Interactions of milk protein-stabilized oil-in-water emulsions with bile salts in a simulated upper intestinal model

The behaviour of milk protein-stabilized emulsions (1.0 wt% protein) as influenced by the addition of bile salts was studied in simulated intestinal conditions (37 °C; pH 7.5; 39 mM K₂HPO₄, 150 mM NaCl, 30 mM CaCl₂; with continuous agitation at ～95 rpm for 2 h). Oil-in-water emulsions (20.0 wt% soy oil) stabilized by lactoferrin or β-lactoglobulin (β-lg) were prepared at pH 6.8 to produce cationic or anionic interfaces respectively. Varying physiological concentrations of bile salts (0.0–25.0 mg/ml) were added to each emulsion. The changes in droplet size, ζ-potential and confocal microstructures were monitored as a function of incubation time. Pre-heat treatment of simulated intestinal buffer containing bile salts was performed to eliminate any residual enzymatic activities.For β-lg-stabilized droplets, ζ-potential significantly changed from ?63.1 ± 0.5 mV to ?37.2 ± 0.3 mV in presence of bile salts due to competitive interfacial displacement of β-lg by bile salts as characterized by SDS-PAGE analysis of the continuous phase. On the other hand, lactoferrin-stabilized emulsion droplets showed considerable aggregation in presence of intestinal electrolytes alone at pH 7.5. The ζ-potential values of lactoferrin emulsion decreased gradually from +53.5 ± 0.6 mV to ?12.2 ± 0.2 mV in presence of bile salts due to certain electrostatic effects (e.g. pH shift towards the isoelectric point, binding of anionic bile salts to cationic interfacial lactoferrin layer) in comparison to the behaviour of β-lg-stabilized droplets.     

Properties of oil-in-water emulsions stabilized by β-lactoglobulin in simulated gastric fluid as influenced by ionic strength and presence of mucin

The effects of ionic strength (0–150 mM NaCl) and the presence of mucin (0.1 wt%) on the properties of oil-in-water emulsions [20.0 wt% soy oil, stabilized by 1.0 wt% β-lactoglobulin (β-lg)] under simulated gastric conditions (with/without 0.32 wt% pepsin at 37 °C, with continuous shaking at approximately 95 rev/min for 2 h) were investigated. Changes in Z-average diameter, ζ-potential and microstructure were determined as a function of incubation time. The emulsions mixed with simulated gastric fluid (SGF) (without added pepsin) were stable at low ionic strength (≤50 mM NaCl) but showed some aggregation at high ionic strength (≥150 mM NaCl). Extensive droplet flocculation with some degree of coalescence was observed in emulsions with 0.32 wt% added pepsin, the flocculation being potentially accelerated in the presence of NaCl. The addition of 0.1 wt% mucin resulted in a greater extent of flocculation, possibly because of non-specific binding of mucin to the positively charged β-lg emulsion droplets. Ionic strength and the presence of mucin had a significant influence on the rate of hydrolysis of β-lg by pepsin. The behaviour of the emulsion in SGF was predominantly driven by electrostatic interactions, which varied as a function of digestion time, ionic strength and the presence of pepsin and mucin.     

Pancreatin-induced coalescence of oil-in-water emulsions in an in vitro duodenal model

The behaviour of cationic lactoferrin-stabilized and anionic β-lactoglobulin (β-lg)-stabilized oil-in-water emulsions (20.0% (w/w) soy oil, 1.0% (w/w) protein) in the presence of simulated intestinal fluid (SIF) containing physiological concentrations of pancreatin (0.0–10.0 mg mL^?1) and/or bile salts (0.0–25.0 mg mL^?1) at 37 °C, pH 7.5 and inorganic salts (39 mm K₂HPO₄, 150 mm NaCl and 30 mm CaCl₂) was investigated. Both emulsions showed a significant degree of coalescence and fatty acid release on mixing with SIF. Appreciably negative ζ-potential values (≥?50 mV) for both types of emulsion droplet at the highest pancreatin/bile salts concentration could be attributed to displacement of and/or binding to the interfacial proteins by bile salts, together with interfacial proteolysis by pancreatin, which enhanced the potential for lipase to act on the hydrophobic lipid core, thus generating free fatty acids and possibly mono- and/or diglycerides at the droplet surface.     

Behaviour of an oil-in-water emulsion stabilized by β-lactoglobulin in an in vitro gastric model

The behaviour of β-lactoglobulin (β-lg)-stabilized emulsions (1.0 wt% protein and 20.0 wt% soy oil) using pepsin digestion under simulated gastric conditions (37 °C, pH 1.2 and 34 mM NaCl ionic strength, with continuous shaking at approximately 95 rev/min for 2 h) was investigated. Changes in particle size, ζ-potential and microstructure were monitored as a function of incubation time in the gastric fluid. Initially, β-lg formed a stable anionic emulsion at pH 7, but the emulsion underwent extensive droplet flocculation, with some coalescence, on mixing with the simulated gastric fluid. The ζ-potential values gradually changed from −57.1 ± 0.5 mV to +17.6 ± 1.2 mV because of pH change and peptic hydrolysis of the interfacial layer. Native β-lg was largely resistant to pepsin attack but, when β-lg was present at the interfacial layer of the oil-in-water emulsion, it was rapidly hydrolysed by pepsin, as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The droplet flocculation and the coalescence observed during hydrolysis were markedly dependent on the digestion time.     

Influence of glycerol and sorbitol on thermally induced droplet aggregation in oil-in-water emulsions stabilized by β-lactoglobulin

The influence of polyol cosolvents (glycerol and sorbitol) on the flocculation stability of hydrocarbon oil-in-water emulsions stabilized by a globular protein was examined. Salt (150 mM NaCl) and polyols (0–40 wt%) were added to n-hexadecane oil-in-water emulsions stabilized by β-lactoglobulin (β-Lg, pH 7.0) either before or after isothermal heat treatments (30–90 °C for 20 min). When salt was added to emulsions before heat treatment, appreciable droplet flocculation was observed below the thermal-denaturation temperature of the adsorbed β-Lg (T_m∼70 °C), and more extensive flocculation was observed above T_m. On the other hand, when salt was added after heat treatment, appreciable droplet flocculation still occurred below T_m, but little flocculation was observed above T_m. Addition of cosolvents to the emulsions increased the temperature where extensive droplet flocculation was first observed when they were heated in the presence of salt, which was attributed to their ability to increase T_m and to reduce the droplet collision frequency, with sorbitol being more effective than glycerol. Our results are interpreted in terms of the influence of the cosolvents on protein conformational stability, protein-protein interactions and the physiochemical properties of aqueous solutions. This study has important implications for the formulation and production of protein stabilized oil-in-water emulsions for industrial applications, such as foods, pharmaceuticals and cosmetics.     

Heat stability of oil-in-water emulsions formed with intact or hydrolysed whey proteins: influence of polysaccharides

The heat stability of emulsions (4 wt% corn oil) formed with whey protein isolate (WPI) or extensively hydrolysed whey protein (WPH) products and containing xanthan gum or guar gum was examined after a retort treatment at 121 °C for 16 min. At neutral pH and low ionic strength, emulsions stabilized with both 0.5 and 4 wt% WPI (intact whey protein) were stable against retorting. The amount of β-lactoglobulin (β-lg) at the droplet surface increased during retorting, especially in the emulsion containing 4 wt% protein, whereas the amount of adsorbed α-lactalbumin (α-la) decreased markedly. Addition of xanthan gum or guar gum caused depletion flocculation of the emulsion droplets, but this flocculation did not lead to their aggregation during heating. In contrast, the droplet size of emulsions formed with WPH increased during heat treatment, indicating that coalescence had occurred. The coalescence during heating was enhanced considerably with increasing concentration of polysaccharide in the emulsions, up to 0.12% and 0.2% for xanthan gum and guar gum, respectively; whey peptides in the WPH emulsions formed weaker and looser, mobile interfacial structures than those formed with intact whey proteins. Consequently, the lack of electrostatic and steric repulsion resulted in the coalescence of flocculated droplets during retort treatment. At higher levels of xanthan gum or guar gum addition, the extent of coalescence decreased gradually, apparently because of the high viscosity of the aqueous phase.     

Utilisation of pectin coating to enhance spray-dry stability of pea protein-stabilised oil-in-water emulsions

In this study, development of pea (Pisum sativum) protein stabilised dry and reconstituted emulsions is presented. Dry emulsions were prepared by spray-drying liquid emulsions in a laboratory spray-dryer. The effect of drying on the physical stability of oil-in-water emulsions containing pea protein-coated and pea protein/pectin-coated oil droplets has been studied. Oil-in-water emulsions (5 wt.% Miglyol 812 N, 0.25 wt.% pea protein, 11% maltodextrin, pH 2.4) were prepared that contained 0 (primary emulsion) or 0.2 wt.% pectin (secondary emulsion). The emulsions were then subjected to spray-drying and reconstitution (pH 2.4). The stability of the emulsions to dry processing was then analysed using oil droplet size, microstructure, Zeta potential, and creaming measurements. Obtained results showed that the secondary emulsions had better stability to droplet aggregation after drying than primary emulsions. To interpret these results, we propound that pectin, an anionic polysaccharide, formed a less charged protective layer around the protein interfacial film surrounding the oil droplets that improved their stability to spray-drying mainly by increasing steric effects.     

Influence of chitosan and NaCl on physicochemical properties of low-acid tuna oil-in-water emulsions stabilized by non-ionic surfactant

An influence of low molecular weight (LMW) chitosan on physicochemical properties and stability of low-acid (pH 6) tuna oil-in-water emulsion stabilized by non-ionic surfactant (Tween 80) was studied. The mean droplet diameter, droplet charge (ζ-potential), creaming stability and microstructure of emulsions (5 wt% oil) were evaluated. The added chitosan was adsorbed on the surface of oil droplets stabilized by Tween 80 through electrostatic interactions. Such addition of chitosan at different concentrations (0–10 wt%) to emulsions showed slight effect on the mean droplet diameter. However, the degree of flocculation was a function of chitosan concentration assessed by emulsions' microstructure and creaming index. The impact of chitosan on the strength of the colloidal interaction between the emulsion droplets increased with increasing chitosan concentration. The mean diameter of droplet in emulsions increased with increasing NaCl because of the electrostatic screening effect. The addition of LMW chitosan could be performed to create tuna oil emulsions with low-acid to neutral character, as well as various physicochemical and stability properties suitable for health food products.     

Effect of chitosan on the stability and properties of modified lecithin stabilized oil-in-water monodisperse emulsion prepared by microchannel emulsification

The effect of chitosan (CHI) on the stability of monodisperse modified lecithin (ML) stabilized soybean oil-in-water (O/W) emulsion was investigated. Monodisperse emulsion droplets with particle size of 24.4 ± 0.7 μm and coefficient of variation below 12% were prepared by microchannel (MC) emulsification using a hydrophilic asymmetric straight-through MC silicon 24 × 24 mm microchip consisting of 23,348 microchannels. The stability of the ML stabilized monodisperse emulsion droplets was investigated as a function of CHI addition at various concentration, pH, ionic strength, thermal treatment and freezing-thawing treatment by means of particle size and ζ-potential measurements as well as microscopic observation. The monodisperse O/W emulsions were diluted with CHI solution at various concentrations to a final droplet concentration of 1 wt% soybean oil, 0.25 wt% ML and 0–0.5 wt% CHI at pH 3. Pronounced droplet aggregation was observed when CHI was present at a concentration range of between 0.01 and 0.04 wt%. Above this concentration range, flocculations were less extensive, indicating some restabilization. ML stabilized emulsions were stable at a wide range of NaCl concentrations (0–1000 mM) and pH (3–8). On the contrary, in the presence of CHI, aggregation of the emulsion droplets was observed when NaCl concentration was above 200 mM and when the pH started to approach the pKa of CHI (i.e. ∼6.2–7.0). Emulsions containing CHI were found to have better stability at high temperature (>70 °C) in comparison to the emulsion stabilized only by ML. With sucrose/sorbitol as cryoprotectant aids, emulsions with the addition of CHI were found to be more resistant to droplet coalescence as compared to those without CHI after freezing at −20 °C for 22 h and thawing at 30 °C for 2 h. The use of CHI may potentially destabilize ML-stabilized O/W emulsions but its stability can be enhanced by selectively choosing the appropriate CHI concentrations and conditions of preparation.     

Physicochemical properties of lactoferrin stabilized oil-in-water emulsions: Effects of pH,salt and heating

Lactoferrin is a globular protein from bovine milk with an unusually high isoelectric point (pI > 8), which may lead to novel functional properties in foods and other products because it is cationic across a wide pH range. In this study, we investigated the influence of pH (2–9), NaCl addition (0–200 mM), CaCl₂ addition (0–200 mM), and thermal processing (30–90 °C, 20 min) on the stability of lactoferrin (LF) stabilized oil-in-water emulsions. At ambient temperature, the emulsions were stable to droplet aggregation at low pH (pH ≤ 6), but exhibited some aggregation at pH ≈ pI (pH 7–9). The thermal stability of the emulsions depended on pH, holding temperature, and thermal history. When LF-coated droplets were heated in distilled water, and then their pH was adjusted in the range 2–9, they were highly unstable to aggregation at pH 7 and 8. On the other hand, when the pH was altered in the range 2–9 first, and then they were heated, the LF-coated droplets were highly unstable to aggregation at pH ≥ 5 when heated above 50 °C. The stability of the emulsions to salt addition depended on pH and salt type, which was attributed to counter-ion binding and electrostatic screening effects. For NaCl, emulsions were stable from 0 to 200 mM at pH 3 and 9, but aggregated at ≥100 mM at pH 6. For CaCl₂, emulsions were stable from 0 to 200 mM at pH 3, but aggregated with ≥150 mM CaCl₂ at pH 6 and 9. These results have important implications for the formulation and production of emulsion-based products using lactoferrin as an emulsifier.     

Influence of chitosan on stability and lipase digestibility of lecithin-stabilized tuna oil-in-water emulsions

The influence of chitosan concentration (0–0.3 wt%) and molecular weight (120, 250 and 342.5 kDa) on the physical stability and lipase digestibility of lecithin-stabilized tuna oil-in-water emulsions was studied. The ζ-potential, droplet size, creaming stability, free fatty acids and glucosamine released was measured for the emulsions when they were subjected to an in vitro digestion model. The ζ-potential of the oil droplets in lecithin-chitosan stabilized emulsions changed from positive (≈+53 mV) to negative and the emulsions were unstable to droplet aggregation for all chitosan concentrations and molecular weights used after being subjected to the digestion model. The amount of free fatty acid and glucosamine released per unit amount of emulsion was higher when pancreatic lipase was included in the digestion model. These results suggest that lecithin-chitosan coated droplets can be degraded by lipase under simulated gastrointestinal conditions. Consequently, chitosan coated lipid droplets may serve as useful carriers for the delivery of bioactive lipophilic nutraceuticals.     

Structuring of lipid phases using controlled heteroaggregation of protein microspheres in water-in-oil emulsions

The effect of heteroaggregation of oppositely charged protein microspheres dispersed within a liquid oil phase on the microstructure and rheological properties of water-in-oil (W/O) emulsions was evaluated. The aqueous phase of the initial W/O emulsions contained either 10% β-lactoglobulin or 10% lactoferrin (pH 7, 100 mM NaCl). At this pH, β-lactoglobulin (BLG) is negatively charged while lactoferrin (LF) is positively charged. The oil phase consisted of a lipophilic non-ionic surfactant (8% polyglycerol polyricinoleate, PGPR) dispersed within soybean oil. Three 40% W/O emulsions were formed containing different types of protein microspheres: (i) BLG: 100% BLG droplets; (ii) LF: 100% LF droplets; and (iii) Mixed: 50% BLG droplets and 50% LF droplets. Prior to heating, the mixed emulsions had a higher shear viscosity, yield stress, and shear modulus than the BLG or LF emulsions, which suggested that electrostatic attraction led to the formation of a three-dimensional network of aggregated droplets. All three W/O emulsions underwent an irreversible fluid-to-solid transition when they were heated above ≈70 °C. This phenomenon was attributed to thermal denaturation of the globular BLG and LF molecules within the aqueous phase promoting aggregation and network formation of the protein microspheres. After heating, the mixed emulsions had a higher shear viscosity, yield stress and shear modulus than the BLG or LF emulsions, suggesting that a stronger droplet network was formed due to electrostatic attraction. Shear rheology measurements of the W/O emulsions showed that the lipid phases formed after heating were non-ideal plastics characterized by a yield stress and shear thinning behavior. These results may facilitate the design of semi-solid or solid foods with reduced saturated- or trans-fat contents suitable for use in commercial products.     

Fabrication of viscous and paste-like materials by controlled heteroaggregation of oppositely charged lipid droplets

This study describes the formation of materials with novel textural characteristics by controlled heteroaggregation of oppositely charged protein-coated lipid droplets. Heteroaggregation was induced by mixing a suspension of β-lactoglobulin (β-Lg)-coated lipid droplets (ζ = −51 mV, d₄₃ ∼ 0.35 μm, 20 wt.%) with a suspension of lactoferrin (LF)-coated lipid droplets (ζ = +32 mV, d₄₃ ∼ 0.35 μm, 20 wt.%) under conditions where the two proteins had opposite charges (pH 7). The mean particle size, flow behaviour, and shear modulus of the materials depended on positive-to-negative particle ratio (0–100%), pH (3–9), ionic strength (0–400 mM), and temperature (30–90 °C). The largest particle sizes, highest viscosities, and largest gel strengths were observed at intermediate particle ratios (40% LF:60% β-Lg), which was attributed to a strong electrostatic attraction between oppositely charged droplets (0 mM NaCl, pH 7, 25 °C). A reduction in particle aggregation, viscosity, and gel strength occurred at intermediate ionic strengths due to screening of the electrostatic attraction between oppositely charged droplets. However, increased aggregation, thickening, and gelation occurred at higher ionic strengths due to screening in electrostatic repulsion between similarly charged droplets. Thermal treatment of the samples (90 °C) promoted a substantial increase in gel strength due to protein denaturation and increased droplet attraction. This study has important implications for the utilisation of controlled particle aggregation to create novel structures in foods, cosmetics, personal care, and other products.     

Emulsions stabilization by lactoferrin nano-particles under in vitro digestion conditions

In recent years there has been a spur of interest in the utilization of nano and micro-particles to fabricate novel food-grade Pickering emulsions. Aligned with increased interest and efforts to promote health through food, this study aimed to extend the understanding of Pickering emulsions stabilized by lactoferrin (LF) nano-particles in respect to their stability and responsiveness to physiological conditions of the human mouth and stomach. Analytical centrifugation revealed that LF nano-particles did not alter mean droplet size of coarse emulsions but significantly (p < 0.05) reduced creaming rates by an order of magnitude. In fine emulsions produced through high pressure homogenization, the use of nano-particles increased mean droplet sizes. This resulted in noted (p < 0.05) differences in stability with emulsions stabilized by LF nano-particles and alginate showing poorest stability. Concomitantly, the use of i-carrageenan and LF nano-particles yielded emulsions with the most reduced creaming (<1 μm/s), even compared to emulsions stabilized by native LF. Interestingly, the use of alginate and i-carrageenan with LF nano-particles also altered emulsion stability to artificial saliva and modulated emulsion behavior under gastric conditions, which was linked to reduced rate of LF gastric proteolysis. Overall, this work establishes a new possibility to incorporate LF in emulsions and demonstrates how LF nano-particles could be harnessed to modulate emulsion destabilization and breakdown in the mouth and stomach.     

Inhibition of droplet flocculation in globular-protein stabilized oil-in-water emulsions by polyols

The influence of neutral cosolvents (polyols) on the stability of hydrocarbon oil-in-water emulsions stabilized by a globular protein was investigated. Glycerol (0–40 wt%) and sorbitol (0–35 wt%) were added to n-hexadecane oil-in-water emulsions stabilized by β-lactoglobulin (β-lg, pH 7.0, 150 mM NaCl), either before or after incubation at 30 °C for 24 h. The stability of the emulsions to flocculation and creaming improved when neutral cosolvents were added, with the effectiveness of the cosolvents depending on their type, concentration and time of addition. Emulsion stability was better for sorbitol than glycerol, improved with increasing cosolvent concentration, and was better when the cosolvents were added immediately after homogenization than when they were added 24 h later. The influence of the cosolvents on emulsion stability is interpreted in terms of their effect on the conformation and interactions of the adsorbed proteins, as well as on the droplet–droplet collision frequency. This study has implications for the development of protein stabilized oil-in-water emulsions for utilization in industrial products.     

Impact of iron encapsulation within the interior aqueous phase of water-in-oil-in-water emulsions on lipid oxidation

Iron (Fe³⁺) was encapsulated within the internal aqueous phase of water-in-oil-in-water (W/O/W) emulsions, and then the impact of this iron on the oxidative stability of fish oil droplets was examined. There was no significant change in lipid droplet diameter in the W/O/W emulsions during 7 days storage, suggesting that the emulsions were stable to lipid droplet flocculation and coalescence, and internal water diffusion/expulsion. The initial iron encapsulation (4 mg/100 g emulsion) within the internal aqueous phase of the water-in-oil (W/O) emulsions was high (>99.75%), although, a small amount leaked out over 7 days storage (≈10 μg/100 g emulsion). When W/O/W emulsions were mixed with fish oil droplets the thiobarbituric acid-reactive substances (TBARS) formed decreased (compared to fish oil droplets alone) by an amount that depended on iron concentration and location, i.e., no added iron < iron in external aqueous phase < iron in internal aqueous phase. These differences were attributed to the impact of W/O droplets on the concentration and location of iron and lipid oxidation reaction products within the system.     

Stabilization of acidic soy protein-based dispersions and emulsions by soy soluble polysaccharides

Soy soluble polysaccharides (SSPS) are shown to prevent destabilization of soy protein isolate (SPI) dispersions and SPI-based oil-in-water (O/W) emulsions under acidic conditions. Addition of SSPS above a critical concentration (0.25 wt%) increased the stability of 0.50 wt% SPI dispersions against aggregation and phase separation under conditions where SPI would normally precipitate (near its isoelectric point). Though SSPS neutralized SPI surface charge via electrostatic interaction, there was increased stability against aggregation due to steric repulsion. At acidic pH, addition of 1 wt% NaCl electrostatically screened protein–polysaccharide complexation which led to SPI precipitation and sedimentation. However, the order of salt addition had a significant impact on charge screening, with salt added before pH adjustment reducing SPI–SSPS complexation whereas it had less effect when added afterwards. Salt penetration efficacy diminished with decreasing pH. O/W emulsions (5 wt% oil) prepared with 0.50 wt% SPI destabilized at pH 4–5 due to protein aggregation, but addition of ≥0.25 wt% SSPS improved emulsion stability by inhibiting protein–protein interactions thus limiting increases in oil droplet diameter over time. Overall, both dispersion and emulsion stability greatly depended on pH, ionic strength and SSPS concentration. These results demonstrated that SSPS could effectively stabilize acidic SPI dispersions and that SPI–SSPS interactions may be used as a tool to improve the kinetic stability of SPI-based O/W emulsions.     

Influence of interfacial composition on oxidative stability of oil-in-water emulsions stabilized by biopolymer emulsifiers

The objective of this research was to evaluate the influence of storage pH (3 and 7) and biopolymer emulsifier type (Whey protein isolate (WPI), Modified starch (MS) and Gum arabic (GA)) on the physical and oxidative stability of rice bran oil-in-water emulsions. All three emulsifiers formed small emulsion droplets (d₃₂ < 0.5 μm) when used at sufficiently high levels: 0.45%, 1% and 10% for WPI, MS and GA, respectively. The droplets were relatively stable to droplet growth throughout storage (d₃₂ < 0.6 μm after 20 days), although there was some evidence of droplet aggregation particularly in the MS-stabilized emulsions. The electrical charge on the biopolymer-coated lipid droplets depended on pH and biopolymer type: −13 and −27 mV at pH 3 and 7 for GA; −2 and −3 mV at pH 3 and 7 for MS; +37 and −38 mV at pH 3 and 7 for WPI. The oxidative stability of the emulsions was monitored by measuring peroxide (primary products) and hexanal (secondary products) formation during storage at 37 °C, for up to 20 days, in the presence of a pro-oxidant (iron/EDTA). Rice bran oil emulsions containing MS- and WPI-coated lipid droplets were relatively stable to lipid oxidation, but those containing GA-coated droplets were highly unstable to oxidation at both pH 3 and 7. The results are interpreted in terms of the impact of the electrical characteristics of the biopolymers on the ability of cationic iron ions to interact with emulsified lipids. These results have important implications for utilizing rice bran oil, and other oxidatively unstable oils, in commercial food and beverage products.     

Synergistic stabilization of heat-treated emulsions containing mixtures of milk proteins

The synergistic effect by which a very small amount of casein can confer stability to a whey protein-stabilized emulsion heated to 90 °C has been investigated. Using β-lactoglobulin (β-lg) as the main emulsifying agent, the extent of heat-induced flocculation increased with ionic strength, with commercial sodium caseinate or β-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 β-lg-stabilized emulsion. With α-lactalbumin (α-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.