Effect of chitosan molecular weight on the stability and rheological properties of β-carotene emulsions stabilized by soybean soluble polysaccharides

In this study, β-carotene emulsions were prepared using a two-stage homogenization process and adsorption of chitosan to anionic droplets coated with soybean soluble polysaccharides (SSPS). Effects of the molecular weights of chitosan on the stability and rheological properties of β-carotene emulsions were investigated. Results demonstrated that the ζ-potential, particle size and rheological properties of emulsions were greatly dependent on the chitosan molecular weight. It was found that the particle size of SSPS-stabilized emulsions increased with the rise of chitosan molecular weight at higher chitosan concentrations (>0.2 wt%). Chitosan molecular weight had a significant impact on the heat and light stability of β-carotene in emulsions. SSPS-stabilized emulsion with the adsorption of medium molecular weight-chitosan (MM-chitosan) was more stable than those with the adsorption of low and high molecular weight-chitosan (LM-chitosan and HM-chitosan). Dynamic oscillatory shear tests indicated that the viscoelasticity could be enhanced by the adsorption of higher molecular weight of chitosan onto the SSPS-coated droplet surfaces.     

Fabrication,characterization and lipase digestibility of food-grade nanoemulsions

The behavior of nanoemulsion-based delivery systems within the gastrointestinal tract determines their functional performance. In this study, the influence of particle radius (30–85 nm) on the in vitro digestion of nanoemulsions containing non-ionic surfactant stabilized lipid (corn oil) droplets was examined using simulated small intestine conditions. Nanoemulsions were prepared by a combination of high-pressure homogenization and solvent (hexane) displacement. Lipid droplets with different sizes were prepared by varying the oil-to-solvent ratio in the disperse phase prior to homogenization. The fraction of free fatty acids (FFA) released from emulsified triacylglycerols (TG) during digestion was measured by an in vitro model (pH-Stat titration). Nanoemulsions exhibited a lag-period before any FFA were released, which was explained by inhibition of lipase adsorption to the oil–water interface by free surfactant. After the lag-period, the digestion rate increased with decreasing oil droplet diameter (increasing specific surface area). The total amount of FFA released from the emulsions increased from 61% to 71% as the mean droplet radius decreased from 86 nm to 30 nm. The incomplete digestion of the emulsified lipids could be explained by inhibition of lipase activity by the release of fatty acids and/or by interactions between lipase and surfactants molecules.     

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.     

Utilization of layer-by-layer interfacial deposition technique to improve freeze–thaw stability of oil-in-water emulsions

The freeze–thaw stability of 5 wt% hydrogenated palm oil-in-water emulsions (pH 3) containing droplets stabilized by sodium dodecyl sulfate (SDS)–chitosan–pectin membranes was studied. The multilayered interfacial membranes were created using an electrostatic layer-by-layer deposition method. The ζ-potential, mean particle diameter, fat destabilization, apparent viscosity and microstructure of the emulsions were used to examine the influence of freezing on their stability. Emulsions containing oil droplets stabilized only by SDS were highly unstable to droplet coalescence when either the oil phase became partially crystallized or the water phase crystallized. Emulsions containing oil droplets stabilized by SDS–chitosan membranes were stable to droplet coalescence, but unstable to droplet flocculation. Emulsions containing droplets stabilized by SDS–chitosan–pectin membranes were stable to both droplet coalescence and flocculation. The interfacial engineering technology utilized in this study could lead to the creation of food emulsions with improved stability to freeze–thaw cycling.     

Flaxseed gums and their adsorption on whey protein-stabilized oil-in-water emulsions

The effect of the addition of flaxseed gum on the physicochemical properties of whey protein-stabilized (WPI) oil-in-water emulsions at pH 3.5 was investigated. Two different varieties (Emerson and McDuff) were tested at concentrations ranging from 0% to 0.33% (w/v), by measuring droplet size, ζ-potential, phase separation behavior, microstructure and apparent viscosity. With addition of flaxseed gum the ζ-potential of the droplets decreased from around +30 mV to a negative value (−10 mV) at concentrations >0.2%. These results indicated that the negatively charged polysaccharide fraction from flaxseed interacted with the protein adsorbed at the interface. An increase in apparent particle size was also noted with increasing flaxseed concentration, with destabilization becoming visually evident at concentrations higher than 0.1% (w/v). Microscopy, rheological data and size distribution analysis demonstrated for the first time that flaxseed gum interacts with protein-stabilized oil droplets at low pH, causing bridging flocculation. No significant differences were noted between flaxseed gums extracted from the Emerson and McDuff varieties. This research demonstrated that the electrostatic interactions between flaxseed gums and protein-stabilized emulsions need to be controlled when designing novel acidic beverages containing these polysaccharides.     

Influence of emulsifier type on in vitro digestibility of lipid droplets by pancreatic lipase

The objective of this study was to investigate the influence of interfacial composition on the in vitro digestion of emulsified lipids coated by various emulsifiers by pancreatic lipase. Sodium caseinate, whey protein isolate (WPI), lecithin and Tween 20 were used to prepare corn oil-in-water emulsions (3 wt% oil). Pancreatic lipase (1.6 mg/mL) and/or bile extract (5.0 mg/mL) were added to each emulsion and the particle charge, droplet aggregation, microstructure and free fatty acids released were measured. In the absence of bile extract, the amount of free fatty acids released per unit volume of emulsion was much lower for lipid droplets coated by Tween 20 (13 ± 16 μmol ml⁻¹) than those coated by lecithin (75 ± 20 μmol ml⁻¹), sodium caseinate (220 ± 24 μmol ml⁻¹) or WPI (212 ± 6 μmol ml⁻¹). In the presence of bile extract, there was an appreciable increase in the amount of free fatty acids released in all the emulsions, with the most appreciable effects being observed in the Tween 20-stabilized emulsions. The stability of the emulsions to droplet flocculation and coalescence during hydrolysis was also strongly dependent on emulsifier type, with the WPI emulsions being the least stable and the Tween 20 emulsions being the most stable. Our results suggest that the access of pancreatic lipase to emulsified fats decreases in the following order: proteins (caseinate and WPI) > phospholipids (lecithin) > non-ionic surfactants (Tween 20). These results may have important consequences for the design of foods with either increased or decreased lipid bioavailability.     

The simulated in vitro infant gastrointestinal digestion of droplets covered with milk fat globule membrane polar lipids concentrate

Milk fat globule membrane polar lipids (MPL) are increasingly used as the surface-active components for emulsions in many infant food products. However, the precise effect of the emulsifier MPL on the digestion of lipids during gastrointestinal digestion has not been elucidated. This study investigated the lipid digestion of droplets covered with MPL with different sizes in a simulated in vitro infant gastrointestinal digestion assay. The well-used surface-active component casein was used as a control. Four types of emulsions were formulated: small and large droplets covered with MPL concentrate (MPL-S and MPL-L, with volumetric means of 0.35 ± 0.01 and 4.04 ± 0.01 μm, respectively), and small and large droplets covered with casein (CN-S and CN-L, with volumetric means of 0.44 ± 0.01 and 4.09 ± 0.03 μm, respectively). The emulsions were subjected to in vitro gastrointestinal digestion using a semidynamic model mimicking infant digestion. Through the determination of particle size evolution, zeta-potential, and microstructure of emulsions, the lipid droplets covered with MPL were found to be more stable than that of the CN-S and CN-L during gastrointestinal digestion. Moreover, although CN-S and CN-L showed a higher initial lipolysis rate at the beginning of gastric digestion, droplets covered by MPL exhibited a significantly higher amount of free fatty acid release during later digestion. The amount of free fatty acid release of the emulsions in both gastric and intestinal digestion could be generally classified as MPL-S ≥ MPL-L > CN-S > CN-L. Our study highlights the crucial role of MPL in the efficient digestion of emulsions and brings new insight for the design of infant food products.     

Influence of Hydrocolloids (Dietary Fibers) on Lipid Digestion of Protein‐Stabilized Emulsions: Comparison of Neutral,Anionic, and Cationic Polysaccharides

The impact of dietary fibers on lipid digestion within the gastrointestinal tract depends on their molecular and physicochemical properties. In this study, the influence of the electrical characteristics of dietary fibers on their ability to interfere with the digestion of protein‐coated lipid droplets was investigated using an in vitro small intestine model. Three dietary fibers were examined: cationic chitosan; anionic alginate; neutral locust bean gum (LBG). The particle size, ζ‐potential, microstructure, and apparent viscosity of β‐lactoglobulin stabilized oil‐in‐water emulsions containing different types and levels of dietary fiber were measured before and after lipid digestion. The rate and extent of lipid digestion depended on polysaccharide type and concentration. At relatively low dietary fiber levels (0.1 to 0.2 wt%), the initial lipid digestion rate was only reduced by chitosan, but the final extent of lipid digestion was unaffected by all 3 dietary fibers. At relatively high dietary fiber levels (0.4 wt%), alginate and chitosan significantly inhibited lipid hydrolysis, whereas LBG did not. The impact of chitosan on lipid digestion was attributed to its ability to promote fat droplet aggregation through bridging flocculation, thereby retarding access of the lipase to the droplet surfaces. The influence of alginate was mainly ascribed to its ability to sequester calcium ions and promote depletion flocculation.     

豌豆分离蛋白-羧甲基纤维素纳静电复合物在乳液中的应用研究

为改善豌豆分离蛋白(PPI)在酸性乳液体系中的应用特性,采用阴离子多糖-羧甲基纤维素钠(CMC)与PPI在酸性条件下形成的静电复合物稳定O/W型乳液。首先研究了3% PPI溶液的溶解度、表面疏水性随CMC浓度(0~0.5%)的变化,在此基础上,分析了PPI-CMC静电复合物对乳液ζ-电位、粒径、粘度、乳析稳定性指数及微观结构的影响。结果表明:在pH4.5条件下,随着CMC浓度由0增加至0.5%,乳状液滴表面负电性不断增强,当CMC浓度≥0.4%时,PPI乳液稳定性明显提高,液滴分散均匀,絮凝程度明显降低,在4 ℃下存放一周,未发现明显分层。因此,通过调控PPI-CMC相互作用可有效改善PPI在酸性乳液体系中的应用特性,研究成果有望为高豌豆蛋白酸性乳品和饮料的开发提供参考。     

Antioxidant and emulsifying properties of potato protein hydrolysate in soybean oil-in-water emulsions

The efficacy of a previously developed antioxidative potato protein hydrolysate (PPH) for the stabilisation of oil droplets and inhibition of lipid oxidation in soybean oil-in-water (O/W) emulsions was investigated. Emulsions (10% lipid, pH 7.0) with PPH-coated oil droplets were less stable than those produced with Tween 20 (P < 0.05). However, the presence of PPH, whether added before or after homogenisation with Tween 20, retarded emulsion oxidation, showing reduced formation of peroxides up to 53.4% and malonaldehyde-equivalent substances up to 70.8% after 7-d storage at 37 °C (P < 0.05), when compared with PPH-free emulsions. In the emulsions stabilised by PPH + Tween 20, 8–15% of PPH was distributed at the interface. Adjustment of the pH from 3 to 7 markedly increased ζ-potential of such emulsions (P < 0.05). Inhibition of lipid oxidation by PPH in soybean O/W emulsions can be attributed to both chemical and physical (shielding) actions.     

Influence of particle size on lipid digestion and β-carotene bioaccessibility in emulsions and nanoemulsions

The interest in incorporating carotenoids, such as β-carotene, into foods and beverages is growing due to their potential health benefits. However, the poor water-solubility and low bioavailability of carotenoids is currently a challenge to their incorporation into many foods. The aim of this work was to study the influence of particle size on lipid digestion and β-carotene bioaccessibility using corn oil-in-water emulsions with different initial droplet diameters: large (d₄₃ ≈ 23 μm); medium (d₄₃ ≈ 0.4 μm); and small (d₄₃ ≈ 0.2 μm). There was a progressive increase in the mean particle size of all the emulsions as they passed through a simulated gastrointestinal tract (GIT) consisting of mouth, stomach, and small intestine phases, which was attributed to droplet coalescence, flocculation, and digestion. The electrical charge on all the lipid particles became highly negative after passage through the GIT due to accumulation of anionic bile salts, phospholipids, and free fatty acids at their surfaces. The rate and extent of lipid digestion increased with decreasing mean droplet diameter (small ≈ medium ? large), which was attributed to the increase in lipid surface area exposed to pancreatic lipase with decreasing droplet size. There was also an appreciable increase in β-carotene bioaccessibility with decreasing droplet diameter (small > medium > large). These results provide useful information for designing emulsion-based delivery systems for carotenoids for food and pharmaceutical uses.     

Role of calcium and calcium-binding agents on the lipase digestibility of emulsified lipids using an in vitro digestion model

Controlling the digestibility of lipids within the gastrointestinal tract is important for developing food and pharmaceutical products. In vitro digestion methods are commonly used to study the influence of formulation composition and microstructure on lipid digestibility. In this paper, we focus on the impact of calcium and calcium-binding agents on the rate of lipid droplet digestion in corn oil-in-water emulsions monitored using a pH-stat method. The rate of fatty acid production increased with increasing calcium, e.g., the free fatty acids released after 20 min digestion was <12% for 0 mM CaCl2, but >95% for 20 mM CaCl2. The ability of calcium to increase the digestion rate was found for three different emulsifiers used to stabilize the initial lipid droplets: lyso-lecithin, caseinate and β-lactoglobulin. For these three systems, the initial rate of lipid digestion increased in the following order lyso-lecithin > β-lactoglobulin > caseinate at both 0 and 20 mM CaCl2, but the rate was considerably faster at higher calcium levels for all systems. The addition of EDTA, a calcium chelating agent, to emulsions containing 20 mM CaCl2 caused an appreciable decrease in lipid digestion rate, reducing the amount of free fatty acids produced after 20 min from around 97% to 32% when the EDTA level was increased from 0 to 5 mM. Finally, we examined the impact of two anionic polysaccharides (pectin and alginate) on the rate of lipid digestion in emulsions containing 20 mM CaCl2. High methoxy pectin, which does not bind calcium strongly, did not have a major effect on the rate of digestion, whereas alginate, which does bind calcium strongly, depressed the rate considerably. This study has important implications for designing and testing delivery systems that control lipid digestion.     

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.     

In vitro study of intestinal lipolysis using pH-stat and gas chromatography

Developing healthy products requires in-depth knowledge of digestion. This study focuses on lipid digestion in relation to emulsion properties typically followed by pH-stat. Although this is a fast and easy method to follow the overall digestion, it provides no details on lipid digestion products. Thus, the aims of the present study were to use gas chromatography (GC) to determine all products present during lipolysis, i.e. monoglycerides (MG), diglycerides (DG) and triglycerides (TG), and to compare this method with the pH-stat method for free fatty acids (FFA). Fine, medium and coarse emulsions stabilized with two different emulsifiers (whey protein isolate (WPI) or gum arabic) were digested under in vitro intestinal conditions. Although the amount of FFA increased for both methods for WPI stabilized emulsions, the amount of FFA was 2-3 times higher when determined by GC compared with pH-stat. GC analysis showed decreasing amounts of MG and DG with increasing droplet size for both emulsions. Molar ratios of FFA/DG and MG/DG were twofold higher for WPI than for gum arabic stabilized emulsions. This indicates that the total production of lipolytic products (i.e. FFA + MG + DG) depends on the droplet size and the emulsifier but their proportions only depend on the emulsifier. Although pH-stat provides a fast measure of FFA release, it is influenced by the emulsifier type at the oil-water interface and therefore care should be taken when interpreting pH-stat results. We suggest combining this method with GC for accurate FFA determination and further evaluation of all lipolytic products.     

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.     

Effects of bacteriostatic emulsifiers on stability of milk-based emulsions

For milk-based emulsion products such as canned coffee or tea, the addition of bacteriostatic emulsifiers is necessary to inhibiting the growth of heat-resistant sporeformers. Since bacteriostatic emulsifiers often cause the destabilization of emulsions, other type of emulsifiers, such as stability-enhancing ones, are necessary for the long-term stability of emulsions. Four milk-based emulsions were prepared from powdered milk combined with several types of emulsifiers. The long-term stability of emulsions, which was detected by the occurrence of a creaming layer after 3 months of storage, differed according to the composition of emulsifiers. To understand the reason for the differences in the stability of emulsions, particle size, distribution, ζ-potential, and the amount of proteins and phospholipids present in the cream layer (separated oil droplets) in the emulsions were measured. Only the amount of proteins adsorbed onto oil droplets was found to be closely related to the difference in emulsion stability, that is, the more proteins adsorbed, the higher the emulsion stability. SDS–PAGE analyses revealed that κ-casein and β-lactoglobulin play an important role in emulsion stability by adsorbing onto the oil droplet surface.     

Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: Proposal for a standardised pH-stat method

The control of lipid digestibility within the human gastrointestinal tract is important for the development of many functional food and pharmaceutical products. The influence of product composition and microstructure on lipid digestibility is typically studied using in vitro digestion methods. This article focuses on the impact of various experimental factors on lipid digestion in oil-in-water emulsions, using a pH-stat method that simulates the small intestine. The rate and extent of lipid digestion were found to increase with: increasing lipase (from 0 to 4.8 mg/ml), decreasing bile extract (from 20 to 0 mg/ml), increasing CaCl₂ (from 0 to 20 mM), decreasing lipid (from 2.5 to 0.1 wt.%); decreasing droplet diameter (from d = 800 to 200 nm), and decreasing fatty acid molecular weight (medium chain triglycerides versus corn oil). These affects are interpreted in terms of the surface area of lipid exposed to the aqueous phase, and factors affecting the accumulation of reaction products (fatty acids) at the oil–water interface. Based on our own and others’ work, we propose a standardised in vitro digestion model to test emulsified lipids, based on pH-stat titration. This study has important implications for designing and testing delivery systems that control lipid digestion using the pH-stat method.     

Improvement of physicochemical stabilities of emulsions containing oil droplets coated by non-globular protein-beet pectin complex membranes

The potential of beet pectin for improving the physical and chemical stabilities of emulsions containing silk fibroin coated droplets was investigated. Five wt.% corn oil-in-water emulsions containing fibroin-coated droplets (0.5 wt.% fibroin) and anionic pectin (0.05 wt.%) were prepared at pH 7. The pH of these emulsions was then adjusted to pH 4, so that the anionic pectin molecules electrostatically deposited to the fibroin-coated droplets. The influence of pH (3 to 7) and sodium chloride concentrations (0 to 500 mM) on the properties of primary (0 wt.% pectin) and secondary (0.05 wt.% pectin) emulsions was studied. Pectin was deposited to the droplet surfaces at pH 3, 4, and 5, but not at pH 6 and 7. In addition, secondary emulsions were stable up to higher ionic strengths (< 500 mM) than primary emulsions (< 200 mM). The addition of beet pectin also prolonged the lag phase of lipid oxidation in the emulsions as determined by the formation of lipid hydroperoxides and headspace hexanal. The controlled electrostatic deposition method utilized in this study could be used to extend the range of application of silk fibroin in the industry.     

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.