Disulfide Bond Formation Affects Stability of Whey Protein Isolate Emulsions

The viscosity and degree of flocculation of 20 wt% n-hexadecane oil-in-water emulsions stabilized by whey protein isolate (1 wt% WPI in 0.05M phosphate buffer, pH 7.0) increased as the emulsions aged. These effects were reduced when N-ethylmaleimide, a sulfhydryl blocking agent, was added to the emulsions immediately after homogenization, but were not completely eliminated. Gel electrophoresis (SDS-PAGE) showed an increase in the extent of intermolecular disulfide bond formation between proteins absorbed at the droplet interface with time. Floes were probably formed initially by noncovalent bonding or bridging flocculation, and then stabilized by disulfide bonds between proteins absorbed to different droplets.     

Characterization of cold-set gels produced from heated emulsions stabilized by whey protein

This paper reports the cold gelation of preheated emulsions stabilized by whey protein, in contrast to, in previous reports, the cold gelation of emulsions formed with preheated whey protein polymers. Emulsions formed with different concentrations of whey protein isolate (WPI) and milk fat were heated at 90 °C for 30 min at low ionic strength and neutral pH. The stable preheated emulsions formed gels through acidification or the addition of CaCl₂ at room temperature. The storage modulus (G′) of the acid-induced gels increased with increasing preheat temperature, decreasing size of the emulsion droplets and increasing fat content. The adsorbed protein denatures and aggregates at the surface of the emulsion droplets during heat treatment, providing the initial step for subsequent formation of the cold-set emulsion gels, suggesting that these preheated emulsion droplets coated by whey protein constitute the structural units responsible for the three-dimensional gel network.     

Deformation and fracture of emulsion-filled gels: Effect of gelling agent concentration and oil droplet size

The effect of the ratio between the modulus of the oil droplets and that of the gel matrix (varied by changing gelling agent concentration and oil droplet size) on the large deformation properties of gelatine, κ-carrageenan and whey protein isolate (WPI) gels was studied at different compression speeds. The effect of gelling agent concentration and oil droplet size on strain-dependency of modulus and viscoelastic properties was also studied. An increase in the concentration of gelling agent resulted in denser gels with more bonds between structural elements. This induced an increase of both Young's modulus and fracture stress for all gels. With increasing gelling agent concentration, polymer gels (gelatine and κ-carrageenan) became less strain-hardening, and the particle gels (WPI) even became strain-softening. The effect of a decrease in the oil droplet size on the Young's modulus was generally according to the Van der Poel theory, unless when the oil droplets were aggregated. Moreover, a decrease in oil droplet size induced a decrease of the fracture strain in gels with non-aggregated bound droplets. The extent of these changes was shown to depend on the gelling agent concentration. The effect of a decrease of the oil droplet size on other fracture parameters and in other gel systems was minor. With decreasing oil droplet size gelatine gels with unbound droplets and WPI gels became more viscous and less elastic.     

Properties of Acidic Whey Protein Gels Containing Emulsified Butterfat

Changes in physical properties of whey protein gels following addition of emulsified fat were investigated. Gels were made by heating mixtures of dialyzed whey protein isolate at pH 4.60 with and without emulsified fat droplets. Addition of emulsified fat improved the gel-like qualities of these systems. Gel strength progressively increased upon addition of emulsified fat up to 30.00% by weight. Mean droplet size 1.85 km produced the greatest reinforcement of gel strength. Gels made with intermediate concentrations of protein were most sensitive to the effect. The elastic moduli and viscosities of whey protein gels at pH 4.60 increased with fat content, whereas syneresis decreased upon addition of fat.     

COLD GELATION OF WHEY PROTEIN EMULSIONS

Stable and homogeneous emulsion‐filled gels were prepared by cold gelation of whey protein isolate (WPI) emulsions. A suspension of heat‐denatured WPI (soluble WPI aggregates) was mixed with a 40% (w/w) oil‐in‐water emulsion to obtain gels with varying concentrations of WPI aggregates and oil. For emulsions stabilized with native WPI, creaming was observed upon mixing of the emulsion with a suspension of WPI aggregates, likely as a result of depletion flocculation induced by the differences in size between the droplets and aggregates. For emulsions stabilized with soluble WPI aggregates, the obtained filled suspension was stable against creaming, and homogeneous emulsion‐filled gels with varying protein and oil concentrations were obtained. Large deformation properties of the emulsion‐filled cold‐set WPI gels were determined by uniaxial compression. With increasing oil concentration, the fracture stress increases slightly, whereas the fracture strain decreases slightly. Small deformation properties were determined by oscillatory rheology. The storage modulus after 16 h of acidification was taken as a measure of the gel stiffness. Experimental results were in good agreement with predictions according to van der Poel's theory for the effect of oil concentration on the stiffness of filled gels. Especially, the influence of the modulus of the matrix on the effect of the oil droplets was in good agreement with van der Poel's theory.     

Rheology and Oxidative Stability of Whey Protein Isolate-Stabilized Menhaden Oil-in-Water Emulsions as a Function of Heat Treatment

ABSTRACT:  Menhaden oil-in-water emulsions (20%, v/v) were stabilized by 2 wt% whey protein isolate (WPI) with 0.2 wt% xanthan gum (XG) in the presence of 10 mM CaCl₂ and 200 μM EDTA at pH 7. Droplet size, lipid oxidation, and rheological properties of the emulsions were investigated as a function of heating temperature and time. During heating, droplet size reached a maximum at 70 °C and then decreased at 90 °C, which can be attributed to both heating effect on increased hydrophobic attractions and the influence of CaCl₂ on decreased electrostatic repulsions. Combination of effects of EDTA and heat treatment contributed to oxidative stability of the heated emulsions. The rheological data indicate that the WPI/XG-stabilized emulsions undergo a state transition from being viscous like to an elastic like upon substantial thermal treatment. Heating below 70 °C or for less than 10 min at 70 °C favors droplet aggregation while heating at 90 °C or for 15 min or longer at 70 °C facilitates WPI adsorption and rearrangement. WPI adsorption leads to the formation of protein network around the droplet surface, which promotes oxidative stability of menhaden oil. Heating also aggravates thermodynamic incompatibility between XG and WPI, which contributes to droplet aggregation and the accumulation of more WPI around the droplet surfaces as well.     

EFFECTS OF OIL DROPLET AND AGAR CONCENTRATION ON GEL STRENGTH AND MICROSTRUCTURE OF O/W EMULSION GEL

The effects of oil droplet size and agar concentration on gel strength and microstructure of emulsion gels were investigated by compression test and cryoscanning electron microscope (Cryo-SEM). At all agar concentrations, the compressive stress values of emulsion gels were lower than those of the oil-free gels. Compressive stress and energy were significantly affected by oil droplet size and agar concentration, but compressive strain was not. SEM observation revealed that the overall volume of void spaces decreased and strand compactness increased with increasing agar concentration. Gels containing oil droplets had some void spaces between the gel network and the oil droplets. The strands of emulsion gels did not cover the oil globules completely, a phenomenon which was also observed for the gel with high agar concentration.     

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.     

Heat-induced Gelation Properties of Salt-Soluble Muscle Proteins as Affected by Non-Meat Proteins

Addition of whey protein concentrate (WPC), whey protein isolate (WPI) or soy protein isolate (SPI) to salt-soluble muscle proteins (SSP) decreased the gel strength. WPI:SSP gels had higher water-holding capacity than SSP, SSP:WPC or SSP:SPI gels. Myosin heavy chain was a principal contributor to gel network formation in SSP, SSP:WPC, SSP:WPI and SSP:SPI systems. The characteristic fibrous network formed by SSP was the dominant feature of the microstructure of SSP:WPC and SSP:WPI gels. SSP:SPI gels had a more aggregated appearance due to the occurrence of clusters of SPI throughout the gel matrix.     

Effects of protein concentration and oil-phase volume fraction on the stability and rheology of menhaden oil-in-water emulsions stabilized by whey protein isolate with xanthan gum

The influences of protein concentration (0.2, 1, 2 wt%) and oil-phase volume fraction (5%, 20%, 40% v/v) on emulsion stability and rheological properties were investigated in whey protein isolate (WPI)-stabilized oil-in-water emulsions containing 0.2 wt% xanthan gum (XG). The data of droplet size, surface charge, creaming index, oxidative stability, and emulsion rheology were obtained. The results showed that increasing WPI concentration significantly affected droplet size, surface charge, and oxidative stability, but had little effect on creaming stability and emulsion rheology. At 0.2 wt% WPI, increasing oil-phase volume fraction greatly increased droplet size but no significant effect on surface charge. At 1 or 2 wt% WPI, increasing oil-phase volume fraction had less influence on droplet size but led to surface charge more negative. Increasing oil-phase volume fraction facilitated the inhibition of lipid oxidation. Meanwhile, oil-phase volume fraction played a dominant role in creaming stability and emulsion viscosity. The rheological data indicated the emulsions may undergo a behavior transition from an entropic polymer gel to an enthalpic particle gel when oil-phase volume fraction increased from 20% to 40% v/v.     

Iron-Catalyzed Oxidation of Menhaden Oil as Affected by Emulsifiers

The ability of Tween 20 and whey protein isolate (WPI) to influence lipid oxidation was investigated by evaluating the effects of emulsifier concentration and physical location on iron-catalyzed oxidation of emulsified Menhaden oil. Addition of Tween 20 or WPI to the aqueous phase of a 0.5 wt% Tween 20 stabilized emulsion increased lipid oxidation as determined by both thiobarbituric acid reactive substances (TBARS) and lipid peroxides. Tween 20 (2.0 wt%) and WPI (0.05–1.0 wt%) combinations inhibited TBARS formation 23–60%. Oxidation of a WPI-stabilized emulsion decreased with decreasing pH (3–7) but in a Tween 20 stabilized emulsion oxidation increased with decreasing pH. The low oxidation rate for the WPI-stabilized emulsion at pH 3 was increased when Tween 20 displaced WPI from the droplet interface. Results indicate that the oxidative stability of emulsifed Menhaden oil could be increased by controlling emulsifier type, location and concentration.     

Impact of whey protein – Beet pectin conjugation on the physicochemical stability of β-carotene emulsions

The aim of the present study was to investigate the impact of whey protein isolate (WPI)-beet pectin conjugation on the physical and chemical properties of oil-in-water emulsions incorporating β-carotene within the oil droplets. Covalent coupling of WPI to beet pectin was achieved by dry heating of WPI-beet pectin mixtures of different weight ratios at 80, 90, 100 °C and 79% relative humidity for incubation times ranging from 1 to 9 h. It was confirmed by SDS-polyacrylamide gel electrophoresis that WPI covalently linked to beet pectin. The physical and chemical stability of β-carotene emulsions was characterized by droplet size and distribution, transmission profiles using novel centrifugal sedimentation technique, microstructure and β-carotene degradation during the storage. Compared with those stabilized by WPI alone and unheated WPI-beet pectin mixtures, β-carotene emulsions stabilized by WPI-beet pectin conjugates had much smaller droplet sizes, more homogenous droplet size distribution, less change in centrifugal transmission profiles and obviously improved freeze–thaw stability, indicating a very substantial improvement in the physical stability. Rheological analysis exhibited that emulsions stabilized by WPI-beet pectin conjugates changed from a shear thinning to more like Newtonian liquid compared those with WPI alone and unheated WPI-beet pectin mixtures. Degradation of β-carotene in emulsion during storage was more obviously retarded by WPI-beet pectin conjugate than WPI and unheated WPI-beet pectin mixture, probably due to a thicker and denser interfacial layer in emulsion droplets. These results implied that protein–polysaccharide conjugates were able to improve the physical stability of β-carotene emulsion and inhibit the deterioration of β-carotene in oil-in-water emulsions.     

不同大分子乳化剂构建番茄红素纳米乳液的体外消化规律比较

通过体外模拟消化,研究以辛烯基琥珀酸酯化（octenyl succinic anhydride,OSA）变性淀粉、乳清分离蛋白（whey protein isolate,WPI）、酪蛋白酸钠（sodium caseinate,SC）为乳化剂构建的番茄红素纳米乳液的消化规律。结果表明,消化过程中纳米乳液的液滴大小、Zeta电位和微观结构取决于乳化剂类型,OSA变性淀粉和蛋白质类乳化剂构建的纳米乳液分别在肠和胃阶段发生水解,液滴聚集,乳液平均粒径增大,同时Zeta电位绝对值达到最小。经胃肠消化后3 种乳化剂构建的番茄红素纳米乳液游离脂肪酸释放率的大小排序为OSA变性淀粉（92.25%）＞SC（86.53%）＞WPI（79.88%）,高于对照组的48.7%,表明纳米乳液包埋体系能有效改善番茄红素的消化特性,且以OSA变性淀粉构建的纳米乳液表现出比蛋白质类乳化剂更高的番茄红素生物利用率,达到（25.60±3.08）%。     

Stability of citral in protein- and gum arabic-stabilized oil-in-water emulsions

Citral is a major flavor component of citrus oils that can undergo chemical degradation leading to loss of aroma and formation of off-flavors. Engineering the interface of emulsion droplets with emulsifiers that inhibit chemical reactions could provide a novel technique to stabilize citral. The objective of this study was to determine if citral was more stable in emulsions stabilized with whey protein isolate (WPI) than gum arabic (GA). Degradation of citral was equal to or less in GA- than WPI-stabilized emulsion at pH 3.0 and 7.0. However, formation of the citral oxidation product, p-cymene was greater in the GA- than WPI-stabilized emulsion at pH 3.0 and 7.0. Emulsions stabilized by WPI had a better creaming stability than those stabilized by GA because the protein emulsifier was able to produce smaller lipid droplets during homogenization. These data suggest that WPI was able to inhibit the oxidative deterioration of citral in oil-in-water emulsions. The ability of WPI to decrease oxidative reactions could be due to the formation of a cationic emulsion droplet interface at pH 3.0 which can repel prooxidative metals and/or the ability of amino acids in WPI to scavenge free radical and chelate prooxidative metals.     

Effect of monoglycerides and diglycerol-esters on viscoelasticity of heat-set whey protein emulsion gels

Summary Effects of small-molecule surfactants (emulsifiers) on the small-deformation viscoelastic properties of heat-set whey protein emulsion gels have been investigated using a controlled stress rheometer. The surfactants used in this investigation were the water-soluble diglycerol monolaurate (DGML) and diglycerol monooleate (DGMO), and the oil-soluble glycerol monooleate (GMO). The elastic modulus of the emulsion gel was found to decrease in the presence of a small amount of surfactant, but then to recover at higher surfactant concentrations. The initial reduction in modulus correlates with protein displacement from the oil droplet surface. The recovery of the storage and loss moduli at higher surfactant concentrations of DGML or DGMO may be due to the depletion flocculation of the emulsion prior to heat-treatment. However, for systems containing high content of GMO in the oil phase, the recovery of the moduli is probably owing mainly to the smaller average particle size. Effects of surface monolayer composition, droplet aggregation and average particle size were discussed. The behaviour obtained here was compared with results for previously investigated whey protein emulsion gel systems containing different emulsifiers.     

Influence of biopolymer emulsifier type on formation and stability of rice bran oil-in-water emulsions: whey protein, gum arabic, and modified starch

Rice bran oil (RBO) is used in foods, cosmetics, and pharmaceuticals due to its desirable health, flavor, and functional attributes. We investigated the effects of biopolymer emulsifier type and environmental stresses on the stability of RBO emulsions. Oil-in-water emulsions (5% RBO, 10 mM citrate buffer) stabilized by whey protein isolate (WPI), gum arabic (GA), or modified starch (MS) were prepared using high-pressure homogenization. The new MS used had a higher number of octenyl succinic anhydride (OSA) groups per starch molecule than conventional MS. The droplet diameters produced by WPI and MS were considerably smaller (d < 300 nm) than those produced by GA (d > 1000 nm). The influence of pH (3 to 8), ionic strength (0 to 500 mM NaCl), and thermal treatment (30 to 90 °C) on the physical stability of the emulsions was examined. Extensive droplet aggregation occurred in WPI-stabilized emulsions around their isoelectric point (4 < pH < 6), at high salt (> 200 mM, pH 7), and at high temperatures (>70 °C, pH 7, 150 mM NaCl), which was attributed to changes in electrostatic and hydrophobic interactions between droplets. There was little effect of pH, ionic strength, and temperature on emulsions stabilized by GA or MS, which was attributed to strong steric stabilization. In summary: WPI produced small droplets at low concentrations, but they had poor stability to environmental stress; GA produced large droplets and needed high concentrations, but they had good stability to stress; new MS produced small droplets at low concentrations, with good stability to stress. Practical Application: This study showed that stable rice bran oil-in-water emulsions can be formed using biopolymer emulsifiers. These emulsions could be used to incorporate RBO into a wide range of food products. We compared the relative performance of whey protein, GA, and a new MS at forming and stabilizing the emulsions. The new OSA MS was capable of forming small stable droplets at relatively low concentrations.     

乳滴粒径和皂皮皂苷浓度对高内相乳液凝胶及其模板油凝胶构建的影响

以天然皂皮皂苷为乳化剂,采用激光散射技术、动态流变学以及激光共聚焦显微技术探究了乳滴粒径皂皮皂苷质量分数对高内相乳液凝胶（HIPE-gels）及其模板制备的油凝胶流变特性和微结构的影响。结果表明： HIPE-gels和油凝胶均表现出剪切稀化特性,油滴间形成非共价物理交联的弹性凝胶结构;随粒径减小,乳滴堆积紧密,赋予HIPE-gels和油凝胶更强的凝胶网络结构和黏弹性;皂皮皂苷质量分数较低（≤1.5%）时,乳滴间的静电排斥作用对HIPE-gels的黏弹性和强度起主导作用,当皂皮皂苷质量分数较高（>1.5%）时,游离皂皮皂苷分子提高了HIPE-gels的凝胶强度,而油凝胶强度随乳滴粒径减小和皂皮皂苷质量分数的增加得到强化。     

EFFECTS OF OIL DROPLETS ON PHYSICAL AND SENSORY PROPERTIES OF O/W EMULSION AGAR GEL

Effects of oil droplets in an agar gel matrix were investigated by mechanical analyses and sensory evaluation. The results for compressive and puncture properties were expressed in terms of relative values. All compressive properties; stress, strain and compressive energy of emulsion gels, as obtained by compression tests, decreased with both increases of oil droplet size and oil volume fraction. The stress, strain and energy of the emulsion gels obtained by puncture tests did not change with an increase in oil droplet size. Results of sensory evaluation showed that the sample containing small oil droplets was harder than that with large oil droplets. On the other hand, the large oil droplet sample was oilier than that with small oil droplets. The sensory evaluations for hardness and oiliness of emulsion gels did not exhibit significant relations to most of the properties of mechanical analyses.     

果胶和热处理对蛋白质乳液凝胶结构特性和复合维生素稳定性的影响

利用大豆分离蛋白的乳化和凝胶特性制备蛋白质乳液凝胶,用以同时传递VE和D-异抗坏血酸钠,重点阐释果胶对凝胶结构特性和复合维生素稳定性的影响规律。结果表明,乳液的分散相油滴粒径随果胶质量分数的增加而增大,蛋白质-果胶复合溶液预加热处理温度越高乳液油滴粒径越大。乳液经葡萄糖酸-δ-内酯诱导形成凝胶,凝胶时间及凝胶强度（G’）受果胶质量分数影响。质构分析表明,果胶质量分数和预加热温度对凝胶硬度无显著影响,但凝胶弹性随果胶质量分数增加呈先增大后减小的趋势。当VE和D-异抗坏血酸钠被包埋于乳液凝胶后,其贮藏稳定性随果胶质量分数的增大而呈下降趋势,并且D-异抗坏血酸钠降解速率高于VE。本研究表明通过控制果胶质量分数可以调节乳液凝胶的结构特性,而不同的凝胶结构可以调控包埋于其中的功能因子的稳定性。     

Preparation and Characterization of Water/Oil/Water Emulsions Stabilized by Polyglycerol Polyricinoleate and Whey Protein Isolate

ABSTRACT: In this study we tried to prepare stable water-in-oil-in-water (W/O/W) emulsions using polyglycerol polyricinoleate (PGPR) as a hydrophobic emulsifier and whey protein isolate (WPI) as a hydrophilic emulsifier. At first, water-in-oil (W/O) emulsions was prepared, and then 40 wt% of this W/O emulsion was homogenized with 60 wt% aqueous solution of different WPI contents (2, 4, and 6 wt% WPI) using a high-pressure homogenizer (14 and 22 MPa) to produce W/O/W emulsions. The mean size of final W/O/W droplets ranged from 3.3 to 9.9 μm in diameter depending on the concentrations of PGPR and WPI. It was shown that most of the W/O/W droplets were small (<5 μm) in size but a small population of large oil droplets (d > 20 μm) was also occasionally observed. W/O/W emulsions prepared at the homogenization pressure of 22 MPa had a larger mean droplet size than that prepared at 14 MPa, and showed a microstructure consisting of mainly approximately 6 to 7-μm droplets. When a water-soluble dye PTSA as a model ingredient was loaded in the inner water phase, all W/O/W emulsions showed a high encapsulation efficiency of the dye (>90%) in the inner water phase. Even after 2 wk of storage, >90% of the encapsulated dye still remained in the inner water phase; however, severe droplet aggregation was observed at relatively high PGPR and WPI concentrations.