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Synergistic stabilization of heat-treated emulsions containing mixtures of milk proteins
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International Dairy Journal 17 (2007) 95–103 www.elsevier.com/locate/idairyj
Synergistic stabilization of heat-treated emulsions containing mixtures of milk proteins Emma L. Parkinson, Eric Dickinson Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK Received 15 September 2005; accepted 15 January 2006
Abstract The synergistic effect by which a very small amount of casein can confer stability to a whey protein-stabilized emulsion heated to 90 1C has been investigated. Using b-lactoglobulin (b-lg) as the main emulsifying agent, the extent of heat-induced flocculation increased with ionic strength, with commercial sodium caseinate or b-casein incorporated. The protective effect of casein was retained for a moderate concentration of ionic calcium. Casein added before heating, or shortly afterwards (i.e. before the emulsion had cooled), offered substantial synergistic protection to the heated b-lg-stabilized emulsion. With a-lactalbumin (a-la) as the primary emulsifying agent, no significant protective effect could be observed. In contrast, casein could confer significant stability to a heat-treated bovine serum albumin emulsion. Quiescent storage stability testing suggests that a combination of limited heating and casein addition could improve the long-term shelf-life of a whey protein-based emulsion. r 2006 Elsevier Ltd. All rights reserved. Keywords: Caseinate; Emulsion stability; Heat denaturation; b-Lactoglobulin; Flocculation; Viscosity; Whey protein
1. Introduction The excellent emulsifying and stabilizing properties of adsorbed layers of individual caseins and sodium caseinate have been well established for many years (Dickinson, 1989, 1999). More recently, experiments have shown (Dickinson & Parkinson, 2004; Parkinson & Dickinson, 2004) that casein polymers are also able to exert a protective stabilizing effect when present in very small amounts (0.03–0.15 wt% of the total emulsion) in whey protein-stabilized emulsions subjected to thermal treatment (e.g., at 90 1C for 3 min). Based on a combination of emulsion viscometry and particle-size measurements, this stabilizing phenomenon has been confirmed for emulsions based on either b-lactoglobulin (b-lg) or a commercial whey protein isolate (WPI) upon the incorporation of very low concentrations of sodium caseinate, b-casein or as1-casein. The exact amount of casein required for inhibition of droplet flocculation on heating depends on Corresponding author. Tel.: +44 113 243 1751; fax: +44 113 343 2982.
E-mail address: [email protected] (E. Dickinson). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.01.010
the purity of the whey protein used; but, in general, the protective effect was found to follow the order b-casein4sodium caseinate4as1-casein. The stabilizing effect of the casein in these mixed milk protein systems is strongly synergistic. This is because the casein polymer appears to be acting in a colloidal stabilizing capacity at a surface concentration very much lower than that at which it could be used as an emulsifying or stabilizing agent simply on its own. Using protein analytical techniques (reverse-phase-high-performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDSPAGE)), we have confirmed experimentally (Parkinson & Dickinson, 2006) that all of the casein present in these systems is indeed located at the surface of the emulsion droplets. Although at first rather surprising, the potential ability of such a low-surface coverage of casein to protect whey protein-coated droplets against heat-induced aggregation can be adequately explained in terms of a model of steric stabilizing casein-like copolymers based on Scheutjens–Fleer self-consistent-field theory (Parkinson, Ettelaie, & Dickinson, 2005).
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E.L. Parkinson, E. Dickinson / International Dairy Journal 17 (2007) 95–103
In the present paper, we address some issues relating to the likely generality and range of applicability of this synergistic stabilization phenomenon. In particular, we report on new experiments designed to provide answers to four specific questions: (1) How does ionic strength or calcium ion concentration affect the ability of casein to protect b-lactoglobulin emulsions against heat-induced destabilization? (2) To what extent does the order of addition of the casein (i.e., before/after homogenization, before/after heating) affect its stabilizing capability? (3) Can casein also be effective in providing synergistic protection to heated emulsions prepared with other individual globular milk proteins, e.g., bovine serum albumin (BSA) or a-lactalbumin? (4) What is the influence of a small amount of added casein on the long-term storage stability of a whey proteinbased emulsion?
2. Materials and methods Spray-dried sodium caseinate (482 wt% dry protein, o6 wt% moisture, according to the manufacturer) containing 800 ppm Ca, was obtained from DeMelkindustrie (Veghel, The Netherlands). Pure b-lg, BSA and a-lactalbumin (a-la) were obtained from Sigma Chemicals (St. Louis, MO, USA). A WPI sample (BiPRO, 90% protein) was supplied by Davisco Foods International (Le Sueur, MN, USA). The b-casein and as1-casein were obtained as freeze-dried samples (498% purity) from the Hannah Research Institute (Ayr, Scotland). Potassium chloride (99%), n-tetradecane (99%), imidazole (99%) and dihydrated calcium chloride (99.5%) were purchased from Sigma Chemicals. Aqueous solutions of (i) b-lg and (ii) sodium caseinate, b-casein or as1-casein were separately prepared by dissolving the proteins in 20 mM imidazole buffer (pH 6.8), containing 0–50 mM KCl and/or 0–5 mM CaCl2, with continuous stirring overnight. Oil-in-water emulsions of constant total protein content (3 wt% protein, 45 vol% oil), but differing relative proportions of casein and whey protein, were prepared by high-pressure homogenization at 300 bar using a home-built jet homogenizer (Burgaud, Dickinson & Nelson, 1990). After a 10 min waiting period following homogenization, the emulsions were heated at 90 1C (70.2 1C) for 3 min without stirring in sealed tubes (capacity 5 mL) contained in a water bath before being rapidly cooled to room temperature by placing the tubes in ice water for a short time. The choice of a small sample volume was to ensure a relatively short thermal equilibration time (90 s). Rheological testing was carried out within 1 h of the original emulsification stage. Large-deformation steadystate shear rheology was performed at 25 1C with a Bohlin CVO rheometer (Bohlin Instruments, Cirencester, UK)
using the concentric cell geometry (C14). The sample (ca. 5 mL) was covered with a thin layer of silicone oil to prevent evaporation, and then allowed to equilibrate at 25 1C for 10 min, prior to viscosity measurement at a shear rate of 5 s1. Numerical values of viscosities quoted below refer to average measurements taken after 2 min of constant shearing at 5 s1. All average values quoted are based on (at least) duplicate results. For the data presented here and elsewhere (Dickinson & Parkinson, 2004; Parkinson & Dickinson, 2004), there was up to 20% variability in the measured emulsion viscosity values, with greater variability typically encountered at the higher degrees of aggregation. Droplet-size distributions of the heated emulsions were determined using a Malvern Mastersizer MS2000 (Malvern Instruments, Malvern, UK) with refractive index ratio ¼ 1.074 and absorbance ¼ 0.005. Varying amounts of calcium chloride dihydrate (CaCl2 2H2O) were added to emulsions prior to emulsification (to ensure uniform distribution of Ca2+). Additions of casein were mainly made after homogenization. The free calcium ion concentration of the emulsions was determined before and after heating (90 1C, 3 min) using a Jenway calcium combination ion-selective electrode (Barloworld Scientific, Dunmow, Essex, UK) and a Jenway 3310 pH meter. Long-term storage stability under ambient conditions was assessed for emulsions containing b-lg (or WPI) alone and b-lg (or WPI)+sodium caseinate. Untreated samples and those that had been heated (and subsequently cooled) were stored in sealed 75 12 mm sample tubes for various lengths of time up to 17 months. The thicknesses of cream and serum layers were measured periodically and expressed as percentages of the total height of the emulsion. 3. Results and discussion 3.1. Effect of ionic strength and calcium ion content on ability of casein to protect b-lg emulsions against heatinduced destabilization The stabilizing effect of small additions of sodium caseinate on a b-lg-stabilized emulsion that was heated at 90 1C for 3 min has been investigated as a function of ionic strength (0–50 mM KCl). The apparent viscosity at 5 s1 and 25 1C was used to monitor the extent of heat-induced flocculation. The results are summarized in Fig. 1. Based on our previous experience with similar systems (Parkinson & Dickinson, 2004), we adopted the arbitrary benchmark criterion of an apparent viscosity above 0.5 Pa s as denoting an emulsion that is deemed ‘unstable’ to heating at 90 1C for 3 min, whilst a viscosity below 0.5 Pa s denotes an emulsion that is ‘stable’ to heating. Also indicated in Fig. 1 are intermediate states where the measured viscosity was 0.5 Pa s after heating. From this diagram, it is clear that emulsion stability improves as the proportion of sodium caseinate incorporated in the emulsion increases and as the ionic strength decreases. We also see that a similar trend is
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40
Viscosity (Pa s)
KCl content (mM)
91
30 20 10
10
10
1
0
46 0.65
5.5
0.72 0.60
0.68
0
0 0
5
10
15
0
20
% Casein in protein mixture Fig. 1. Stability map of b-lg-stabilized emulsions (3 wt% protein, 45 vol% oil, pH 6.8) with different KCl contents in the presence of small additions of sodium caseinate or b-casein (expressed as a percentage of the total protein present) after heating for 3 min at 90 1C. Depending on the measured apparent viscosity after heating, samples may be assigned to different categories: m, unstable with caseinate or b-casein; D, unstable with caseinate, stable with b-casein; ., unstable with caseinate, intermediate state with b-casein; ’, intermediate state with caseinate, stable with b-casein; O, stable with caseinate and b-casein.
apparent for the case of equivalent small additions of b-casein to a similar b-lg emulsion heated (90 1C, 3 min) at varying ionic strengths, i.e., the stability was found to be minimal for low casein additions and for high ionic strengths. For pure b-casein, however, it appears that less casein polymer (p5% addition) is required to confer stability than with commercial sodium caseinate (p10% addition). Casein is a calcium-sensitive protein, and this sensitivity is temperature dependent (Dalgleish & Parker, 1980; Parker & Dalgleish, 1981). Specific calcium ion binding at neutral pH lowers the net negative charge on the protein (and hence at the surface of protein-coated emulsion droplets), since the positively charged Ca2+ ions bind to the anionic phosphoseryl residues on b- and as1-casein (5 and 8 per molecule, respectively). This lowered net charge reduces the level of electrostatic repulsion between protein-coated layers, thereby promoting an increased degree of emulsion flocculation (Dickinson & Davies, 1999). The level of steric stabilization may also be diminished by the consequent partial collapse of the adsorbed casein layer on calcium ion addition (Dalgleish, 1997), and so a combination of these effects could act to prevent the stabilizing action of small additions of casein to a b-lg-stabilized emulsion. Some preliminary experiments were carried out with a 3 wt% b-lg-stabilized emulsion (45 vol% oil, pH 6.8) to determine the concentration of CaCl2 required to cause the same extent of heat-induced aggregation (indicated by the relative increase in apparent viscosity) as when 30 mM KCl was present. It can be seen from Fig. 2 that the viscosity after heating increased strongly with calcium content in the range 3–4 mM, and for 5 mM Ca2+ the viscosity was too high to measure in the rheometer with the C14 geometry. Replacement of 5% of the b-lg by sodium
3
3.5
4
5
Calcium chloride content (mM) Fig. 2. Influence of CaCl2 addition on the apparent viscosity at 5 s1 and 25 1C of b-lg-stabilized emulsions (3 wt% total protein, 45 vol% oil, pH 6.8) heated at 90 1C for 3 min. Filled (black) columns represent emulsions stabilized by 3 wt% b-lg. Unfilled (white) columns represent emulsions stabilized by 2.85% b-lg+0.15% caseinate. Each number at the top of the column indicates the mean particle size d43 (in mm) as measured by static light-scattering (Mastersizer).
caseinate (i.e., so that 0.15 wt% of the emulsion was composed of caseinate) was found to lower the apparent viscosity and the mean particle size d43 measured after heating. Fig. 2 also tells us that a Ca2+ concentration somewhere between 3.5 and 4 mM was required to give the same magnitude of viscosity increase for a b-lg-stabilized emulsion as was found in the presence of 30 mM KCl. The total ionic strength I from added salts is given by I¼
1X mi z2i , 2 i
(1)
where z is the valency of each ionic species i, and m the molar ionic concentration. The same concentration of divalent ions (Ca2+) therefore makes a substantially greater contribution to the ionic strength (and hence it affects the double-layer thickness and emulsion stability to a greater extent) than the monovalent ions (K+ and Cl). To assess whether the emulsion viscosity increase on heating had mainly changed because of a general ionic strength effect, or as a result of a specific calcium-binding effect, or due to a combination of the two, the CaCl2 and KCl were added together at intermediate concentrations to some b-lg-stabilized emulsion samples. The inferred state of aggregation induced on heating in the absence of casein is shown by the viscosity data in Fig. 3. The viscosity increase of the b-lg-stabilized emulsion in the presence of 4 mM Ca2+ can be seen to be intermediate in value between that for the 2 mM Ca2++20 mM KCl emulsion and that for the 2 mM Ca2++30 mM KCl emulsion. However, the extent of viscosity increase does not simply correlate with the change in overall ionic strength (as defined by Eq. (1)). If this were to be the case, the viscosity of the 4 mM Ca2+ sample would be expected to lie between the viscosities of the 2 mM Ca2+ and the 2 mM Ca2++10 mM K+ samples. The fact that aggregation proceeds to a greater extent than would be expected on the basis of the ionic strength alone
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Viscosity (Pa s)
Viscosity (Pa s)
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1 0.1 0.01
Concentration Ca2+ (mM)
2
2
2
2
4
Concentration K (mM)
0
10
20
30
0
Total ionic strength (mM)
6
16
26
36
12
+
Fig. 3. Influence of the combined presence of CaCl2 and KCl on the viscosity increase on heating (90 1C, 3 min) of a b-lg-stabilized emulsion (3 wt% protein, 45 vol% oil, pH 6.8). The total ionic strength is that calculated from Eq. (1).
indicates that the heat-induced flocculation increase of the 4 mM Ca2+ emulsion is due to a specific binding effect on top of the general background ionic strength effect. The concentration of 4 mM CaCl2 in our systems corresponds to around 2–3 calcium ions per b-lg molecule. This is despite there being no casein present in the emulsions whose behaviour is considered in Fig. 3. These results confirm work by Agboola and Dalgleish (1995) who found that Ca2+ could also bind to b-lg (although more weakly) as well as to casein; this binding was attributed to an electrostatic interaction with the aspartic and glutamic acid side-chains. In mixed milk protein systems, therefore, calcium ions can bind to both the caseins and the whey proteins when in the adsorbed state. In our own systems, the protective ability of as1-casein might be expected to be compromised to a greater degree by Ca2+-binding than that of b-casein, since the former contains a greater number of phosphoserine residues. However, the amount of free Ca2+ measured with a calcium-sensitive electrode in our b-lgstabilized emulsions in the presence of 0.15 wt% sodium caseinate, 0.15 wt% b-casein or 0.15 wt% as1-casein was found to be very low (o0.1 mM)—especially once the samples had been heated. This means that the calcium present in our emulsion systems was predominantly in the bound state. In practice, therefore, at the low levels of casein present in our systems, it appears that the as1-casein does not bind significantly more Ca2+ than either b-casein or sodium caseinate (within the experimental error). Consequently, as illustrated in Fig. 4, the order of efficiency of the protective stabilizing effect for the two pure caseins (b-casein4as1-casein) remains the same as in the absence of added ionic calcium. Once sufficient casein is present to completely inhibit the viscosity increase (5% casein addition), both b-casein and as1-casein lower the viscosity down to the value recorded before any heating of the emulsion (i.e.,o0.5 Pa s). The proportion of b-casein or as1-casein required to inhibit the heat-induced flocculation was found to increase
10
1
0.1 0 (A)
0 (B)
1
2.5
5
% Casein (of total protein) Fig. 4. Effect of 4 mM CaCl2 on the apparent viscosity of a 3 wt% b-lgstabilized emulsion (45 vol% oil, pH 6.8) after heating at 90 1C for 3 min. The first two (grey) columns represent b-lg-stabilized emulsions in the absence of casein: (A) 0 mM Ca2+ (d 43 ¼ 4:1 mm); (B) 4 mM Ca2+ (d 43 ¼ 90 mm). Filled (black) columns represent the addition of b-casein and unfilled (white) columns represent the addition of as1-casein in the presence of 4 mM Ca2+.
in the presence of 4 mM Ca2+ to a value of 5% replacement of b-lg (from 0.75% and 2.5% replacement for b-casein and as1-casein, respectively, in the absence of calcium). A plausible explanation for the increased amount of casein required to eliminate emulsion sensitivity to heating in the presence of Ca2+ ions is the partial collapse of the casein tails, as already mentioned above. But, the fact that the casein addition does improve the heat stability under these conditions indicates that even the partially collapsed casein layer must still form a reasonably thick protective layer at the interface, as suggested by Horne and Leaver (1995). Nevertheless, the reduced steric stabilizing capacity of casein polymer tails (and loops) due to the presence of the bound Ca2+ ions means that more of them per unit area are required to protect the emulsion droplets against heat-induced aggregation. As the systems studied here contain only a small fraction of casein (0–0.15 wt%), this means that any phosphoserine residues on the caseins would have become fully saturated with Ca2+. Therefore, following Agboola and Dalgleish (1995), it seems likely that the involvement of the weaker binding sites on the more numerous adsorbed b-lg molecules is also responsible for the enhanced viscosity increase of the emulsion containing the added CaCl2, as compared with the emulsion of equivalent ionic strength in the absence of any specific Ca2+-binding effect. 3.2. Effect of order of addition of casein on its synergistic stabilizing capability Previous work by Kim, Decker, and McClements (2004) on b-lg-stabilized emulsions has shown that the exact time when additional protein (b-lg) is added can be an important factor affecting the extent of subsequent aggregation. Therefore, it is possible that the stage/time at which casein is included in the mixed systems studied
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here could also be important in relation to the degree of flocculation induced by heating. In our previously reported experiments on emulsions containing a commercial WPI ingredient and sodium caseinate in the absence of salt (Dickinson & Parkinson, 2004), we mixed the two aqueous protein solutions in the appropriate ratio before homogenization so that the two protein types were free to adsorb together at the oil–water interface during emulsification. Here, we study the effect of replacing 5% WPI by caseinate (i.e., 0.15 wt% caseinate) at various stages during the processes of emulsion preparation, heating, cooling and short-term storage. This particular concentration of caseinate was chosen as it had been demonstrated previously (Dickinson & Parkinson, 2004) to be sufficient to give complete inhibition of heat-induced aggregation, when the system was heated at 90 1C for 3 min. Fig. 5 shows that, so long as the sodium caseinate was added to the WPI-stabilized emulsion before heating (samples B–F), the viscosity remained low, indicating that the casein addition prevents any heat-induced aggregation of the WPI-stabilized emulsion. Comparing samples B and C, addition of caseinate (0.15 wt%) before or after homogenization had no significant effect on the final viscosity obtained after heating. Interestingly, when casein was added to the emulsion immediately after heating (sample G), the viscosity did increase slightly, but it was still much lower than that for the emulsion heated in the absence of caseinate (sample A). Presumably, therefore, the casein must still be able to disrupt some aggregates that form on heating when added at this late stage, or alternatively its presence can prevent further aggregation on cooling of the emulsion. As shown in Fig. 5, the viscosity progressively increased (samples G–I) as the time elapsed after heating, at which the casein was added, was extended to 30 min. Subsequent storage of sample I for a further 60 min (sample J) led to recovery of the original 0.9 0.8
99
viscosity obtained in the absence of casein (sample A), suggesting that the ageing of the adsorbed protein layer changes the interactions between the droplets. These findings are consistent with those of Kim et al. (2004), who found that addition of extra b-lg to a 24 h-old b-lgstabilized emulsion could not lower the extent of aggregation as was found to be possible with protein addition to the fresh emulsion. Not unexpectedly, therefore, it would appear that, once the droplets have already become extensively aggregated, they cannot be readily redispersed through the addition of a small amount of casein. Fig. 6 shows the effect of the order of casein addition on the heat-induced flocculation of a b-lg-stabilized emulsion in the presence of 30 mM KCl. We see that sodium caseinate does have some protective effect, as compared with the pure b-lg-stabilized emulsion (sample A), when added before homogenization (sample B), but the effect is even greater when the caseinate is added after homogenization (sample C). One possible explanation is that the homogenization procedure somehow alters the conformation of the casein, so that the disordered protein behaves less effectively as a steric stabilizer; but this seems unlikely due to the excellent protection offered by casein(ate) to the WPI emulsion before homogenization in the absence of added KCl (Fig. 5). A more plausible alternative explanation lies in the difference in the electrostatic contribution to the droplet–droplet interactions. Addition of electrolyte reduces the thickness of the electrical double-layer and therefore diminishes the magnitude of the electrostatic repulsive force that helps to keep emulsion droplets apart. Increase in ionic strength therefore affects the delicate balance of the interdroplet attractive and repulsive forces (Parkinson et al., 2005) with the result that casein is unable to completely inhibit the viscosity increase on heating, when 30 mM KCl is present. The fact that addition of casein after homogenization provides more effective stabilization than addition before homogenization may be explained by the casein adsorbing on top of the established layer of b-lg molecules, and
0.6 0.5 0.4 0.3 0.2 0.1 0.0 A
B
C
D
E
Casein before heating
F
G
H
I
J
Casein after heating
Fig. 5. Effect on the apparent viscosity after heating (90 1C, 3 min) of the time at which sodium caseinate (0.15 wt%) is added to a WPI-stabilized emulsion (3 wt% protein, 45 vol% oil, pH 6.8): A, no caseinate added; B, before homogenization (HG); C, immediately after HG; D, 10 min after HG; E, 30 min after HG; F, 1 h after HG; G, immediately after heat treatment (emulsion still hot); H, 10 min after heat treatment (emulsion cold); I, 30 min after heat treatment (emulsion cold); J, 30 min after heat treatment, and ageing for 1 h.
Viscosity (Pa s)
Viscosity (Pa s)
0.7 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
A
B
C
D
Fig. 6. Effect on the apparent viscosity after heating (90 1C, 3 min) of the time at which sodium caseinate (0.15 wt%) is added to a b-lg-stabilized emulsion (3 wt% protein, 45 vol% oil, pH 6.80) containing 30 mM KCl added after homogenization (HG): A, no caseinate added; B, before HG; C, immediately after HG; D, 10 min after heating (emulsion cooled).
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therefore extending even further into the aqueous phase, and making the copolymer an even more effective steric stabilizer (Parkinson et al., 2005). On the other hand, once the emulsion droplets have already aggregated via strong b-lgb-lg bonds in the absence of casein, and the emulsion has been cooled, the addition of casein is unable to break up these flocs, and so the viscosity remains high (sample D in Fig. 6). In the pure b-lg-based emulsions investigated here, around 70% of the protein was in the non-adsorbed state before heating, and this reduced to around 30% after heating (Parkinson & Dickinson, 2004). Based on the work of Euston and coworkers (Euston, Finnigan, & Hirst, 2000), we would expect the non-adsorbed whey protein fraction to make an overwhelming contribution to the heat-induced emulsion flocculation mechanism. Indeed, it has been found (Parkinson, 2005) that, when all the nonadsorbed protein was removed from an emulsion sample (3 wt% b-lg, 45 vol% oil, 30 mM KCl) by centrifugation before heating, the viscosity increase after heating was very low (three orders of magnitude smaller than when the nonadsorbed protein was present). By trial and error, it was found that the addition of ca. 250 mM KCl to this centrifuged emulsion was sufficient to cause a similar degree of viscosity increase as with the standard pure b-lg emulsion (30 mM KCl) containing 70% non-adsorbed protein. Repeating this same experiment with 2.94 wt% b-lg+0.06 wt% caseinate (i.e., just 2% b-lg replaced by caseinate) led to the complete elimination of the heatinduced viscosity increase of the centrifuged emulsion containing 250 mM KCl (Parkinson, 2005). So, although the non-adsorbed b-lg can contribute very importantly to the heat-induced aggregation of whey protein emulsions, the protective effect of caseinate does operate in the absence of any non-adsorbed whey protein. This is again consistent with our view that it is casein in the adsorbed state which is predominantly responsible for the casein stabilizing mechanism, and not some sort of complex between a component of the caseinate (e.g., k-casein) and unadsorbed b-lg in the continuous phase. In summarizing this section, we conclude that the ability of casein added to a WPI-based or b-lg-based emulsion to exert a protective effect on its heat stability (90 1C, 3 min) depends on the stage at which the disordered protein is added. Generally speaking, if the casein is added before heating, the degree of heat-induced aggregation is reduced compared to an emulsion prepared in the absence of casein. However, depending on the age of the emulsion, some increase in stability can still be achieved when casein is added after heating. This suggests that additional flocculation may be taking place on cooling of the emulsions, and that the introduction of caseinate even at this late stage prevents further droplet–droplet association. We note that a somewhat analogous effect occurs when the rigidity of a b-lg-stabilized (emulsion) gel increases strongly on cooling as a result of enhanced hydrogen bonding and disulphide cross-linking (Dickinson & Hong, 1995, 1997).
3.3. Effect of casein in inhibiting heat-induced destabilization of emulsions made with other globular milk proteins When bovine serum albumin (BSA) was used instead of b-lg as the primary emulsifier under the same conditions (3 wt% protein, 45 vol% oil, 30 mM KCl, pH 6.8, 90 1C 3 min), the degree of heat-induced aggregation, as measured by the increase in viscosity, was higher (16 Pa s) than for the equivalent b-lg-stabilized system (1 Pa s). Lowering the KCl concentration to 10 mM in the BSAbased emulsion was found to reduce by 50% the viscosity increase in the absence of casein (i.e. to 8 Pa s). At this ionic strength, the potential stabilizing effect of added casein was investigated for sodium caseinate, b-casein and as1-casein. Fig. 7 shows that, although the different casein ingredients were all able to provide effective protection, higher concentrations (0.3–0.6 wt% protein) were required to completely inhibit the viscosity increase than for the equivalent b-lg-stabilized system. Even when the ionic strength was lowered to 2.5 mM (so that the original viscosity after heating in the absence of casein was just 1 Pa s), we found that 40.3 wt% addition of casein (i.e., replacement of 10% of the BSA by caseinate) was still required to provide complete inhibition of the heat-induced aggregation (results not shown). In addition, it seems that with BSA, the protective effect is less sensitive to the casein type; Fig. 7 shows only minor differences in viscosity for the systems containing added as1-casein (A), b-casein (B), or sodium caseinate (C). The difference in sensitivity to added casein of the heat stability of BSA-based and b-lg-based emulsions may simply be related to differences in globular protein molecular mass. The b-lactoglobulin monomer (1.8 104 Da) gives an adsorbed layer thickness of approximately 2 nm (Atkinson, Dickinson, Horne, & Richardson, 1995; Caldwell, Li, Li, & Dalgleish, 1992), whereas the larger size of the BSA molecule (6.6 104 Da) gives this globular protein an adsorbed layer thickness of approximately 4 nm (Lu, Su, & Thomas, 1999). For
10 Viscosity (Pa s)
100
8 6 4 2 C 0
B 0 5 10 % Casein (in total p ro
A 20 tein)
Fig. 7. Influence of casein type and concentration on apparent viscosity of BSA-stabilized emulsion (45 vol% oil, pH 6.8, 3 wt% total protein, 10 mM KCl) following heating at 90 1C for 3 min: (A) as1-casein; (B) b-casein; (C) sodium caseinate.
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reasons explored in detail elsewhere (Parkinson et al., 2005), this could mean that a greater density of casein polymer tails are required to confer heat-induced stability to BSA-stabilized emulsion droplets. When a-la was used instead of b-lg as the primary emulsifying agent, and the stability experiments in 30 mM KCl were repeated, no heat-induced flocculation after 3 min at 90 1C could be detected by either light scattering or rheological measurements. Even increasing the heating time to 10 min (30 mM KCl, pH 6.8, 90 1C) caused no apparent aggregation of the emulsion (Fig. 8). A comparable viscosity increase to that of a b-lg and WPI emulsion heated for 3 min at 90 1C (in the range 1–10 Pa s) could only be induced by increasing the temperature to 99 1C, the ionic strength to 100 mM, and the heating period to 6 min. However, Fig. 8 shows that the addition of 0.3 wt% sodium caseinate (i.e., 10% replacement of a-la by casein) in this case does not protect the a-la-stabilized emulsion from the resulting heatinduced viscosity increase. Presumably under these severe conditions of high temperature and ionic strength, the casein tails are unable to provide sufficient steric repulsion to prevent aggregation of the emulsion droplets. This qualitative difference in behaviour between a-la and b-lg (or BSA) may not be too surprising, given the extensive literature on the thermal aggregation and gelation of these different globular milk proteins in aqueous solution (Singh & Havea, 2003). One obvious difference from b-lg (and BSA) is that the a-la molecule has no free SH group. As a result, partially unfolded a-la molecules are unable to cross-link covalently amongst themselves (Schokker, Singh, & Creamer, 2000), although oligomers can be formed in the presence of b-lg due to disulphide exchange reactions (Livney, Verespej, & Dalgleish, 2003).
whey protein emulsion system. The physical stability of a b-lg-stabilized emulsion before and after heating (90 1C, 3 min), and in the presence and absence of sodium caseinate (1% replacement, or 0.03 wt% of the total emulsion), was studied by observing the visible development of phase separation on extended storage at room temperature (20–25 1C). Fig. 9 shows the proportion (vol%) of the sample present as the serum layer after 1, 3, 8 and 17 months of quiescent storage, and Fig. 10 shows photographs of the samples after 17 months of storage. It is clear that the pure b-lg-stabilized emulsion was more susceptible to long-term phase separation than the equivalent emulsion containing just 0.03 wt% caseinate, either with or without prior heat treatment. However, as these emulsions were stored in air-tight tubes without preservative present, the role of microbial activity in the latter samples cannot be ruled out. Furthermore, whilst the b-lg-stabilized emulsion was clearly visibly affected by heat treatment (as shown by the higher proportion of serum layer in the heated emulsion compared to the untreated emulsion), it can be seen that there was relatively little difference in the appearance of heated and untreated emulsion samples containing 2.97 wt% b-lg+0.03 wt% caseinate. Qualitatively similar results were also found with emulsions made with WPI instead of b-lg. So it seems that heating in the presence of casein may have actually led to an increase in the stability of the emulsion after several months storage at room temperature, as also reported by Srinivasan, Singh, and Munro (2002). These findings might be explained by the increased viscosity of the continuous phase on heating—and also the larger surface load, which would mean that the effective density of the droplets is increased, and so the rate of creaming is slowed. From these results, we can conclude that adding just 0.03 wt%
3.4. Effect of small additions of casein on long-term stability of a b-lg-stabilized emulsion
B
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Fig. 8. Influence of heating conditions, ionic strength and addition of sodium caseinate on the viscosity of a-lactalbumin-stabilized emulsion (45 vol% oil, 3 wt% total protein, pH 6.8): (A) 3 wt% a-la, 30 mM KCl, 90 1C, 10 min; (B) 3 wt% a-la, 100 mM KCl, 99 1C, 6 min; (C) 2.7 wt% a-la+0.3 wt% caseinate, 100 mM KCl, 99 1C, 6 min.
Fig. 9. Extent of phase separation following quiescent storage of b-lg-stabilized emulsions (3 wt% protein, 45 vol% oil, 30 mM KCl, pH 6.8) before and after heating (90 1C) and with or without 1% replacement of b-lg by sodium caseinate (i.e., 0.03 wt% caseinate): A, non-heated emulsion in the absence of caseinate; B, heated emulsion in the absence of caseinate; C, non-heated emulsion in the presence of caseinate; D, heated emulsion in the presence of caseinate.
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Fig. 10. Effect of heat treatment (90 1C, 3 min) and addition of 0.03 wt% sodium caseinate (1% of total protein present) on visual appearance of emulsion samples (3 wt% b-lg, 45 vol% oil, pH 6.8, 30 mM KCl) stored quiescently in tubes of height 75 mm at ambient temperature for 17 months: A, 3 wt% b-lg untreated emulsion; B, 3 wt% b-lg heat-treated emulsion; C, 2.97 wt% b-lg+0.03 wt% caseinate untreated emulsion; D, 2.97 wt% b-lg+0.03 wt% caseinate heat-treated emulsion.
caseinate to a concentrated heat-treated b-lg-stabilized emulsion can improve its long-term storage stability. 4. Conclusions The synergistic protective effect of small additions of casein to a b-lg-stabilized emulsion (3 wt% protein, 45 vol% n-tetradecane, pH 6.8), as established previously (Parkinson and Dickinson, 2004) at low ionic strength (0–50 mM KCl), has been found to be sensitive to changes in various conditions. In the absence of any added casein, b-lg-stabilized emulsions were less stable in the presence of Ca2+ ions (as shown by the increase in viscosity on heating). Comparable experiments with K+ and Ca2+ ions in combination has shown that some destabilization due to specific calcium binding can be inferred. This conclusion is reinforced by the very low measured values of free ionic calcium in the heated emulsions. Nevertheless, moderate Ca2+ addition was found not to inhibit the protective effect of the caseins, with the same order of efficiency operating (b-casein4sodium caseinate4as1-casein). However, slightly more casein was required for full heat stability than in the absence of added CaCl2. Since most dairy products do contain calcium salts, the colloidal stabilizing capacity of casein in the presence of a moderate content of ionic calcium seems technologically noteworthy. This synergistic protective effect due to small additions of casein is fairly robust to processing changes relating to the stage at which the disordered protein is incorporated in the whey protein-based emulsion formulation. For a WPIstabilized emulsion with no salt, protection was evident at 0.15 wt% sodium caseinate addition so long as the casein was added before the heating stage, either before or after homogenization. Furthermore, a partial protective effect was also observed when sodium caseinate was added
immediately after heating, indicating that casein may prevent droplet aggregation that occurs on cooling. However, ageing of the emulsion after heating (before casein addition) diminishes the protective effect of the casein polymer. This implies that, once droplets have become aggregated by denatured whey protein, they cannot be redispersed simply by means of casein addition. The impressive protective effect at very low casein contents, as found with b-lg and WPI emulsions, did not appear to extend to all systems based on globular milk protein ingredients. With BSA as primary emulsifier, significantly more caseinate was found to be required (0.3 wt% protein or 10% replacement of BSA by caseinate) for complete protection from heat-induced changes. For a-la, no protective effect of casein at these same levels of addition could be found, possibly due to the more extreme conditions required to induce the viscosity increase. Finally, we have shown that casein addition can inhibit phase separation of b-lg-stabilized emulsions during long-term storage (up to 1.5 years). Furthermore, this enhanced stability may be increased further by thermal processing. The extra stabilizing effect of heating in the presence of casein opens up the possibility of using sodium caseinate to add to whey protein-stabilized emulsions for improvement in shelf-life of products such as imitation creams and whipped toppings. Acknowledgment E.P. gratefully acknowledges receipt of a BBSRC Industrial CASE Studentship in collaboration with the Campden and Chorleywood Food Research Association (CCFRA). References Agboola, S. O., & Dalgleish, D. G. (1995). Calcium-induced destabilization of oil-in-water emulsions stabilized by caseinate or by b-lactoglobulin. Journal of Food Science, 60, 399–404. Atkinson, P. J., Dickinson, E., Horne, D. S., & Richardson, R. M. (1995). Neutron reflectivity of adsorbed b-casein and b-lactoglobulin at the air–water interface. Journal of the Chemical Society, Faraday Transactions, 91, 2847–2854. Burgaud, I., Dickinson, E., & Nelson, P. V. (1990). An improved high-pressure homogenizer for making fine emulsions on a small scale. International Journal of Food Science and Technology, 25, 39–46. Caldwell, K. D., Li, J., Li, J.-T., & Dalgleish, D. G. (1992). Adsorption behaviour of milk proteins on polystyrene latex. A study based on sedimentation field-flow fractionation and dynamic light scattering. Journal of Chromatography, 604, 63–71. Dalgleish, D. G. (1997). Adsorption of protein and the stability of emulsions. Trends in Food Science and Technology, 8, 1–6. Dalgleish, D. G., & Parker, T. G. (1980). Binding of calcium ions to bovine as1-casein and precipitability of the protein calcium ion complexes. Journal of Dairy Research, 47, 113–122. Dickinson, E. (1989). Surface and emulsifying properties of caseins. Journal of Dairy Research, 56, 471–477. Dickinson, E. (1999). Caseins in emulsions: Interfacial properties and interactions. International Dairy Journal, 9, 305–312.
ARTICLE IN PRESS E.L. Parkinson, E. Dickinson / International Dairy Journal 17 (2007) 95–103 Dickinson, E., & Davies, E. (1999). Influence of ionic calcium on stability of sodium caseinate emulsions. Colloids and Surfaces B, 12, 203–212. Dickinson, E., & Hong, S.-T. (1995). Influence of water-soluble nonionic emulsifier on the rheology of heat-set protein-stabilized emulsion gels. Journal of Agricultural and Food Chemistry, 43, 2560–2566. Dickinson, E., & Hong, S.-T. (1997). Influence of an anionic surfactant on the rheology of heat-set b-lactoglobulin-stabilized emulsion gels. Colloids and Surfaces A, 127, 1–10. Dickinson, E., & Parkinson, E. L. (2004). Heat-induced aggregation of milk protein-stabilized emulsions: Sensitivity to processing and composition. International Dairy Journal, 14, 635–645. Euston, S. R., Finnigan, S. R., & Hirst, R. L. (2000). Aggregation kinetics of heated whey protein-stabilized emulsions. Food Hydrocolloids, 14, 155–161. Horne, D. S., & Leaver, J. (1995). Milk proteins on surfaces. Food Hydrocolloids, 9, 91–95. Kim, H.-J., Decker, E. A., & McClements, D. J. (2004). Influence of free protein on flocculation stability of b-lactoglobulin-stabilized oil-inwater emulsions at neutral pH and ambient temperature. Langmuir, 20, 10394–10398. Livney, Y. D., Verespej, E., & Dalgleish, D. G. (2003). Steric effects governing disulfide bond interchange during thermal aggregation in solutions of b-lactoglobulin B and a-lactalbumin. Journal of Agricultural and Food Chemistry, 51, 8098–8106. Lu, J. R., Su, T. J., & Thomas, R. K. (1999). Structural conformation of bovine serum albumin layers at the air–water interface studied by neutron reflection. Journal of Colloid and Interface Science, 213, 426–437.
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Parker, T. G., & Dalgleish, D. G. (1981). Binding of calcium ions to bovine b-casein. Journal of Dairy Research, 48, 71–76. Parkinson, E.L. (2005). Protective effect of casein on the thermal stability of whey protein-stabilized emulsions. Ph.D. thesis, University of Leeds. Parkinson, E. L., & Dickinson, E. (2004). Inhibition of heat-induced aggregation of a b-lactoglobulin-stabilized emulsion by very small additions of casein. Colloids and Surfaces B, 39, 23–30. Parkinson, E. L., & Dickinson, E. (2006). Understanding the stabilizing property of casein in heated milk protein emulsions. In P. A. Williams, & G. O. Phillips (Eds.), Gums and stabilizers for the food industry, vol. 13. Cambridge, UK: Royal Society of Chemistry (in press). Parkinson, E. L., Ettelaie, R., & Dickinson, E. (2005). Using selfconsistent-field theory to understand enhanced steric stabilization by casein-like copolymers at low surface coverage in mixed protein layers. Biomacromolecules, 6, 3018–3029. Schokker, E. P., Singh, H., & Creamer, L. K. (2000). Heat-induced aggregation of b-lactoglobulin A and B with a-lactalbumin. International Dairy Journal, 10, 843–853. Singh, H., Havea, P., 2003. Thermal denaturation, aggregation and gelation of whey proteins. In P. F. Fox, & P. L. H. McSweeney (Eds.), Advanced dairy chemistry, (3rd ed.) vol. 1: Proteins, pp. 1261–1287. New York, USA: Kluwer/Plenum. Srinivasan, M., Singh, H., & Munro, P. A. (2002). Formation and stabilization of sodium caseinate emulsions: Influence of retorting (121 1C for 15 min) before or after emulsification. Food Hydrocolloids, 16, 153–160.
International Dairy Journal 17 (2007) 95–103 www.elsevier.com/locate/idairyj
Synergistic stabilization of heat-treated emulsions containing mixtures of milk proteins Emma L. Parkinson, Eric Dickinson Procter Department of Food Science, University of Leeds, Leeds LS2 9JT, UK Received 15 September 2005; accepted 15 January 2006
Abstract The synergistic effect by which a very small amount of casein can confer stability to a whey protein-stabilized emulsion heated to 90 1C has been investigated. Using b-lactoglobulin (b-lg) as the main emulsifying agent, the extent of heat-induced flocculation increased with ionic strength, with commercial sodium caseinate or b-casein incorporated. The protective effect of casein was retained for a moderate concentration of ionic calcium. Casein added before heating, or shortly afterwards (i.e. before the emulsion had cooled), offered substantial synergistic protection to the heated b-lg-stabilized emulsion. With a-lactalbumin (a-la) as the primary emulsifying agent, no significant protective effect could be observed. In contrast, casein could confer significant stability to a heat-treated bovine serum albumin emulsion. Quiescent storage stability testing suggests that a combination of limited heating and casein addition could improve the long-term shelf-life of a whey protein-based emulsion. r 2006 Elsevier Ltd. All rights reserved. Keywords: Caseinate; Emulsion stability; Heat denaturation; b-Lactoglobulin; Flocculation; Viscosity; Whey protein
1. Introduction The excellent emulsifying and stabilizing properties of adsorbed layers of individual caseins and sodium caseinate have been well established for many years (Dickinson, 1989, 1999). More recently, experiments have shown (Dickinson & Parkinson, 2004; Parkinson & Dickinson, 2004) that casein polymers are also able to exert a protective stabilizing effect when present in very small amounts (0.03–0.15 wt% of the total emulsion) in whey protein-stabilized emulsions subjected to thermal treatment (e.g., at 90 1C for 3 min). Based on a combination of emulsion viscometry and particle-size measurements, this stabilizing phenomenon has been confirmed for emulsions based on either b-lactoglobulin (b-lg) or a commercial whey protein isolate (WPI) upon the incorporation of very low concentrations of sodium caseinate, b-casein or as1-casein. The exact amount of casein required for inhibition of droplet flocculation on heating depends on Corresponding author. Tel.: +44 113 243 1751; fax: +44 113 343 2982.
E-mail address: [email protected] (E. Dickinson). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.01.010
the purity of the whey protein used; but, in general, the protective effect was found to follow the order b-casein4sodium caseinate4as1-casein. The stabilizing effect of the casein in these mixed milk protein systems is strongly synergistic. This is because the casein polymer appears to be acting in a colloidal stabilizing capacity at a surface concentration very much lower than that at which it could be used as an emulsifying or stabilizing agent simply on its own. Using protein analytical techniques (reverse-phase-high-performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDSPAGE)), we have confirmed experimentally (Parkinson & Dickinson, 2006) that all of the casein present in these systems is indeed located at the surface of the emulsion droplets. Although at first rather surprising, the potential ability of such a low-surface coverage of casein to protect whey protein-coated droplets against heat-induced aggregation can be adequately explained in terms of a model of steric stabilizing casein-like copolymers based on Scheutjens–Fleer self-consistent-field theory (Parkinson, Ettelaie, & Dickinson, 2005).
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In the present paper, we address some issues relating to the likely generality and range of applicability of this synergistic stabilization phenomenon. In particular, we report on new experiments designed to provide answers to four specific questions: (1) How does ionic strength or calcium ion concentration affect the ability of casein to protect b-lactoglobulin emulsions against heat-induced destabilization? (2) To what extent does the order of addition of the casein (i.e., before/after homogenization, before/after heating) affect its stabilizing capability? (3) Can casein also be effective in providing synergistic protection to heated emulsions prepared with other individual globular milk proteins, e.g., bovine serum albumin (BSA) or a-lactalbumin? (4) What is the influence of a small amount of added casein on the long-term storage stability of a whey proteinbased emulsion?
2. Materials and methods Spray-dried sodium caseinate (482 wt% dry protein, o6 wt% moisture, according to the manufacturer) containing 800 ppm Ca, was obtained from DeMelkindustrie (Veghel, The Netherlands). Pure b-lg, BSA and a-lactalbumin (a-la) were obtained from Sigma Chemicals (St. Louis, MO, USA). A WPI sample (BiPRO, 90% protein) was supplied by Davisco Foods International (Le Sueur, MN, USA). The b-casein and as1-casein were obtained as freeze-dried samples (498% purity) from the Hannah Research Institute (Ayr, Scotland). Potassium chloride (99%), n-tetradecane (99%), imidazole (99%) and dihydrated calcium chloride (99.5%) were purchased from Sigma Chemicals. Aqueous solutions of (i) b-lg and (ii) sodium caseinate, b-casein or as1-casein were separately prepared by dissolving the proteins in 20 mM imidazole buffer (pH 6.8), containing 0–50 mM KCl and/or 0–5 mM CaCl2, with continuous stirring overnight. Oil-in-water emulsions of constant total protein content (3 wt% protein, 45 vol% oil), but differing relative proportions of casein and whey protein, were prepared by high-pressure homogenization at 300 bar using a home-built jet homogenizer (Burgaud, Dickinson & Nelson, 1990). After a 10 min waiting period following homogenization, the emulsions were heated at 90 1C (70.2 1C) for 3 min without stirring in sealed tubes (capacity 5 mL) contained in a water bath before being rapidly cooled to room temperature by placing the tubes in ice water for a short time. The choice of a small sample volume was to ensure a relatively short thermal equilibration time (90 s). Rheological testing was carried out within 1 h of the original emulsification stage. Large-deformation steadystate shear rheology was performed at 25 1C with a Bohlin CVO rheometer (Bohlin Instruments, Cirencester, UK)
using the concentric cell geometry (C14). The sample (ca. 5 mL) was covered with a thin layer of silicone oil to prevent evaporation, and then allowed to equilibrate at 25 1C for 10 min, prior to viscosity measurement at a shear rate of 5 s1. Numerical values of viscosities quoted below refer to average measurements taken after 2 min of constant shearing at 5 s1. All average values quoted are based on (at least) duplicate results. For the data presented here and elsewhere (Dickinson & Parkinson, 2004; Parkinson & Dickinson, 2004), there was up to 20% variability in the measured emulsion viscosity values, with greater variability typically encountered at the higher degrees of aggregation. Droplet-size distributions of the heated emulsions were determined using a Malvern Mastersizer MS2000 (Malvern Instruments, Malvern, UK) with refractive index ratio ¼ 1.074 and absorbance ¼ 0.005. Varying amounts of calcium chloride dihydrate (CaCl2 2H2O) were added to emulsions prior to emulsification (to ensure uniform distribution of Ca2+). Additions of casein were mainly made after homogenization. The free calcium ion concentration of the emulsions was determined before and after heating (90 1C, 3 min) using a Jenway calcium combination ion-selective electrode (Barloworld Scientific, Dunmow, Essex, UK) and a Jenway 3310 pH meter. Long-term storage stability under ambient conditions was assessed for emulsions containing b-lg (or WPI) alone and b-lg (or WPI)+sodium caseinate. Untreated samples and those that had been heated (and subsequently cooled) were stored in sealed 75 12 mm sample tubes for various lengths of time up to 17 months. The thicknesses of cream and serum layers were measured periodically and expressed as percentages of the total height of the emulsion. 3. Results and discussion 3.1. Effect of ionic strength and calcium ion content on ability of casein to protect b-lg emulsions against heatinduced destabilization The stabilizing effect of small additions of sodium caseinate on a b-lg-stabilized emulsion that was heated at 90 1C for 3 min has been investigated as a function of ionic strength (0–50 mM KCl). The apparent viscosity at 5 s1 and 25 1C was used to monitor the extent of heat-induced flocculation. The results are summarized in Fig. 1. Based on our previous experience with similar systems (Parkinson & Dickinson, 2004), we adopted the arbitrary benchmark criterion of an apparent viscosity above 0.5 Pa s as denoting an emulsion that is deemed ‘unstable’ to heating at 90 1C for 3 min, whilst a viscosity below 0.5 Pa s denotes an emulsion that is ‘stable’ to heating. Also indicated in Fig. 1 are intermediate states where the measured viscosity was 0.5 Pa s after heating. From this diagram, it is clear that emulsion stability improves as the proportion of sodium caseinate incorporated in the emulsion increases and as the ionic strength decreases. We also see that a similar trend is
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% Casein in protein mixture Fig. 1. Stability map of b-lg-stabilized emulsions (3 wt% protein, 45 vol% oil, pH 6.8) with different KCl contents in the presence of small additions of sodium caseinate or b-casein (expressed as a percentage of the total protein present) after heating for 3 min at 90 1C. Depending on the measured apparent viscosity after heating, samples may be assigned to different categories: m, unstable with caseinate or b-casein; D, unstable with caseinate, stable with b-casein; ., unstable with caseinate, intermediate state with b-casein; ’, intermediate state with caseinate, stable with b-casein; O, stable with caseinate and b-casein.
apparent for the case of equivalent small additions of b-casein to a similar b-lg emulsion heated (90 1C, 3 min) at varying ionic strengths, i.e., the stability was found to be minimal for low casein additions and for high ionic strengths. For pure b-casein, however, it appears that less casein polymer (p5% addition) is required to confer stability than with commercial sodium caseinate (p10% addition). Casein is a calcium-sensitive protein, and this sensitivity is temperature dependent (Dalgleish & Parker, 1980; Parker & Dalgleish, 1981). Specific calcium ion binding at neutral pH lowers the net negative charge on the protein (and hence at the surface of protein-coated emulsion droplets), since the positively charged Ca2+ ions bind to the anionic phosphoseryl residues on b- and as1-casein (5 and 8 per molecule, respectively). This lowered net charge reduces the level of electrostatic repulsion between protein-coated layers, thereby promoting an increased degree of emulsion flocculation (Dickinson & Davies, 1999). The level of steric stabilization may also be diminished by the consequent partial collapse of the adsorbed casein layer on calcium ion addition (Dalgleish, 1997), and so a combination of these effects could act to prevent the stabilizing action of small additions of casein to a b-lg-stabilized emulsion. Some preliminary experiments were carried out with a 3 wt% b-lg-stabilized emulsion (45 vol% oil, pH 6.8) to determine the concentration of CaCl2 required to cause the same extent of heat-induced aggregation (indicated by the relative increase in apparent viscosity) as when 30 mM KCl was present. It can be seen from Fig. 2 that the viscosity after heating increased strongly with calcium content in the range 3–4 mM, and for 5 mM Ca2+ the viscosity was too high to measure in the rheometer with the C14 geometry. Replacement of 5% of the b-lg by sodium
3
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Calcium chloride content (mM) Fig. 2. Influence of CaCl2 addition on the apparent viscosity at 5 s1 and 25 1C of b-lg-stabilized emulsions (3 wt% total protein, 45 vol% oil, pH 6.8) heated at 90 1C for 3 min. Filled (black) columns represent emulsions stabilized by 3 wt% b-lg. Unfilled (white) columns represent emulsions stabilized by 2.85% b-lg+0.15% caseinate. Each number at the top of the column indicates the mean particle size d43 (in mm) as measured by static light-scattering (Mastersizer).
caseinate (i.e., so that 0.15 wt% of the emulsion was composed of caseinate) was found to lower the apparent viscosity and the mean particle size d43 measured after heating. Fig. 2 also tells us that a Ca2+ concentration somewhere between 3.5 and 4 mM was required to give the same magnitude of viscosity increase for a b-lg-stabilized emulsion as was found in the presence of 30 mM KCl. The total ionic strength I from added salts is given by I¼
1X mi z2i , 2 i
(1)
where z is the valency of each ionic species i, and m the molar ionic concentration. The same concentration of divalent ions (Ca2+) therefore makes a substantially greater contribution to the ionic strength (and hence it affects the double-layer thickness and emulsion stability to a greater extent) than the monovalent ions (K+ and Cl). To assess whether the emulsion viscosity increase on heating had mainly changed because of a general ionic strength effect, or as a result of a specific calcium-binding effect, or due to a combination of the two, the CaCl2 and KCl were added together at intermediate concentrations to some b-lg-stabilized emulsion samples. The inferred state of aggregation induced on heating in the absence of casein is shown by the viscosity data in Fig. 3. The viscosity increase of the b-lg-stabilized emulsion in the presence of 4 mM Ca2+ can be seen to be intermediate in value between that for the 2 mM Ca2++20 mM KCl emulsion and that for the 2 mM Ca2++30 mM KCl emulsion. However, the extent of viscosity increase does not simply correlate with the change in overall ionic strength (as defined by Eq. (1)). If this were to be the case, the viscosity of the 4 mM Ca2+ sample would be expected to lie between the viscosities of the 2 mM Ca2+ and the 2 mM Ca2++10 mM K+ samples. The fact that aggregation proceeds to a greater extent than would be expected on the basis of the ionic strength alone
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Fig. 3. Influence of the combined presence of CaCl2 and KCl on the viscosity increase on heating (90 1C, 3 min) of a b-lg-stabilized emulsion (3 wt% protein, 45 vol% oil, pH 6.8). The total ionic strength is that calculated from Eq. (1).
indicates that the heat-induced flocculation increase of the 4 mM Ca2+ emulsion is due to a specific binding effect on top of the general background ionic strength effect. The concentration of 4 mM CaCl2 in our systems corresponds to around 2–3 calcium ions per b-lg molecule. This is despite there being no casein present in the emulsions whose behaviour is considered in Fig. 3. These results confirm work by Agboola and Dalgleish (1995) who found that Ca2+ could also bind to b-lg (although more weakly) as well as to casein; this binding was attributed to an electrostatic interaction with the aspartic and glutamic acid side-chains. In mixed milk protein systems, therefore, calcium ions can bind to both the caseins and the whey proteins when in the adsorbed state. In our own systems, the protective ability of as1-casein might be expected to be compromised to a greater degree by Ca2+-binding than that of b-casein, since the former contains a greater number of phosphoserine residues. However, the amount of free Ca2+ measured with a calcium-sensitive electrode in our b-lgstabilized emulsions in the presence of 0.15 wt% sodium caseinate, 0.15 wt% b-casein or 0.15 wt% as1-casein was found to be very low (o0.1 mM)—especially once the samples had been heated. This means that the calcium present in our emulsion systems was predominantly in the bound state. In practice, therefore, at the low levels of casein present in our systems, it appears that the as1-casein does not bind significantly more Ca2+ than either b-casein or sodium caseinate (within the experimental error). Consequently, as illustrated in Fig. 4, the order of efficiency of the protective stabilizing effect for the two pure caseins (b-casein4as1-casein) remains the same as in the absence of added ionic calcium. Once sufficient casein is present to completely inhibit the viscosity increase (5% casein addition), both b-casein and as1-casein lower the viscosity down to the value recorded before any heating of the emulsion (i.e.,o0.5 Pa s). The proportion of b-casein or as1-casein required to inhibit the heat-induced flocculation was found to increase
10
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0.1 0 (A)
0 (B)
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5
% Casein (of total protein) Fig. 4. Effect of 4 mM CaCl2 on the apparent viscosity of a 3 wt% b-lgstabilized emulsion (45 vol% oil, pH 6.8) after heating at 90 1C for 3 min. The first two (grey) columns represent b-lg-stabilized emulsions in the absence of casein: (A) 0 mM Ca2+ (d 43 ¼ 4:1 mm); (B) 4 mM Ca2+ (d 43 ¼ 90 mm). Filled (black) columns represent the addition of b-casein and unfilled (white) columns represent the addition of as1-casein in the presence of 4 mM Ca2+.
in the presence of 4 mM Ca2+ to a value of 5% replacement of b-lg (from 0.75% and 2.5% replacement for b-casein and as1-casein, respectively, in the absence of calcium). A plausible explanation for the increased amount of casein required to eliminate emulsion sensitivity to heating in the presence of Ca2+ ions is the partial collapse of the casein tails, as already mentioned above. But, the fact that the casein addition does improve the heat stability under these conditions indicates that even the partially collapsed casein layer must still form a reasonably thick protective layer at the interface, as suggested by Horne and Leaver (1995). Nevertheless, the reduced steric stabilizing capacity of casein polymer tails (and loops) due to the presence of the bound Ca2+ ions means that more of them per unit area are required to protect the emulsion droplets against heat-induced aggregation. As the systems studied here contain only a small fraction of casein (0–0.15 wt%), this means that any phosphoserine residues on the caseins would have become fully saturated with Ca2+. Therefore, following Agboola and Dalgleish (1995), it seems likely that the involvement of the weaker binding sites on the more numerous adsorbed b-lg molecules is also responsible for the enhanced viscosity increase of the emulsion containing the added CaCl2, as compared with the emulsion of equivalent ionic strength in the absence of any specific Ca2+-binding effect. 3.2. Effect of order of addition of casein on its synergistic stabilizing capability Previous work by Kim, Decker, and McClements (2004) on b-lg-stabilized emulsions has shown that the exact time when additional protein (b-lg) is added can be an important factor affecting the extent of subsequent aggregation. Therefore, it is possible that the stage/time at which casein is included in the mixed systems studied
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here could also be important in relation to the degree of flocculation induced by heating. In our previously reported experiments on emulsions containing a commercial WPI ingredient and sodium caseinate in the absence of salt (Dickinson & Parkinson, 2004), we mixed the two aqueous protein solutions in the appropriate ratio before homogenization so that the two protein types were free to adsorb together at the oil–water interface during emulsification. Here, we study the effect of replacing 5% WPI by caseinate (i.e., 0.15 wt% caseinate) at various stages during the processes of emulsion preparation, heating, cooling and short-term storage. This particular concentration of caseinate was chosen as it had been demonstrated previously (Dickinson & Parkinson, 2004) to be sufficient to give complete inhibition of heat-induced aggregation, when the system was heated at 90 1C for 3 min. Fig. 5 shows that, so long as the sodium caseinate was added to the WPI-stabilized emulsion before heating (samples B–F), the viscosity remained low, indicating that the casein addition prevents any heat-induced aggregation of the WPI-stabilized emulsion. Comparing samples B and C, addition of caseinate (0.15 wt%) before or after homogenization had no significant effect on the final viscosity obtained after heating. Interestingly, when casein was added to the emulsion immediately after heating (sample G), the viscosity did increase slightly, but it was still much lower than that for the emulsion heated in the absence of caseinate (sample A). Presumably, therefore, the casein must still be able to disrupt some aggregates that form on heating when added at this late stage, or alternatively its presence can prevent further aggregation on cooling of the emulsion. As shown in Fig. 5, the viscosity progressively increased (samples G–I) as the time elapsed after heating, at which the casein was added, was extended to 30 min. Subsequent storage of sample I for a further 60 min (sample J) led to recovery of the original 0.9 0.8
99
viscosity obtained in the absence of casein (sample A), suggesting that the ageing of the adsorbed protein layer changes the interactions between the droplets. These findings are consistent with those of Kim et al. (2004), who found that addition of extra b-lg to a 24 h-old b-lgstabilized emulsion could not lower the extent of aggregation as was found to be possible with protein addition to the fresh emulsion. Not unexpectedly, therefore, it would appear that, once the droplets have already become extensively aggregated, they cannot be readily redispersed through the addition of a small amount of casein. Fig. 6 shows the effect of the order of casein addition on the heat-induced flocculation of a b-lg-stabilized emulsion in the presence of 30 mM KCl. We see that sodium caseinate does have some protective effect, as compared with the pure b-lg-stabilized emulsion (sample A), when added before homogenization (sample B), but the effect is even greater when the caseinate is added after homogenization (sample C). One possible explanation is that the homogenization procedure somehow alters the conformation of the casein, so that the disordered protein behaves less effectively as a steric stabilizer; but this seems unlikely due to the excellent protection offered by casein(ate) to the WPI emulsion before homogenization in the absence of added KCl (Fig. 5). A more plausible alternative explanation lies in the difference in the electrostatic contribution to the droplet–droplet interactions. Addition of electrolyte reduces the thickness of the electrical double-layer and therefore diminishes the magnitude of the electrostatic repulsive force that helps to keep emulsion droplets apart. Increase in ionic strength therefore affects the delicate balance of the interdroplet attractive and repulsive forces (Parkinson et al., 2005) with the result that casein is unable to completely inhibit the viscosity increase on heating, when 30 mM KCl is present. The fact that addition of casein after homogenization provides more effective stabilization than addition before homogenization may be explained by the casein adsorbing on top of the established layer of b-lg molecules, and
0.6 0.5 0.4 0.3 0.2 0.1 0.0 A
B
C
D
E
Casein before heating
F
G
H
I
J
Casein after heating
Fig. 5. Effect on the apparent viscosity after heating (90 1C, 3 min) of the time at which sodium caseinate (0.15 wt%) is added to a WPI-stabilized emulsion (3 wt% protein, 45 vol% oil, pH 6.8): A, no caseinate added; B, before homogenization (HG); C, immediately after HG; D, 10 min after HG; E, 30 min after HG; F, 1 h after HG; G, immediately after heat treatment (emulsion still hot); H, 10 min after heat treatment (emulsion cold); I, 30 min after heat treatment (emulsion cold); J, 30 min after heat treatment, and ageing for 1 h.
Viscosity (Pa s)
Viscosity (Pa s)
0.7 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
A
B
C
D
Fig. 6. Effect on the apparent viscosity after heating (90 1C, 3 min) of the time at which sodium caseinate (0.15 wt%) is added to a b-lg-stabilized emulsion (3 wt% protein, 45 vol% oil, pH 6.80) containing 30 mM KCl added after homogenization (HG): A, no caseinate added; B, before HG; C, immediately after HG; D, 10 min after heating (emulsion cooled).
ARTICLE IN PRESS E.L. Parkinson, E. Dickinson / International Dairy Journal 17 (2007) 95–103
therefore extending even further into the aqueous phase, and making the copolymer an even more effective steric stabilizer (Parkinson et al., 2005). On the other hand, once the emulsion droplets have already aggregated via strong b-lgb-lg bonds in the absence of casein, and the emulsion has been cooled, the addition of casein is unable to break up these flocs, and so the viscosity remains high (sample D in Fig. 6). In the pure b-lg-based emulsions investigated here, around 70% of the protein was in the non-adsorbed state before heating, and this reduced to around 30% after heating (Parkinson & Dickinson, 2004). Based on the work of Euston and coworkers (Euston, Finnigan, & Hirst, 2000), we would expect the non-adsorbed whey protein fraction to make an overwhelming contribution to the heat-induced emulsion flocculation mechanism. Indeed, it has been found (Parkinson, 2005) that, when all the nonadsorbed protein was removed from an emulsion sample (3 wt% b-lg, 45 vol% oil, 30 mM KCl) by centrifugation before heating, the viscosity increase after heating was very low (three orders of magnitude smaller than when the nonadsorbed protein was present). By trial and error, it was found that the addition of ca. 250 mM KCl to this centrifuged emulsion was sufficient to cause a similar degree of viscosity increase as with the standard pure b-lg emulsion (30 mM KCl) containing 70% non-adsorbed protein. Repeating this same experiment with 2.94 wt% b-lg+0.06 wt% caseinate (i.e., just 2% b-lg replaced by caseinate) led to the complete elimination of the heatinduced viscosity increase of the centrifuged emulsion containing 250 mM KCl (Parkinson, 2005). So, although the non-adsorbed b-lg can contribute very importantly to the heat-induced aggregation of whey protein emulsions, the protective effect of caseinate does operate in the absence of any non-adsorbed whey protein. This is again consistent with our view that it is casein in the adsorbed state which is predominantly responsible for the casein stabilizing mechanism, and not some sort of complex between a component of the caseinate (e.g., k-casein) and unadsorbed b-lg in the continuous phase. In summarizing this section, we conclude that the ability of casein added to a WPI-based or b-lg-based emulsion to exert a protective effect on its heat stability (90 1C, 3 min) depends on the stage at which the disordered protein is added. Generally speaking, if the casein is added before heating, the degree of heat-induced aggregation is reduced compared to an emulsion prepared in the absence of casein. However, depending on the age of the emulsion, some increase in stability can still be achieved when casein is added after heating. This suggests that additional flocculation may be taking place on cooling of the emulsions, and that the introduction of caseinate even at this late stage prevents further droplet–droplet association. We note that a somewhat analogous effect occurs when the rigidity of a b-lg-stabilized (emulsion) gel increases strongly on cooling as a result of enhanced hydrogen bonding and disulphide cross-linking (Dickinson & Hong, 1995, 1997).
3.3. Effect of casein in inhibiting heat-induced destabilization of emulsions made with other globular milk proteins When bovine serum albumin (BSA) was used instead of b-lg as the primary emulsifier under the same conditions (3 wt% protein, 45 vol% oil, 30 mM KCl, pH 6.8, 90 1C 3 min), the degree of heat-induced aggregation, as measured by the increase in viscosity, was higher (16 Pa s) than for the equivalent b-lg-stabilized system (1 Pa s). Lowering the KCl concentration to 10 mM in the BSAbased emulsion was found to reduce by 50% the viscosity increase in the absence of casein (i.e. to 8 Pa s). At this ionic strength, the potential stabilizing effect of added casein was investigated for sodium caseinate, b-casein and as1-casein. Fig. 7 shows that, although the different casein ingredients were all able to provide effective protection, higher concentrations (0.3–0.6 wt% protein) were required to completely inhibit the viscosity increase than for the equivalent b-lg-stabilized system. Even when the ionic strength was lowered to 2.5 mM (so that the original viscosity after heating in the absence of casein was just 1 Pa s), we found that 40.3 wt% addition of casein (i.e., replacement of 10% of the BSA by caseinate) was still required to provide complete inhibition of the heat-induced aggregation (results not shown). In addition, it seems that with BSA, the protective effect is less sensitive to the casein type; Fig. 7 shows only minor differences in viscosity for the systems containing added as1-casein (A), b-casein (B), or sodium caseinate (C). The difference in sensitivity to added casein of the heat stability of BSA-based and b-lg-based emulsions may simply be related to differences in globular protein molecular mass. The b-lactoglobulin monomer (1.8 104 Da) gives an adsorbed layer thickness of approximately 2 nm (Atkinson, Dickinson, Horne, & Richardson, 1995; Caldwell, Li, Li, & Dalgleish, 1992), whereas the larger size of the BSA molecule (6.6 104 Da) gives this globular protein an adsorbed layer thickness of approximately 4 nm (Lu, Su, & Thomas, 1999). For
10 Viscosity (Pa s)
100
8 6 4 2 C 0
B 0 5 10 % Casein (in total p ro
A 20 tein)
Fig. 7. Influence of casein type and concentration on apparent viscosity of BSA-stabilized emulsion (45 vol% oil, pH 6.8, 3 wt% total protein, 10 mM KCl) following heating at 90 1C for 3 min: (A) as1-casein; (B) b-casein; (C) sodium caseinate.
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reasons explored in detail elsewhere (Parkinson et al., 2005), this could mean that a greater density of casein polymer tails are required to confer heat-induced stability to BSA-stabilized emulsion droplets. When a-la was used instead of b-lg as the primary emulsifying agent, and the stability experiments in 30 mM KCl were repeated, no heat-induced flocculation after 3 min at 90 1C could be detected by either light scattering or rheological measurements. Even increasing the heating time to 10 min (30 mM KCl, pH 6.8, 90 1C) caused no apparent aggregation of the emulsion (Fig. 8). A comparable viscosity increase to that of a b-lg and WPI emulsion heated for 3 min at 90 1C (in the range 1–10 Pa s) could only be induced by increasing the temperature to 99 1C, the ionic strength to 100 mM, and the heating period to 6 min. However, Fig. 8 shows that the addition of 0.3 wt% sodium caseinate (i.e., 10% replacement of a-la by casein) in this case does not protect the a-la-stabilized emulsion from the resulting heatinduced viscosity increase. Presumably under these severe conditions of high temperature and ionic strength, the casein tails are unable to provide sufficient steric repulsion to prevent aggregation of the emulsion droplets. This qualitative difference in behaviour between a-la and b-lg (or BSA) may not be too surprising, given the extensive literature on the thermal aggregation and gelation of these different globular milk proteins in aqueous solution (Singh & Havea, 2003). One obvious difference from b-lg (and BSA) is that the a-la molecule has no free SH group. As a result, partially unfolded a-la molecules are unable to cross-link covalently amongst themselves (Schokker, Singh, & Creamer, 2000), although oligomers can be formed in the presence of b-lg due to disulphide exchange reactions (Livney, Verespej, & Dalgleish, 2003).
whey protein emulsion system. The physical stability of a b-lg-stabilized emulsion before and after heating (90 1C, 3 min), and in the presence and absence of sodium caseinate (1% replacement, or 0.03 wt% of the total emulsion), was studied by observing the visible development of phase separation on extended storage at room temperature (20–25 1C). Fig. 9 shows the proportion (vol%) of the sample present as the serum layer after 1, 3, 8 and 17 months of quiescent storage, and Fig. 10 shows photographs of the samples after 17 months of storage. It is clear that the pure b-lg-stabilized emulsion was more susceptible to long-term phase separation than the equivalent emulsion containing just 0.03 wt% caseinate, either with or without prior heat treatment. However, as these emulsions were stored in air-tight tubes without preservative present, the role of microbial activity in the latter samples cannot be ruled out. Furthermore, whilst the b-lg-stabilized emulsion was clearly visibly affected by heat treatment (as shown by the higher proportion of serum layer in the heated emulsion compared to the untreated emulsion), it can be seen that there was relatively little difference in the appearance of heated and untreated emulsion samples containing 2.97 wt% b-lg+0.03 wt% caseinate. Qualitatively similar results were also found with emulsions made with WPI instead of b-lg. So it seems that heating in the presence of casein may have actually led to an increase in the stability of the emulsion after several months storage at room temperature, as also reported by Srinivasan, Singh, and Munro (2002). These findings might be explained by the increased viscosity of the continuous phase on heating—and also the larger surface load, which would mean that the effective density of the droplets is increased, and so the rate of creaming is slowed. From these results, we can conclude that adding just 0.03 wt%
3.4. Effect of small additions of casein on long-term stability of a b-lg-stabilized emulsion
B
Viscosity (Pa s)
10
C
1
0.1
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Serum layer (% of sample)
Finally, let us consider the shelf-life implications of incorporating a small amount of casein into a heat-treated
101
40
30
20
10
AB C D
AB C D
AB C D
AB C D
1 month
3 months
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0 0.01
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Fig. 8. Influence of heating conditions, ionic strength and addition of sodium caseinate on the viscosity of a-lactalbumin-stabilized emulsion (45 vol% oil, 3 wt% total protein, pH 6.8): (A) 3 wt% a-la, 30 mM KCl, 90 1C, 10 min; (B) 3 wt% a-la, 100 mM KCl, 99 1C, 6 min; (C) 2.7 wt% a-la+0.3 wt% caseinate, 100 mM KCl, 99 1C, 6 min.
Fig. 9. Extent of phase separation following quiescent storage of b-lg-stabilized emulsions (3 wt% protein, 45 vol% oil, 30 mM KCl, pH 6.8) before and after heating (90 1C) and with or without 1% replacement of b-lg by sodium caseinate (i.e., 0.03 wt% caseinate): A, non-heated emulsion in the absence of caseinate; B, heated emulsion in the absence of caseinate; C, non-heated emulsion in the presence of caseinate; D, heated emulsion in the presence of caseinate.
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Fig. 10. Effect of heat treatment (90 1C, 3 min) and addition of 0.03 wt% sodium caseinate (1% of total protein present) on visual appearance of emulsion samples (3 wt% b-lg, 45 vol% oil, pH 6.8, 30 mM KCl) stored quiescently in tubes of height 75 mm at ambient temperature for 17 months: A, 3 wt% b-lg untreated emulsion; B, 3 wt% b-lg heat-treated emulsion; C, 2.97 wt% b-lg+0.03 wt% caseinate untreated emulsion; D, 2.97 wt% b-lg+0.03 wt% caseinate heat-treated emulsion.
caseinate to a concentrated heat-treated b-lg-stabilized emulsion can improve its long-term storage stability. 4. Conclusions The synergistic protective effect of small additions of casein to a b-lg-stabilized emulsion (3 wt% protein, 45 vol% n-tetradecane, pH 6.8), as established previously (Parkinson and Dickinson, 2004) at low ionic strength (0–50 mM KCl), has been found to be sensitive to changes in various conditions. In the absence of any added casein, b-lg-stabilized emulsions were less stable in the presence of Ca2+ ions (as shown by the increase in viscosity on heating). Comparable experiments with K+ and Ca2+ ions in combination has shown that some destabilization due to specific calcium binding can be inferred. This conclusion is reinforced by the very low measured values of free ionic calcium in the heated emulsions. Nevertheless, moderate Ca2+ addition was found not to inhibit the protective effect of the caseins, with the same order of efficiency operating (b-casein4sodium caseinate4as1-casein). However, slightly more casein was required for full heat stability than in the absence of added CaCl2. Since most dairy products do contain calcium salts, the colloidal stabilizing capacity of casein in the presence of a moderate content of ionic calcium seems technologically noteworthy. This synergistic protective effect due to small additions of casein is fairly robust to processing changes relating to the stage at which the disordered protein is incorporated in the whey protein-based emulsion formulation. For a WPIstabilized emulsion with no salt, protection was evident at 0.15 wt% sodium caseinate addition so long as the casein was added before the heating stage, either before or after homogenization. Furthermore, a partial protective effect was also observed when sodium caseinate was added
immediately after heating, indicating that casein may prevent droplet aggregation that occurs on cooling. However, ageing of the emulsion after heating (before casein addition) diminishes the protective effect of the casein polymer. This implies that, once droplets have become aggregated by denatured whey protein, they cannot be redispersed simply by means of casein addition. The impressive protective effect at very low casein contents, as found with b-lg and WPI emulsions, did not appear to extend to all systems based on globular milk protein ingredients. With BSA as primary emulsifier, significantly more caseinate was found to be required (0.3 wt% protein or 10% replacement of BSA by caseinate) for complete protection from heat-induced changes. For a-la, no protective effect of casein at these same levels of addition could be found, possibly due to the more extreme conditions required to induce the viscosity increase. Finally, we have shown that casein addition can inhibit phase separation of b-lg-stabilized emulsions during long-term storage (up to 1.5 years). Furthermore, this enhanced stability may be increased further by thermal processing. The extra stabilizing effect of heating in the presence of casein opens up the possibility of using sodium caseinate to add to whey protein-stabilized emulsions for improvement in shelf-life of products such as imitation creams and whipped toppings. Acknowledgment E.P. gratefully acknowledges receipt of a BBSRC Industrial CASE Studentship in collaboration with the Campden and Chorleywood Food Research Association (CCFRA). References Agboola, S. O., & Dalgleish, D. G. (1995). Calcium-induced destabilization of oil-in-water emulsions stabilized by caseinate or by b-lactoglobulin. Journal of Food Science, 60, 399–404. Atkinson, P. J., Dickinson, E., Horne, D. S., & Richardson, R. M. (1995). Neutron reflectivity of adsorbed b-casein and b-lactoglobulin at the air–water interface. Journal of the Chemical Society, Faraday Transactions, 91, 2847–2854. Burgaud, I., Dickinson, E., & Nelson, P. V. (1990). An improved high-pressure homogenizer for making fine emulsions on a small scale. International Journal of Food Science and Technology, 25, 39–46. Caldwell, K. D., Li, J., Li, J.-T., & Dalgleish, D. G. (1992). Adsorption behaviour of milk proteins on polystyrene latex. A study based on sedimentation field-flow fractionation and dynamic light scattering. Journal of Chromatography, 604, 63–71. Dalgleish, D. G. (1997). Adsorption of protein and the stability of emulsions. Trends in Food Science and Technology, 8, 1–6. Dalgleish, D. G., & Parker, T. G. (1980). Binding of calcium ions to bovine as1-casein and precipitability of the protein calcium ion complexes. Journal of Dairy Research, 47, 113–122. Dickinson, E. (1989). Surface and emulsifying properties of caseins. Journal of Dairy Research, 56, 471–477. Dickinson, E. (1999). Caseins in emulsions: Interfacial properties and interactions. International Dairy Journal, 9, 305–312.
ARTICLE IN PRESS E.L. Parkinson, E. Dickinson / International Dairy Journal 17 (2007) 95–103 Dickinson, E., & Davies, E. (1999). Influence of ionic calcium on stability of sodium caseinate emulsions. Colloids and Surfaces B, 12, 203–212. Dickinson, E., & Hong, S.-T. (1995). Influence of water-soluble nonionic emulsifier on the rheology of heat-set protein-stabilized emulsion gels. Journal of Agricultural and Food Chemistry, 43, 2560–2566. Dickinson, E., & Hong, S.-T. (1997). Influence of an anionic surfactant on the rheology of heat-set b-lactoglobulin-stabilized emulsion gels. Colloids and Surfaces A, 127, 1–10. Dickinson, E., & Parkinson, E. L. (2004). Heat-induced aggregation of milk protein-stabilized emulsions: Sensitivity to processing and composition. International Dairy Journal, 14, 635–645. Euston, S. R., Finnigan, S. R., & Hirst, R. L. (2000). Aggregation kinetics of heated whey protein-stabilized emulsions. Food Hydrocolloids, 14, 155–161. Horne, D. S., & Leaver, J. (1995). Milk proteins on surfaces. Food Hydrocolloids, 9, 91–95. Kim, H.-J., Decker, E. A., & McClements, D. J. (2004). Influence of free protein on flocculation stability of b-lactoglobulin-stabilized oil-inwater emulsions at neutral pH and ambient temperature. Langmuir, 20, 10394–10398. Livney, Y. D., Verespej, E., & Dalgleish, D. G. (2003). Steric effects governing disulfide bond interchange during thermal aggregation in solutions of b-lactoglobulin B and a-lactalbumin. Journal of Agricultural and Food Chemistry, 51, 8098–8106. Lu, J. R., Su, T. J., & Thomas, R. K. (1999). Structural conformation of bovine serum albumin layers at the air–water interface studied by neutron reflection. Journal of Colloid and Interface Science, 213, 426–437.
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Parker, T. G., & Dalgleish, D. G. (1981). Binding of calcium ions to bovine b-casein. Journal of Dairy Research, 48, 71–76. Parkinson, E.L. (2005). Protective effect of casein on the thermal stability of whey protein-stabilized emulsions. Ph.D. thesis, University of Leeds. Parkinson, E. L., & Dickinson, E. (2004). Inhibition of heat-induced aggregation of a b-lactoglobulin-stabilized emulsion by very small additions of casein. Colloids and Surfaces B, 39, 23–30. Parkinson, E. L., & Dickinson, E. (2006). Understanding the stabilizing property of casein in heated milk protein emulsions. In P. A. Williams, & G. O. Phillips (Eds.), Gums and stabilizers for the food industry, vol. 13. Cambridge, UK: Royal Society of Chemistry (in press). Parkinson, E. L., Ettelaie, R., & Dickinson, E. (2005). Using selfconsistent-field theory to understand enhanced steric stabilization by casein-like copolymers at low surface coverage in mixed protein layers. Biomacromolecules, 6, 3018–3029. Schokker, E. P., Singh, H., & Creamer, L. K. (2000). Heat-induced aggregation of b-lactoglobulin A and B with a-lactalbumin. International Dairy Journal, 10, 843–853. Singh, H., Havea, P., 2003. Thermal denaturation, aggregation and gelation of whey proteins. In P. F. Fox, & P. L. H. McSweeney (Eds.), Advanced dairy chemistry, (3rd ed.) vol. 1: Proteins, pp. 1261–1287. New York, USA: Kluwer/Plenum. Srinivasan, M., Singh, H., & Munro, P. A. (2002). Formation and stabilization of sodium caseinate emulsions: Influence of retorting (121 1C for 15 min) before or after emulsification. Food Hydrocolloids, 16, 153–160.