low values on cooling back to ambient temperature whilst shearing at 40 Pa. That is, the category III emulsion is already in the gel state even before heating above 30 8C. On the basis of these criteria, we can summarize our temperature-dependent rheological experiments for sodium caseinate emulsions of different composition with respect to the added GDL and CaCl2 concentrations in terms of the state diagram in Fig. 6. Three different symbols distinguish (I) emulsions that do not gel on heating, (II) emul-
Fig. 6. Diagram showing state of sodium caseinate-stabilized emulsions (45 vol.% oil, 8.1 wt.% protein in aqueous phase) with respect to heat-induced gelation as a function of added GDL concentration and added CaCl2 concentration in aqueous phase: j, category I; \, category II (potentially reversible emulsion gel); m, category III; /, solution exhibits visible gelation before emulsification.
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sions that do form gels on heating with some potentially reversible character, and (III) emulsions that are already gelled before heating and become very much thicker after heating. Also indicated in Fig. 6 are compositions for which the protein solution was already gelled before emulsification (no emulsions were prepared in these cases). Fig. 7 shows an alternative representation of the state diagram in terms of a plot of emulsion pH versus CaCl2 concentration in the aqueous phase. As discussed above, the measured pH is dependent not only on the GDL concentration but also the CaCl2 concentration. We can see from Fig. 7 that there are clear boundaries in composition space defining the limits of potentially reversible heatinduced gelation (category II behaviour). The triangular region of heat-induced emulsion gel behaviour is centred on a composition with pH :/5.8 and [CaCl2] :/25 mM. According to the criteria adopted, no category II systems can be formed at calcium ion concentrations below 10
Fig. 7. State diagram of sodium caseinate-stabilized emulsions (45 vol.% oil, 8.1 wt.% protein in aqueous phase) as a function of final emulsion pH and added CaCl2 concentration in aqueous phase: j, category I; \, category II (potentially reversible emulsion gel); m, category III. The boundary lines are drawn to guide the eye.
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mM or pH values outside the range 5.4 B/pH 0/ 6.4. Some of the changes in aggregation behaviour with pH and CaCl2 concentration indicated in Fig. 7 may be attributable to differing adsorbed layer compositions arising from the different sensitivities of the casein monomers (especially as1 and b) towards self-association in the presence of calcium ions [9,10]. This could explain, for instance, the ‘unexpected’ liquid-like character, at ambient temperatures, of the emulsions made at low pH, whereas the equivalent systems appear solid-like at neutral pH. It should be noted, however, that the ‘thermoreversibility’ referred to here, in the context of the category II emulsions, relates to the large-strain apparent viscosity behaviour and not to the smallstrain complex modulus behaviour. What this means is that the flocculated network of proteincoated droplets formed on heating is not spontaneously disrupted on cooling under the influence of Brownian motion. Rather the dissociation of aggregated droplets requires the application of external hydrodynamic forces (as during the viscosity measurement) in order to break down the emulsion gel network and to convert the system back to the fully dispersed liquid-like emulsion on cooling. As the state diagram in Fig. 7 is based largely on steady-state viscometry, its boundaries are undoubtedly influenced by the conditions (i.e. stress) set in the viscosity measurements. Were the viscosities to have been measured at lower stresses, some category II emulsions would have been allocated to category III. Conversely, if an even higher stress had been used, some category III emulsions might have been put in category II. However, the general form of the state diagram should not be affected by such considerations */ nor the position of the low pH boundary. Presumably as a result of its proximity to the casein’s isoelectric point, the location of the border between category II and category III at pH :/5.4 was found to be uninfluenced by the viscometry flow conditions. The sensitivity of the potentially reversible character to the stress applied during viscosity measurement is illustrated in Fig. 8 for the case of
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Fig. 8. Sensitivity of apparent viscosity versus temperature plot to the shear stress applied during viscosity measurement. Four consecutive heating/cooling cycles over the range 25 /45 8C were carried out with different applied stresses: m, 1st cycle (10 Pa); 2, 2nd cycle (20 Pa); ', 3rd cycle (30 Pa); \, 4th cycle (40 Pa). Solid and dotted lines refer to heating and cooling, respectively.
an emulsion to which 0.30 wt.% GDL and 20 mM CaCl2 were added prior to emulsification. Four temperature cycles between 25 and 45 8C were consecutively carried out on this same sample, with a 40 Pa shear stress applied for 1 h before each cycle. In this case, category II behaviour was indicated by a jump in apparent viscosity by at least an order of magnitude on heating from 40 to 45 8C. Fig. 8 shows that the measured viscosities on heating are relatively insensitive to the applied stress in the range 10/40 Pa. However, on cooling back to 25 8C during the 1st cycle, the viscosity measured at stress 10 Pa remains consistently constant at around the high value induced by the heating to 45 8C. So, it would appear that a stress of 10 Pa is insufficient to disrupt the droplet / droplet interactions leading to the heat-induced emulsion gel network. On the other hand, during the 2nd cycle involving a 20 Pa applied stress, there was observed to be a substantial reduction in viscosity on cooling, with the viscosity returning almost to its original value at 25 8C. The stresses
of 30 and 40 Pa applied during the 3rd and 4th cycles, respectively, were sufficient to allow complete thermoreversibility. Finally, we note that the likely molecular origin of this heat-induced emulsion gelation behaviour in terms of casein /casein interactions in bulk solution and at the surface of emulsion droplets has been discussed previously [7]. Hydrophobic interaction provides the main molecular driving force for the self-association of the caseins and hence, the aggregation of casein-coated droplets, but this can be moderated by changes in electrostatic interactions caused by pH-sensitive hydrogen ion equilibria involving the ionizable aminoacid side-chains and by specific calcium-ion binding to the phosphoserine residues [10,18]. This calcium binding is promoted by increase in temperature [19]. Moreover, it is well established that hydrophobic interactions become stronger with increasing temperature over the temperature range explored in this study. In the case of sodium caseinate, it would appear that some calcium ion binding combined with pH reduction is required to provide the optimum balance of intermolecular forces conducive to heat-induced reversible gelation.
Acknowledgements C.E. acknowledges receipt of a BBSRC Research Studentship in collaboration with Unilever Research (Colworth House).
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