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Thermoreversible gelation of caseinate-stabilized emulsions at around body temperature
ARTICLE IN PRESS
International Dairy Journal 13 (2003) 679–684
Thermoreversible gelation of caseinate-stabilized emulsions at around body temperature Caroline Eliot, Eric Dickinson* Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK Received 3 January 2003; accepted 8 March 2003
Abstract We have formulated food-grade protein-stabilized emulsions (30 vol% vegetable oil, 4 wt% sodium caseinate) which exhibit heatinduced gelation at around body temperature. Prior to emulsification, these systems have the continuous phase pH adjusted to between 6.8 and 5.3 and various concentrations of calcium chloride added. The minimum CaCl2 content required to cause gelation on heating decreases with decreasing pH, and the gelation temperature also decreases with decreasing pH. Under certain conditions the small-deformation rheological change associated with the heat-induced gelation has been found to be reversible on back-cooling to ambient. The systems have also been studied with regards to viscometry and phase separation. Emulsion compositions associated with depletion flocculation by excess non-adsorbed protein are shown to be sensitive to both the ionic calcium content and the pH. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Calcium ions; Caseinate; Emulsion gel; Reversible flocculation; Rheology
1. Introduction Aggregated casein gels are commonly encountered in food processing. In making yoghurt and other fermented dairy products, the action of lactic acid bacteria lowers the pH from ca. 6.7 to o4.5. This acidification converts a liquid-like dispersion of modified casein particles into a soft solid-like aggregated network (Horne, 1999). While commonly the primary particles involved are the native casein micelles of bovine milk, dairy-type particle gels can also be made from aggregated casein-coated emulsion droplets (Chen, Dickinson, & Edwards, 1999). A gel is typically defined by one or more of its rheological properties. In small-deformation measurements, an oscillating force is applied at a fixed frequency o and a fixed maximum strain. The response lags the applied force and is out of phase by a phase angle d: The complex shear modulus G n consists of a real (in-phase) component, G 0 ¼ G n cos d; called the elastic or storage modulus, which reflects the solid-like properties of the system, and an imaginary (out-of-phase) component, *Corresponding author. Tel.: +44-113-343-2956; fax: +44-113-343-2082. E-mail address: [email protected] (E. Dickinson).
G 0 ¼ G n sin d; called the viscous or loss modulus, which reflects its liquid-like properties. Both G 0 and G 00 are dependent on o: The ratio of the two moduli, G 00 =G0 ; then defines the tangent of the phase angle d: Thus, when fluid-like properties dominate over solid-like properties, G00 is greater than G 0 (leading to tan d > 1 and d > 45 ). Also, a ‘gel’ may be conveniently characterized (Dickinson & McClements, 1995) by (i) a storage modulus G 0 which exhibits a pronounced plateau region extending to time scales at least of the order of seconds and/or (ii) a loss modulus G00 which is considerably smaller than G 0 in the plateau region. In a system that gels on heating, the change from fluid to gel state, as the temperature is ‘ramped up’, is typically followed by recording the elastic modulus as a function of time. Detection of the ‘gel point’ can be said to occur when G0 or G n becomes greater than a pre-assigned threshold value significantly above the background noise (Ross-Murphy, 1994). Sodium caseinate is a widely used food ingredient. The constituent surface-active caseins (as1-, as2-, b- and k-) adsorb rapidly at the oilwater interface during emulsification, providing long-term stability to oil-inwater emulsions via a combination of electrostatic and steric stabilization (Dickinson, 1997, 1999). For an emulsion prepared at neutral pH with sodium caseinate
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as sole emulsifying agent, the adsorbed polymers mainly contributing to the colloid stability are as1- and b-casein (Dickinson, Whyman, & Dalgleish, 1987). Lowering the pH towards the protein isoelectric point converts the interdroplet steric repulsion into a net attraction, thereby inducing droplet flocculation and hence transforming the concentrated casein-stabilized emulsion into an aggregated emulsion gel (Chen et al., 1999). The aggregation and self-assembly behaviour of caseinate systems is very sensitive to temperature, pH and ionic calcium content (Horne, 1998). A hydrocarbon oil-in-water emulsion based on pure as1-casein, or sodium caseinate with a controlled amount of additional ionic calcium present, exhibits gelation (Casanova & Dickinson, 1998; Dickinson & Casanova, 1999; Dickinson & Eliot, 2003a, b) when heated from ambient temperature to ca. 45 C. Under certain conditions this gelation possesses thermoreversible character (Dickinson & Casanova, 1999; Eliot, Radford, & Dickinson, 2003). As a preparation for intended consumer-based texture testing investigations, this paper describes the formulation and rheological properties of a casein-stabilized thermoreversible emulsion gel based on a vegetable oil.
2. Materials and methods Sodium caseinate (90.0% protein, 5.0% moisture, 800 ppm calcium ions) was obtained from DeMelkindustrie (Veghel, Netherlands). Dihydrate calcium chloride (147.0 g mol1, 99.5%), acidulant glucono-dlactone (GDL) (99.0%), sodium azide and standard buffer solutions for pH meter calibration were supplied by Sigma Chemicals (St Louis, MO). Vegetable oil of low melting point was purchased from the local supermarket. Aqueous solutions of sodium caseinate (5.3 wt%) and 0.01 wt% sodium azide were prepared with stirring for 16 h at room temperature, followed by 30 min at 70 C, and ca. 2 h at room temperature. The pH was adjusted by addition of an appropriate amount of GDL added as a powder, followed by stirring for 16 h at room temperature. The ionic calcium level was carefully adjusted by dropwise addition of 1 m CaCl2 solution at 5 C with vigorous stirring. A notional molecular weight of 22 kDa was assumed for the caseinate in calculating the GDL/protein and calcium/protein molar ratios. Oil-in-water emulsions (4 wt% protein, 30 vol% oil) were prepared by jet homogenization (Burgaud, Dickinson, & Nelson, 1990) of vegetable oil and 5.3 wt% sodium caseinate solution in the volume ratio 30:70. To ensure that the temperature of any freshly prepared emulsion was below 30 C, 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. For 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 which could have been misinterpreted as larger droplets in the initial size distribution. But in most cases flocculation was apparent from the bimodal shape of the particle-size distribution. The flocculated state of these emulsions was confirmed by obtaining a monomodal distribution following addition of excess of non-ionic surfactant Tween 20 (polyoxyethylene sorbitan monolaurate, p50% lauric acid, 1.23 kDa), obtained from Sigma Chemicals. The pH of solutions and emulsions was measured using a Jenway 3310 pH meter, calibrated using pH 4 and pH 7 reference solutions (70.01). Large-deformation steady-state shear rheology was performed at 25 C with a double-gap DG24/27 measurement geometry on a Bohlin CVO with another portion of the same sample. Gravity creaming of emulsion samples (containing sodium azide as antimicrobial agent) was monitored visually over a period of 48 h (2571 C) as described previously (Dickinson, Ritzoulis, & Povey, 1999). Small-deformation oscillatory shear rheology was carried out using a Bohlin CVO-R, CVO or CS50 rheometer with a C14 concentric cylinder measuring geometry. Prior to the measurements the emulsions were subjected to a pre-shearing treatment at a constant stress of 40 Pa at 25 C for 10 or 20 min. The rheometers were set with a target strain of 0.5%, and storage and loss moduli were recorded at a constant frequency of 0.1 Hz, with temperature increasing from 25 C to 65 C at 1 C per min. For some emulsion compositions which exhibited heat-induced gelation at body temperature, the reversibility of the gelation was investigated by applying a 25 C-37 C-25 C temperature profile, followed by an appropriate waiting time at 25 C, before applying a second identical temperature cycle.
3. Results and discussion 3.1. Effect of ionic calcium and pH on emulsion stability An important instability mechanism in emulsions is depletion flocculation induced by non-adsorbed species such as polymers or small particles. Depletion flocculation of casein(ate)-based emulsions, and its associated influence on creaming and serum separation, can arise from excess surface-active material in the form of casein(ate) sub-micelles (Dickinson & Golding, 1997; Dickinson, Golding, & Povey, 1997). The flocculated emulsions exhibit pseudoplastic flow behaviour (shearthinning), and the strength of the depletion interaction is sensitively affected by the concentration of
ARTICLE IN PRESS C. Eliot, E. Dickinson / International Dairy Journal 13 (2003) 679–684
Viscosity (Pa s)
10
1
6.5 6.0 5.5 5.0 0
5 10 calcium/protein molar ratio
15
Fig. 2. Stability diagram for caseinate-based emulsions (30 vol% vegetable oil, 4 wt% protein, pH 6.8) stored for 48 h at 25 C with various concentrations of GDL and CaCl2. Filled symbols (m) denote compositions stable with respect to flocculation. Open symbols (&, J) denote states of flocculation: (&) depletion by excess caseinate; (J) acid-induced and/or calcium-induced electrostatic/bridging flocculation; ( ) near-boundary limit of stability. The dotted line denotes compositions without added GDL. The dashed line denotes the onset of acid-induced gelation (pH o5.5).
distributions) as a result of calcium-induced bridging flocculation (Dickinson & Davies, 1999). The upper row of symbols in Fig. 2 (along the dotted curve line) denotes the set of GDL-free samples with varying contents of Ca2+. This illustrates that the measured pH is not only lowered by the presence of the GDL, but that it also decreases with increasing CaCl2 concentration. Reduction in pH is found to diminish or enhance emulsion stability depending on the acidification extent and the CaCl2 content. At medium CaCl2 concentrations (R ¼ 3 9), lowering the pH leads to a broadened stability region, presumably due to an enhancement of the non-adsorbed protein aggregational state, which reduces the number density of the depleted species in the continuous phase, inhibiting depletion flocculation. Independent of the calcium concentration change, a further decrease in pH to o5.5 causes instability via acid-induced gelation occurring in the proximity of the protein’s isoelectric point. 3.2. Rheology of heat-induced emulsion gels
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non-adsorbed protein. However, it has also been established (Dickinson & Golding, 1998; Dickinson et al., 2001) that incorporating a moderate concentration of calcium ions (5–8 mm) inhibits the flocculation and its associated serum separation. Another important stability factor is pH, as illustrated in Fig. 1. The lowering of the pH on addition of GDL leads to a change in the rheological character of the caseinatebased oil-in-water emulsion (30 vol% vegetable oil, 4 wt% protein) from ‘depletion flocculated’ shear-thinning behaviour to ‘stable’ Newtonian behaviour. By analogy with arguments developed in connection with calcium ion sensitivity (Dickinson et al., 2001), this inhibition of flocculation upon acidification can be attributed to effects of pH on (i) the surface coverage and structure of adsorbed protein at the oilwater interface and (ii) the state of aggregation of the nonadsorbed protein which influences the size and number density of the depleting species (caseinate sub-micelles). Assessing the relative importance of these two factors requires further experimental information (light scattering from solutions, surface coverage, etc.). Fig. 2 shows the influence of ionic calcium and pH on the stability of a sodium caseinate-based emulsion system with excess protein in the aqueous phase. The solid triangular symbols denote stable emulsions, with no obvious creaming after 2 days, a steady monomodal particle-size distribution, and near-Newtonian to Newtonian rheology. At low calcium ion molar ratios (0pRp4), the non-acidified emulsions are monomodal in particle-size distribution, but unstable towards depletion flocculation induced by non-adsorbed caseinate. For 5pRp7; the samples are stable towards creaming over the 48 h observational period, as a result of an inhibition of the depletion flocculation by the added calcium ions (Dickinson & Golding, 1998; Dickinson, Radford, & Golding, 2003). A further increase in Ca2+ concentration (RX8) leads to instability (visibly precipitated aggregates and distinctly bimodal particle-size
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Fig. 1. Effect of pH on the rheology of sodium caseinate-stabilized emulsions (30 vol% vegetable oil, 4 wt% protein) at 25 C. Apparent viscosity is plotted against shear stress: (’) pH 7.0; (&) pH 6.6; (m) pH 6.3; and (J) pH 5.8.
Recent light-scattering measurements (Semenova, Belyakova, & Antipova, unpublished) of aqueous sodium caseinate at pH values below neutral in the presence of ionic calcium have shown a dramatic increase in the state of aggregation of the protein on heating from 35 C to 45 C. Hence, it is not surprising that concentrated, pH-adjusted, calcium-enriched, liquid-like caseinate-stabilized emulsions (45 vol% ntetradecane, 5 wt% protein) have been found to exhibit gelation on heating over a similar temperature range (Eliot et al., 2003). Furthermore, under certain
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conditions, the solgel transition has been regarded as having some reversible character on back-cooling. Also, the extent of heat-induced gelation has been found (Dickinson & Eliot, 2003a, b) to be enhanced by a moderate lowering of the pH. Fig. 3 illustrates that this same type of behaviour is exhibited here by a caseinatebased emulsion system of lower volume fraction based on vegetable oil (30 vol% oil, 4 wt% protein). On increasing the CaCl2/caseinate molar ratio of the GDL-free emulsion from 6.2 to 8.3, the complex modulus G n of the resulting emulsion is converted from temperature-independent behaviour into one exhibiting an increase in modulus of several orders of magnitude on raising to temperatures near and above normal body temperature (37 C) (Fig. 3a). This trend becomes even more pronounced for an emulsion of CaCl2/caseinate molar ratio 10.4, with a 1–2 order of magnitude increase in G n occurring over the temperature range 34–38 C. Fig. 3b shows similar heat-induced G n behaviour for acidified samples at a GDL/protein molar ratio of 5.8 for the same three CaCl2/protein molar ratios. The substantial increase in G n rises at a lower temperature than in the GDL-free samples (36–37 C, 31–32 C, and 29–30 C,
1000 100 G* (Pa)
10 1 0.1
respectively), and at a greater rate. As previously stated, the addition of CaCl2 itself leads to a lowering of the pH of the sodium caseinate solution, and so accordingly of the caseinate-based emulsion. Hence, the results in Fig. 3 relate to systems where pH is affected by both the addition of GDL and by the ionic calcium content, although the former is still clearly more important. Fig. 4 summarizes the temperature-dependent rheology of sets of caseinate-stabilized oil-in-water emulsions (30 vol% vegetable oil, 4 wt% protein) containing different amounts of GDL and CaCl2. The state diagram of emulsion pH versus calcium/caseinate molar ratio is qualitatively similar to that recently reported for the more concentrated hydrocarbon system (Eliot et al., 2003). Fig. 4 identifies three different categories of emulsion composition—A–C. The complex modulus G n (at 0.1 Hz) of a category A emulsion (high pH, low Ca2+ content) is temperature insensitive. The category B emulsion exhibits a large increase in Gn ; from a low value (o0.1 Pa) at p25 C to a value at least an order of magnitude higher at around body temperature 35–40 C, when heated at 1 C min1. The category C emulsion (pH o5.5) typically exhibits gelation below 35 C. We can see from Fig. 4 that heat-induced gelation at X35 C requires a moderately high Ca2+/caseinate molar ratio (RE4). As recently reported for the concentrated hydrocarbon oil emulsion (Eliot et al., 2003), the minimum CaCl2 content required to cause gelation on heating becomes reduced with decreasing pH. Furthermore, there is a lowering of the gelation temperature with decreasing pH. The ‘thermoreversibility’ defined previously (Dickinson & Eliot, 2003a) in the context of 45 vol% n-tetradecane emulsions (5 wt% sodium caseinate)
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G* (Pa)
pH
100 10 1
6.0 5.5
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35
45 55 Temperature (degree C)
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Fig. 3. Temperature-dependent complex modulus Gn at 0.1 Hz of caseinate-stabilized emulsions (30 vol% vegetable oil, 4 wt% protein) heated from 25 C to 65 C as a function of added CaCl2 concentration: (n) Ca2+/protein molar ratio=6.2; (’) Ca2+/protein molar ratio=8.3; (J) Ca2+/protein molar ratio=10.4: (a) without prior acidification and (b) prior acidification with GDL/protein molar ratio=5.8.
5
10
15
calcium/protein molar ratio Fig. 4. State diagram of caseinate-stabilized emulsions (30 vol% vegetable oil, 4 wt% protein) as a function of final emulsion pH and added CaCl2 content: ( ) liquid-like (category A) emulsions remaining liquid when heated to 43 C; (,) liquid-like (category B) emulsions becoming converted to emulsion gels with complex modulus Gn (0.1 Hz) of value >10 Pa on heating to 35–43 C; () ‘solid-like’ emulsions at ambient temperature or those forming gels below 35 C (category C).
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4. Conclusions By appropriate adjustment of pH and ionic calcium content, a vegetable oil-in-water emulsion based on
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relates to large-strain apparent viscosity behaviour. This means that the flocculated network of protein-coated droplets formed on heating was not spontaneously disrupted on cooling under the influence of Brownian motion alone. To redisperse the flocculated droplets and to disrupt the emulsion gel network, thereby converting the system back again into a liquid-like emulsion on cooling, a substantial hydrodynamic flow field had to be applied (as induced during viscometry measurements at applied stresses of 30–40 Pa). The special conditions giving ‘thermoreversible’ gelation are deemed to be associated with a delicate balance of intermolecular forces, involving calcium-ion binding and side-chain ionization moderating the hydrophobic interactions. The same temperature sensitivity has also been observed recently (Semenova, Belyakova, & Antipova, unpublished) in dilute caseinate solutions under similar conditions of pH and ionic calcium content. This delicate balance of intermolecular forces confers the optimum sensitivity of interdroplet interactions towards heating, as well as the phenomenon of shear-induced aggregate dispersion and associated gel disruption on cooling. In the absence of any substantial applied shear stress, the rate at which the gel-like character is reversed on cooling is very sensitive to the concentrations of GDL and ionic calcium, and to the heating cycle characteristics. This is illustrated in Fig. 5 for the case of two different caseinate-stabilized vegetable oil-in-water emulsion samples (30 vol% oil, 4 wt% protein) with (a) [Ca2+]/[protein]=10.4 and [GDL]/[protein]=2.4 (pH=5.9) and (b) [Ca2+]/[protein]=8.3 and [GDL]/ [protein]=3.6 (pH=5.8). In both cases, on heating from 25 C to 37 C, there is an increase in the storage modulus G 0 by three orders of magnitude to B102 Pa, accompanied by a G0 G 00 crossover around 1–30 Pa. Nevertheless, on lowering the temperature back down to 25 C, the storage modulus for system (a) remains >102 Pa for several hours, with G 0 twice as large as G00 ; whereas G0 for system (b) (having the lower Ca2+/protein ratio) falls to o0.5 Pa within 1 h, with a G 00 G 0 crossover at around 0.2–0.3 Pa. In the latter case, this thermoreversibility is repeated over a second cycle, albeit with a slightly lower rate of gel melting. Hence we can see from the data in Fig. 5 that the combination of a small change in pH (B0.1 unit) and ionic calcium content (B4 mm) can lead to a very large change in the rheology of the emulsion system and in the degree of (thermo)reversibility of the gelation process. This confirms the high sensitivity of the strength of the droplet–droplet interactions to the pH and the Ca2+/protein molar ratio.
683
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20
0.01 0
10000
(b)
20000 Time (s)
30000
Fig. 5. Kinetics of quiescent thermoreversibility of caseinate-stabilized emulsion gels (30 vol% vegetable oil, 4 wt% protein). Storage modulus G0 (0.1 Hz) (thick line) is plotted against time and temperature T (thin line) in the range from 25 C to 37 C: (a) Ca2+/protein molar ratio=10.4, pH=5.9 and (b) Ca2+/protein molar ratio=8.3, pH=5.8.
sodium caseinate has been formulated that exhibits thermoreversible gelation at around normal body temperature. Further experimental study of the rheology and thermoreversibility of these emulsion gel systems is currently underway under conditions of zero applied stress using the technique of diffusing wave spectroscopy (Eliot, Dickinson, & Horne, unpublished). Additionally, these emulsions are being incorporated into prototype model food products for analysis by taste panels as a way of assessing the likely consumer response to the textural changes determined rheologically.
Acknowledgements C.E. acknowledges receipt of a BBSRC Research Studentship and financial support from Unilever Research Colworth (UK).
References Burgaud, I., Dickinson, E., & Nelson, P. V. (1990). An improved highpressure homogenizer for making fine emulsions on a small scale. International Journal of Food Science Technology, 25, 39–46. Casanova, H., & Dickinson, E. (1998). Rheology and flocculation of oil-in-water emulsions made with mixtures of as1-casein+b-casein. Journal of Colloid and Interface Science, 206, 82–89.
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Chen, J., Dickinson, E., & Edwards, M. (1999). Rheology of acidtreated sodium caseinate stabilized emulsion gels. Journal of Texture Studies, 30, 377–396. Dickinson, E. (1997). Properties of emulsions stabilized with milk proteins: Overview of some recent developments. Journal of Dairy Science, 80, 2607–2619. Dickinson, E. (1999). Caseins in emulsions: Interfacial properties and interactions. International Dairy Journal, 9, 305–312. Dickinson, E., & Casanova, H. (1999). A thermoreversible emulsion gel based on sodium caseinate. Food Hydrocolloids, 13, 285–289. Dickinson, E., & Davies, E. (1999). Influence of ionic calcium on stability of sodium caseinate emulsions. Colloids and Surfaces B, 12, 203–212. Dickinson, E., & Eliot, C. (2003a). Defining the conditions for reversible heat-induced gelation of a caseinate-stabilized emulsion. Colloids and Surfaces B, 29, 89–97. Dickinson, E., & Eliot, C. (2003b). Aggregated casein gels: interactions, rheology and microstructure. In: P. Fisher, I. Marti, & E. J. Windhab (Eds.), Proceedings of the third international symposium on Food Rheology and Structure, (pp. 37–44). Zurich: . Laboratory of Food Process Engineering, ETH, 9–13 February. Dickinson, E., & Golding, M. (1997). Depletion flocculation of emulsions containing unadsorbed sodium caseinate. Food Hydrocolloids, 11, 13–18. Dickinson, E., & Golding, M. (1998). Influence of calcium ions on creaming and rheology of emulsions containing sodium caseinate. Colloids and Surfaces A, 144, 167–177. Dickinson, E., Golding, M., & Povey, M. J. W. (1997). Creaming and flocculation of oil-in-water emulsions containing sodium caseinate. Journal of Colloid and Interface Science, 185, 515–529.
Dickinson, E., & McClements, D. J. (Eds.). (1995). Advances in food colloids (p. 304). Glasgow: Blackie Academic & Professional. Dickinson, E., Radford, S. J., & Golding, M. (2003). Stability and rheology of emulsions containing sodium caseinate: Combined effects of ionic calcium and non-ionic surfactant. Food Hydrocolloids, 17, 211–220. Dickinson, E., Ritzoulis, C., & Povey, M. J. W. (1999). Stability of emulsions containing both sodium caseinate and Tween 20. Journal of Colloid and Interface Science, 212, 466–473. Dickinson, E., Semenova, M. G., Belyakova, L. E., Antipova, A. S., Il’in, M. M., Tsapkina, E. N., & Ritzoulis, C. (2001). Analysis of light scattering data on the calcium ion sensitivity of caseinate solution thermodynamics: Relationship to emulsion flocculation. Journal of Colloid and Interface Science, 239, 87–97. Dickinson, E., Whyman, R. H., & Dalgleish, D. G. (1987). Colloidal properties of model oil-in-water food emulsions stabilized separately by as1-casein, b-casein and k-casein. In E. Dickinson (Ed.), Food emulsions and foams (pp. 40–51). London: Royal Society of Chemistry. Eliot, C., Radford, S. J., & Dickinson, E. (2003). Effect of ionic calcium on the flocculation and gelation of sodium caseinate oil-inwater emulsions. In E. Dickinson, & T. van Vliet (Eds.), Food colloids, biopolymers and materials (pp. 234–242). Cambridge: Royal Society of Chemistry. Horne, D. S. (1998). Casein interactions: Casting light on the black boxes, the structure in dairy products. International Dairy Journal, 8, 171–177. Horne, D. S. (1999). Formation and structure of acidified milk gels. International Dairy Journal, 9, 261–268. Ross-Murphy, S. B. (1994). Rheological characterization of polymer gels and networks. Polymer Gels and Networks, 2, 229–237.
International Dairy Journal 13 (2003) 679–684
Thermoreversible gelation of caseinate-stabilized emulsions at around body temperature Caroline Eliot, Eric Dickinson* Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK Received 3 January 2003; accepted 8 March 2003
Abstract We have formulated food-grade protein-stabilized emulsions (30 vol% vegetable oil, 4 wt% sodium caseinate) which exhibit heatinduced gelation at around body temperature. Prior to emulsification, these systems have the continuous phase pH adjusted to between 6.8 and 5.3 and various concentrations of calcium chloride added. The minimum CaCl2 content required to cause gelation on heating decreases with decreasing pH, and the gelation temperature also decreases with decreasing pH. Under certain conditions the small-deformation rheological change associated with the heat-induced gelation has been found to be reversible on back-cooling to ambient. The systems have also been studied with regards to viscometry and phase separation. Emulsion compositions associated with depletion flocculation by excess non-adsorbed protein are shown to be sensitive to both the ionic calcium content and the pH. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Calcium ions; Caseinate; Emulsion gel; Reversible flocculation; Rheology
1. Introduction Aggregated casein gels are commonly encountered in food processing. In making yoghurt and other fermented dairy products, the action of lactic acid bacteria lowers the pH from ca. 6.7 to o4.5. This acidification converts a liquid-like dispersion of modified casein particles into a soft solid-like aggregated network (Horne, 1999). While commonly the primary particles involved are the native casein micelles of bovine milk, dairy-type particle gels can also be made from aggregated casein-coated emulsion droplets (Chen, Dickinson, & Edwards, 1999). A gel is typically defined by one or more of its rheological properties. In small-deformation measurements, an oscillating force is applied at a fixed frequency o and a fixed maximum strain. The response lags the applied force and is out of phase by a phase angle d: The complex shear modulus G n consists of a real (in-phase) component, G 0 ¼ G n cos d; called the elastic or storage modulus, which reflects the solid-like properties of the system, and an imaginary (out-of-phase) component, *Corresponding author. Tel.: +44-113-343-2956; fax: +44-113-343-2082. E-mail address: [email protected] (E. Dickinson).
G 0 ¼ G n sin d; called the viscous or loss modulus, which reflects its liquid-like properties. Both G 0 and G 00 are dependent on o: The ratio of the two moduli, G 00 =G0 ; then defines the tangent of the phase angle d: Thus, when fluid-like properties dominate over solid-like properties, G00 is greater than G 0 (leading to tan d > 1 and d > 45 ). Also, a ‘gel’ may be conveniently characterized (Dickinson & McClements, 1995) by (i) a storage modulus G 0 which exhibits a pronounced plateau region extending to time scales at least of the order of seconds and/or (ii) a loss modulus G00 which is considerably smaller than G 0 in the plateau region. In a system that gels on heating, the change from fluid to gel state, as the temperature is ‘ramped up’, is typically followed by recording the elastic modulus as a function of time. Detection of the ‘gel point’ can be said to occur when G0 or G n becomes greater than a pre-assigned threshold value significantly above the background noise (Ross-Murphy, 1994). Sodium caseinate is a widely used food ingredient. The constituent surface-active caseins (as1-, as2-, b- and k-) adsorb rapidly at the oilwater interface during emulsification, providing long-term stability to oil-inwater emulsions via a combination of electrostatic and steric stabilization (Dickinson, 1997, 1999). For an emulsion prepared at neutral pH with sodium caseinate
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as sole emulsifying agent, the adsorbed polymers mainly contributing to the colloid stability are as1- and b-casein (Dickinson, Whyman, & Dalgleish, 1987). Lowering the pH towards the protein isoelectric point converts the interdroplet steric repulsion into a net attraction, thereby inducing droplet flocculation and hence transforming the concentrated casein-stabilized emulsion into an aggregated emulsion gel (Chen et al., 1999). The aggregation and self-assembly behaviour of caseinate systems is very sensitive to temperature, pH and ionic calcium content (Horne, 1998). A hydrocarbon oil-in-water emulsion based on pure as1-casein, or sodium caseinate with a controlled amount of additional ionic calcium present, exhibits gelation (Casanova & Dickinson, 1998; Dickinson & Casanova, 1999; Dickinson & Eliot, 2003a, b) when heated from ambient temperature to ca. 45 C. Under certain conditions this gelation possesses thermoreversible character (Dickinson & Casanova, 1999; Eliot, Radford, & Dickinson, 2003). As a preparation for intended consumer-based texture testing investigations, this paper describes the formulation and rheological properties of a casein-stabilized thermoreversible emulsion gel based on a vegetable oil.
2. Materials and methods Sodium caseinate (90.0% protein, 5.0% moisture, 800 ppm calcium ions) was obtained from DeMelkindustrie (Veghel, Netherlands). Dihydrate calcium chloride (147.0 g mol1, 99.5%), acidulant glucono-dlactone (GDL) (99.0%), sodium azide and standard buffer solutions for pH meter calibration were supplied by Sigma Chemicals (St Louis, MO). Vegetable oil of low melting point was purchased from the local supermarket. Aqueous solutions of sodium caseinate (5.3 wt%) and 0.01 wt% sodium azide were prepared with stirring for 16 h at room temperature, followed by 30 min at 70 C, and ca. 2 h at room temperature. The pH was adjusted by addition of an appropriate amount of GDL added as a powder, followed by stirring for 16 h at room temperature. The ionic calcium level was carefully adjusted by dropwise addition of 1 m CaCl2 solution at 5 C with vigorous stirring. A notional molecular weight of 22 kDa was assumed for the caseinate in calculating the GDL/protein and calcium/protein molar ratios. Oil-in-water emulsions (4 wt% protein, 30 vol% oil) were prepared by jet homogenization (Burgaud, Dickinson, & Nelson, 1990) of vegetable oil and 5.3 wt% sodium caseinate solution in the volume ratio 30:70. To ensure that the temperature of any freshly prepared emulsion was below 30 C, 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. For 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 which could have been misinterpreted as larger droplets in the initial size distribution. But in most cases flocculation was apparent from the bimodal shape of the particle-size distribution. The flocculated state of these emulsions was confirmed by obtaining a monomodal distribution following addition of excess of non-ionic surfactant Tween 20 (polyoxyethylene sorbitan monolaurate, p50% lauric acid, 1.23 kDa), obtained from Sigma Chemicals. The pH of solutions and emulsions was measured using a Jenway 3310 pH meter, calibrated using pH 4 and pH 7 reference solutions (70.01). Large-deformation steady-state shear rheology was performed at 25 C with a double-gap DG24/27 measurement geometry on a Bohlin CVO with another portion of the same sample. Gravity creaming of emulsion samples (containing sodium azide as antimicrobial agent) was monitored visually over a period of 48 h (2571 C) as described previously (Dickinson, Ritzoulis, & Povey, 1999). Small-deformation oscillatory shear rheology was carried out using a Bohlin CVO-R, CVO or CS50 rheometer with a C14 concentric cylinder measuring geometry. Prior to the measurements the emulsions were subjected to a pre-shearing treatment at a constant stress of 40 Pa at 25 C for 10 or 20 min. The rheometers were set with a target strain of 0.5%, and storage and loss moduli were recorded at a constant frequency of 0.1 Hz, with temperature increasing from 25 C to 65 C at 1 C per min. For some emulsion compositions which exhibited heat-induced gelation at body temperature, the reversibility of the gelation was investigated by applying a 25 C-37 C-25 C temperature profile, followed by an appropriate waiting time at 25 C, before applying a second identical temperature cycle.
3. Results and discussion 3.1. Effect of ionic calcium and pH on emulsion stability An important instability mechanism in emulsions is depletion flocculation induced by non-adsorbed species such as polymers or small particles. Depletion flocculation of casein(ate)-based emulsions, and its associated influence on creaming and serum separation, can arise from excess surface-active material in the form of casein(ate) sub-micelles (Dickinson & Golding, 1997; Dickinson, Golding, & Povey, 1997). The flocculated emulsions exhibit pseudoplastic flow behaviour (shearthinning), and the strength of the depletion interaction is sensitively affected by the concentration of
ARTICLE IN PRESS C. Eliot, E. Dickinson / International Dairy Journal 13 (2003) 679–684
Viscosity (Pa s)
10
1
6.5 6.0 5.5 5.0 0
5 10 calcium/protein molar ratio
15
Fig. 2. Stability diagram for caseinate-based emulsions (30 vol% vegetable oil, 4 wt% protein, pH 6.8) stored for 48 h at 25 C with various concentrations of GDL and CaCl2. Filled symbols (m) denote compositions stable with respect to flocculation. Open symbols (&, J) denote states of flocculation: (&) depletion by excess caseinate; (J) acid-induced and/or calcium-induced electrostatic/bridging flocculation; ( ) near-boundary limit of stability. The dotted line denotes compositions without added GDL. The dashed line denotes the onset of acid-induced gelation (pH o5.5).
distributions) as a result of calcium-induced bridging flocculation (Dickinson & Davies, 1999). The upper row of symbols in Fig. 2 (along the dotted curve line) denotes the set of GDL-free samples with varying contents of Ca2+. This illustrates that the measured pH is not only lowered by the presence of the GDL, but that it also decreases with increasing CaCl2 concentration. Reduction in pH is found to diminish or enhance emulsion stability depending on the acidification extent and the CaCl2 content. At medium CaCl2 concentrations (R ¼ 3 9), lowering the pH leads to a broadened stability region, presumably due to an enhancement of the non-adsorbed protein aggregational state, which reduces the number density of the depleted species in the continuous phase, inhibiting depletion flocculation. Independent of the calcium concentration change, a further decrease in pH to o5.5 causes instability via acid-induced gelation occurring in the proximity of the protein’s isoelectric point. 3.2. Rheology of heat-induced emulsion gels
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non-adsorbed protein. However, it has also been established (Dickinson & Golding, 1998; Dickinson et al., 2001) that incorporating a moderate concentration of calcium ions (5–8 mm) inhibits the flocculation and its associated serum separation. Another important stability factor is pH, as illustrated in Fig. 1. The lowering of the pH on addition of GDL leads to a change in the rheological character of the caseinatebased oil-in-water emulsion (30 vol% vegetable oil, 4 wt% protein) from ‘depletion flocculated’ shear-thinning behaviour to ‘stable’ Newtonian behaviour. By analogy with arguments developed in connection with calcium ion sensitivity (Dickinson et al., 2001), this inhibition of flocculation upon acidification can be attributed to effects of pH on (i) the surface coverage and structure of adsorbed protein at the oilwater interface and (ii) the state of aggregation of the nonadsorbed protein which influences the size and number density of the depleting species (caseinate sub-micelles). Assessing the relative importance of these two factors requires further experimental information (light scattering from solutions, surface coverage, etc.). Fig. 2 shows the influence of ionic calcium and pH on the stability of a sodium caseinate-based emulsion system with excess protein in the aqueous phase. The solid triangular symbols denote stable emulsions, with no obvious creaming after 2 days, a steady monomodal particle-size distribution, and near-Newtonian to Newtonian rheology. At low calcium ion molar ratios (0pRp4), the non-acidified emulsions are monomodal in particle-size distribution, but unstable towards depletion flocculation induced by non-adsorbed caseinate. For 5pRp7; the samples are stable towards creaming over the 48 h observational period, as a result of an inhibition of the depletion flocculation by the added calcium ions (Dickinson & Golding, 1998; Dickinson, Radford, & Golding, 2003). A further increase in Ca2+ concentration (RX8) leads to instability (visibly precipitated aggregates and distinctly bimodal particle-size
681
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100
Shear Stress (Pa)
Fig. 1. Effect of pH on the rheology of sodium caseinate-stabilized emulsions (30 vol% vegetable oil, 4 wt% protein) at 25 C. Apparent viscosity is plotted against shear stress: (’) pH 7.0; (&) pH 6.6; (m) pH 6.3; and (J) pH 5.8.
Recent light-scattering measurements (Semenova, Belyakova, & Antipova, unpublished) of aqueous sodium caseinate at pH values below neutral in the presence of ionic calcium have shown a dramatic increase in the state of aggregation of the protein on heating from 35 C to 45 C. Hence, it is not surprising that concentrated, pH-adjusted, calcium-enriched, liquid-like caseinate-stabilized emulsions (45 vol% ntetradecane, 5 wt% protein) have been found to exhibit gelation on heating over a similar temperature range (Eliot et al., 2003). Furthermore, under certain
ARTICLE IN PRESS C. Eliot, E. Dickinson / International Dairy Journal 13 (2003) 679–684
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conditions, the solgel transition has been regarded as having some reversible character on back-cooling. Also, the extent of heat-induced gelation has been found (Dickinson & Eliot, 2003a, b) to be enhanced by a moderate lowering of the pH. Fig. 3 illustrates that this same type of behaviour is exhibited here by a caseinatebased emulsion system of lower volume fraction based on vegetable oil (30 vol% oil, 4 wt% protein). On increasing the CaCl2/caseinate molar ratio of the GDL-free emulsion from 6.2 to 8.3, the complex modulus G n of the resulting emulsion is converted from temperature-independent behaviour into one exhibiting an increase in modulus of several orders of magnitude on raising to temperatures near and above normal body temperature (37 C) (Fig. 3a). This trend becomes even more pronounced for an emulsion of CaCl2/caseinate molar ratio 10.4, with a 1–2 order of magnitude increase in G n occurring over the temperature range 34–38 C. Fig. 3b shows similar heat-induced G n behaviour for acidified samples at a GDL/protein molar ratio of 5.8 for the same three CaCl2/protein molar ratios. The substantial increase in G n rises at a lower temperature than in the GDL-free samples (36–37 C, 31–32 C, and 29–30 C,
1000 100 G* (Pa)
10 1 0.1
respectively), and at a greater rate. As previously stated, the addition of CaCl2 itself leads to a lowering of the pH of the sodium caseinate solution, and so accordingly of the caseinate-based emulsion. Hence, the results in Fig. 3 relate to systems where pH is affected by both the addition of GDL and by the ionic calcium content, although the former is still clearly more important. Fig. 4 summarizes the temperature-dependent rheology of sets of caseinate-stabilized oil-in-water emulsions (30 vol% vegetable oil, 4 wt% protein) containing different amounts of GDL and CaCl2. The state diagram of emulsion pH versus calcium/caseinate molar ratio is qualitatively similar to that recently reported for the more concentrated hydrocarbon system (Eliot et al., 2003). Fig. 4 identifies three different categories of emulsion composition—A–C. The complex modulus G n (at 0.1 Hz) of a category A emulsion (high pH, low Ca2+ content) is temperature insensitive. The category B emulsion exhibits a large increase in Gn ; from a low value (o0.1 Pa) at p25 C to a value at least an order of magnitude higher at around body temperature 35–40 C, when heated at 1 C min1. The category C emulsion (pH o5.5) typically exhibits gelation below 35 C. We can see from Fig. 4 that heat-induced gelation at X35 C requires a moderately high Ca2+/caseinate molar ratio (RE4). As recently reported for the concentrated hydrocarbon oil emulsion (Eliot et al., 2003), the minimum CaCl2 content required to cause gelation on heating becomes reduced with decreasing pH. Furthermore, there is a lowering of the gelation temperature with decreasing pH. The ‘thermoreversibility’ defined previously (Dickinson & Eliot, 2003a) in the context of 45 vol% n-tetradecane emulsions (5 wt% sodium caseinate)
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G* (Pa)
pH
100 10 1
6.0 5.5
0.1
5.0 0
0.01 25
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35
45 55 Temperature (degree C)
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Fig. 3. Temperature-dependent complex modulus Gn at 0.1 Hz of caseinate-stabilized emulsions (30 vol% vegetable oil, 4 wt% protein) heated from 25 C to 65 C as a function of added CaCl2 concentration: (n) Ca2+/protein molar ratio=6.2; (’) Ca2+/protein molar ratio=8.3; (J) Ca2+/protein molar ratio=10.4: (a) without prior acidification and (b) prior acidification with GDL/protein molar ratio=5.8.
5
10
15
calcium/protein molar ratio Fig. 4. State diagram of caseinate-stabilized emulsions (30 vol% vegetable oil, 4 wt% protein) as a function of final emulsion pH and added CaCl2 content: ( ) liquid-like (category A) emulsions remaining liquid when heated to 43 C; (,) liquid-like (category B) emulsions becoming converted to emulsion gels with complex modulus Gn (0.1 Hz) of value >10 Pa on heating to 35–43 C; () ‘solid-like’ emulsions at ambient temperature or those forming gels below 35 C (category C).
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4. Conclusions By appropriate adjustment of pH and ionic calcium content, a vegetable oil-in-water emulsion based on
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relates to large-strain apparent viscosity behaviour. This means that the flocculated network of protein-coated droplets formed on heating was not spontaneously disrupted on cooling under the influence of Brownian motion alone. To redisperse the flocculated droplets and to disrupt the emulsion gel network, thereby converting the system back again into a liquid-like emulsion on cooling, a substantial hydrodynamic flow field had to be applied (as induced during viscometry measurements at applied stresses of 30–40 Pa). The special conditions giving ‘thermoreversible’ gelation are deemed to be associated with a delicate balance of intermolecular forces, involving calcium-ion binding and side-chain ionization moderating the hydrophobic interactions. The same temperature sensitivity has also been observed recently (Semenova, Belyakova, & Antipova, unpublished) in dilute caseinate solutions under similar conditions of pH and ionic calcium content. This delicate balance of intermolecular forces confers the optimum sensitivity of interdroplet interactions towards heating, as well as the phenomenon of shear-induced aggregate dispersion and associated gel disruption on cooling. In the absence of any substantial applied shear stress, the rate at which the gel-like character is reversed on cooling is very sensitive to the concentrations of GDL and ionic calcium, and to the heating cycle characteristics. This is illustrated in Fig. 5 for the case of two different caseinate-stabilized vegetable oil-in-water emulsion samples (30 vol% oil, 4 wt% protein) with (a) [Ca2+]/[protein]=10.4 and [GDL]/[protein]=2.4 (pH=5.9) and (b) [Ca2+]/[protein]=8.3 and [GDL]/ [protein]=3.6 (pH=5.8). In both cases, on heating from 25 C to 37 C, there is an increase in the storage modulus G 0 by three orders of magnitude to B102 Pa, accompanied by a G0 G 00 crossover around 1–30 Pa. Nevertheless, on lowering the temperature back down to 25 C, the storage modulus for system (a) remains >102 Pa for several hours, with G 0 twice as large as G00 ; whereas G0 for system (b) (having the lower Ca2+/protein ratio) falls to o0.5 Pa within 1 h, with a G 00 G 0 crossover at around 0.2–0.3 Pa. In the latter case, this thermoreversibility is repeated over a second cycle, albeit with a slightly lower rate of gel melting. Hence we can see from the data in Fig. 5 that the combination of a small change in pH (B0.1 unit) and ionic calcium content (B4 mm) can lead to a very large change in the rheology of the emulsion system and in the degree of (thermo)reversibility of the gelation process. This confirms the high sensitivity of the strength of the droplet–droplet interactions to the pH and the Ca2+/protein molar ratio.
683
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20
0.01 0
10000
(b)
20000 Time (s)
30000
Fig. 5. Kinetics of quiescent thermoreversibility of caseinate-stabilized emulsion gels (30 vol% vegetable oil, 4 wt% protein). Storage modulus G0 (0.1 Hz) (thick line) is plotted against time and temperature T (thin line) in the range from 25 C to 37 C: (a) Ca2+/protein molar ratio=10.4, pH=5.9 and (b) Ca2+/protein molar ratio=8.3, pH=5.8.
sodium caseinate has been formulated that exhibits thermoreversible gelation at around normal body temperature. Further experimental study of the rheology and thermoreversibility of these emulsion gel systems is currently underway under conditions of zero applied stress using the technique of diffusing wave spectroscopy (Eliot, Dickinson, & Horne, unpublished). Additionally, these emulsions are being incorporated into prototype model food products for analysis by taste panels as a way of assessing the likely consumer response to the textural changes determined rheologically.
Acknowledgements C.E. acknowledges receipt of a BBSRC Research Studentship and financial support from Unilever Research Colworth (UK).
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Dickinson, E., & McClements, D. J. (Eds.). (1995). Advances in food colloids (p. 304). Glasgow: Blackie Academic & Professional. Dickinson, E., Radford, S. J., & Golding, M. (2003). Stability and rheology of emulsions containing sodium caseinate: Combined effects of ionic calcium and non-ionic surfactant. Food Hydrocolloids, 17, 211–220. Dickinson, E., Ritzoulis, C., & Povey, M. J. W. (1999). Stability of emulsions containing both sodium caseinate and Tween 20. Journal of Colloid and Interface Science, 212, 466–473. Dickinson, E., Semenova, M. G., Belyakova, L. E., Antipova, A. S., Il’in, M. M., Tsapkina, E. N., & Ritzoulis, C. (2001). Analysis of light scattering data on the calcium ion sensitivity of caseinate solution thermodynamics: Relationship to emulsion flocculation. Journal of Colloid and Interface Science, 239, 87–97. Dickinson, E., Whyman, R. H., & Dalgleish, D. G. (1987). Colloidal properties of model oil-in-water food emulsions stabilized separately by as1-casein, b-casein and k-casein. In E. Dickinson (Ed.), Food emulsions and foams (pp. 40–51). London: Royal Society of Chemistry. Eliot, C., Radford, S. J., & Dickinson, E. (2003). Effect of ionic calcium on the flocculation and gelation of sodium caseinate oil-inwater emulsions. In E. Dickinson, & T. van Vliet (Eds.), Food colloids, biopolymers and materials (pp. 234–242). Cambridge: Royal Society of Chemistry. Horne, D. S. (1998). Casein interactions: Casting light on the black boxes, the structure in dairy products. International Dairy Journal, 8, 171–177. Horne, D. S. (1999). Formation and structure of acidified milk gels. International Dairy Journal, 9, 261–268. Ross-Murphy, S. B. (1994). Rheological characterization of polymer gels and networks. Polymer Gels and Networks, 2, 229–237.