Interactions between Whey Protein Isolate and Soy Protein Fractions at Oil–Water Interfaces: Effects of Heat and Concentration of Protein in the Aqueous Phase

ABSTRACT:  The 2 main storage proteins of soy—glycinin (11S) and β-conglycinin (7S)—exhibit unique behaviors during processing, such as gelling, emulsifying, or foaming. The objective of this work was to observe the interactions between soy protein isolates enriched in 7S or 11S and whey protein isolate (WPI) in oil–water emulsion systems. Soy oil emulsion droplets were stabilized by either soy proteins (7S or 11S rich fractions) or whey proteins, and then whey proteins or soy proteins were added to the aqueous phase. Although the emulsifying behavior of these proteins has been studied separately, the effect of the presence of mixed protein systems at interfaces on the bulk properties of the emulsions has yet to be characterized. The particle size distribution and viscosity of the emulsions were measured before and after heating at 80 and 90 °C for 10 min. In addition, SDS-PAGE electrophoresis was carried out to determine if protein adsorption or exchanges at the interface occurred after heating. When WPI was added to soy protein emulsions, gelling occurred with heat treatment at WPI concentrations >2.5%. In addition, whey proteins were found adsorbed at the oil–water interface together with 7S or 11S proteins. When 7S or 11S fractions were added to WPI-stabilized emulsions, no gelation occurred at concentrations up to 2.5% soy protein. In this case also, 7S or 11S formed complexes at the interface with whey proteins during heating.     

The effect of chitosan on the properties of emulsions stabilized by whey proteins

The influence of the cationic amino polysaccharide chitosan content (0–0.5%) on particle size distribution, creaming stability, apparent viscosity, and microstructure of oil-in-water emulsions (40% of rapeseed oil) containing whey protein isolate (WPI) (4%) at pH 3 was investigated. The emulsifying properties, apparent viscosity and phase separation behaviour of aqueous WPI/chitosan mixture at pH 3 were also studied. The interface tension data showed that WPI/chitosan mixture had a slightly higher emulsifying activity than had whey protein alone. An increase in chitosan content resulted in a decreased average particle size, higher viscosity and increased creaming stability of emulsions. The microstructure analysis indicated that increasing concentration of chitosan resulted in the formation of a flocculated droplet network. This behaviour of acidic model emulsions containing WPI and chitosan was explained by a flocculation phenomenon.     

乳清分离蛋白-葡聚糖接枝物的乳化性能研究

A294和褐变程度的变化证实乳清分离蛋白与葡聚糖在干热处理条件下确实发生了以美拉德反应为机理的接枝反应。由于亲水性葡聚糖的共价接入,导致接枝物在O/W乳液中能够快速且更为紧密地吸附在油-水界面上,在油滴表面形成厚的界面膜,从而提高了乳清分离蛋白的乳化性能。在pH3~10范围内,接枝产物G150的乳化活性明显优于原蛋白;对接枝物样品溶液在90°C保温处理10min后,可以进一步提高其乳化活性;高盐体系中接枝产物G150的乳化活性并未发生明显的下降。     

Ability of whey protein isolate and/or fish gelatin to inhibit physical separation and lipid oxidation in fish oil-in-water beverage emulsion

The effect of pH on the capability of whey protein isolate (WPI) and fish gelatin (FG), alone and in conjugation, to form and stabilize fish oil-in-water emulsions was examined. Using layer-by-layer interfacial deposition technique for WPI–FG conjugate, a total of 1% protein was used to prepare 10% fish oil emulsions. The droplets size distributions and electrical charge, surface protein concentration, flow and dynamic rheological properties and physiochemical stability of emulsions were characterize at two different pH of 3.4 and 6.8 which were selected based on the ranges of citrus and milk beverages pHs, respectively. Emulsions prepared with WPI–FG conjugate had superior physiochemical stability compare to the emulsions prepared with individual proteins. Higher rate of coalescence was associated with reduction in net charge and consequent decrease of the repulsion between coated oil droplets due to the proximity of pH to the isoelectric point of proteins. The noteworthy shear thinning viscosity, as an indication of flocculation onset, was associated with whey protein stabilized fish oil emulsion prepared at pH of 3.4 and gelatin stabilized fish oil emulsion made at pH of 6.8. At pH 3.4, it appeared that lower surface charge and higher surface area of WPI stabilized emulsions promoted lipid oxidation and production of hexanal.     

Water‐soluble myofibrillar proteins prepared by high‐pressure homogenisation: a comparison study on the composition and functionality

To expand utilisation of meat in various products, the characterisation and functionalities of water‐soluble myofibrillar proteins (WSMP) induced by high‐pressure homogenisation (HPH) were determined by comparison with those of soy protein isolate (SPI) and whey protein isolate (WPI). WSMP had high contents of protein (87.40%), which was mainly composed of myosin, actin and tropomyosin. The essential amino acids of WSMP achieved the FAO/WHO/UNO (2007) standards for preschool children, and the contents of lysine and sulphur‐containing amino acids of WSMP were higher than those of SPI, making it desirable for children formulations. WSMP showed higher surface hydrophobicity while its water solubility was similar to that of SPI, but lower than that of WPI. WSMP demonstrated superior water/oil absorption capacities and emulsifying properties. The fibrous structure and high hydrophobic activity characteristics of WSMP were able to stabilise oil droplets with submicron droplet size, consequently responsible for its excellent emulsifying properties.     

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.     

Partition and digestive stability of α-tocopherol and resveratrol/naringenin in whey protein isolate emulsions

Co-encapsulation of multiple bioactive components is an emerging field that shows promise as an approach to develop functional foods. Hydrophobic components are generally dissolved in the inner oil phase of protein-stabilised emulsions. Some components may co-adsorb to oil droplet surfaces, due to the ligand-binding properties of proteins. In this study, α-tocopherol and resveratrol/naringenin were co-encapsulated in emulsions stabilised by whey protein isolate (WPI). α-Tocopherol was totally encapsulated and its partitioning inside oil droplets was about 3.3 times that bound by free WPI in the aqueous phase. The total encapsulation efficiency for resveratrol or naringenin was 52% and 58%, respectively. Addition of resveratrol improved digestive stability of α-tocopherol, but naringenin did not. Co-encapsulation with α-tocopherol had no significant influence on the digestive stability of resveratrol/naringenin. The data gathered here should be useful for the delivery of bioactive components with different solubilities.     

Whey protein-stabilized emulsion properties in relation to thermal modification of the continuous phase

The aim of the present study was to investigate the effect of the protein heat treatment on the physical properties of whey protein concentrate dispersions and the resulting protein-stabilized 30% O/W emulsions. Protein preheating influenced protein particles sizes, viscosity, density, emulsifying capacity and conductivity of protein dispersions. Emulsion stability was improved by protein heat treatment. The emulsions showed bimodal particle size distributions and higher particle sizes with increasing temperature. Thermal modification of the continuous phase influenced flow characteristics of the emulsions and did not affect emulsion conductivity. Heating of whey proteins may generate stable nano-particles and/or aggregates that are able to improve stability of the emulsions. These small structures may stabilize the emulsions by increasing viscosity of the continuous phase and by taking part in formation of the second protein layer on the oil droplets.     

慢消化椰油乳液系列体系的构建及其微流变特性和体外消化特性对比

本文以椰子油为芯材,乳清分离蛋白(Whey protein isolate,WPI)为壁材制备单层椰油乳液,再以单层椰油乳液为芯材,分别以羧甲基纤维素钠(Carboxmethylcellulo sesodium,CMC)、纤维素纳米晶体(Cellulose nanocrystals,CNC)、壳聚糖(Chitosan,CNI)、微晶纤维素(Microcrystalline cellulose,MCC)为壁材制备四种双层椰油乳液,进而探究各乳液体系的微流变特性和体外消化特性。结果显示,WPI-CNC稳定的椰油乳液体系粘弹性最高(P<0.05),乳液中的粒子不能自由运动,乳液的固液平衡值最低(P<0.05),乳液中粒子运动的速率低;WPI-CNC稳定的椰油乳液有最低的肠释放率,且释放速率最为缓慢;除WPI-CNC稳定的椰油乳液外,各乳液体系经胃相消化后均出现明显聚集,小肠消化后聚集程度增加;WPI、WPI-CNC、WPI-CMC稳定的椰油乳液经过口腔、胃、肠消化后平均粒径依次增加,粒径分布出现多峰现象;肠消化后,各乳液表面负电位增大。综上,椰油乳液的流变学特性显著影响其体外消化率,WPI-CNC稳定的椰油乳液体外消化率最低且消化最慢。     

Comparison of emulsifying properties of milk fat globule membrane materials isolated from different dairy by-products

Emulsifying properties of milk fat globule membrane (MFGM) materials isolated from reconstituted buttermilk (BM; i.e., BM-MFGM) and BM whey (i.e., whey-MFGM), individually or in mixtures with BM powder (BMP) were compared with those of a commercial dairy ingredient (Lacprodan PL-20; Arla Foods Ingredients Group P/S, Viby, Denmark), a material rich in milk polar lipids and proteins. The particle size distribution, viscosity, interfacial protein, and polar lipids load of oil-in-water emulsions prepared using soybean oil were examined. Pronounced droplet aggregation was observed with emulsions stabilized with whey-MFGM or with a mixture of whey-MFGM and BMP. No aggregation was observed for emulsions stabilized with BM-MFGM, Lacprodan PL-20, or a mixture of BM-MFGM and BMP. The surface protein load and polar lipids load were lowest in emulsions with BM-MFGM. The highest protein load and polar lipids load were observed for emulsions made with a mixture of whey-MFGM and BMP. The differences in composition of MFGM materials, such as in whey proteins, caseins, MFGM-specific proteins, polar lipids, minerals, and especially their possible interactions determine their emulsifying properties.     

乙醇诱导改性对乳清分离蛋白结构及乳化特性的影响

本实验以未经乙醇溶液处理的乳清分离蛋白(Whey protein isolate,WPI)作为对照组,研究不同浓度乙醇(10%、30%、50%和70%,v/v)处理对WPI结构特性和乳化特性的影响。结果表明,乙醇诱导改性会使WPI的二级结构和三级结构发生显著变化。当乙醇浓度低于50%时可以使蛋白结构展开,而乙醇浓度高于50%时会使WPI分子之间通过二硫键形成聚合物。另外,与对照组相比,乙醇改性能够赋予WPI更高的表面疏水性和更佳的乳化特性(包括乳化活性和乳化稳定性),且乙醇浓度为50%时改善效果最为显著(P<0.05)。上述研究结果表明,乙醇诱导改性是一种有效的改善WPI乳化特性的改性方法,从而为蛋白质的改性奠定了理论基础。     

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.     

Behaviour of oil droplets during spray drying of milk-protein-stabilised oil-in-water emulsions

The effects of spray drying on the behaviour of oil droplets in oil-in-water emulsions (12.0%, w/w, maltodextrin; 20.0%, w/w, soya oil) stabilised with either sodium caseinate or whey protein isolate (WPI) were examined as a function of protein concentration (0.5–5.0%, w/w). Spray drying and redispersion caused a shift in the droplet size distribution to larger values for all emulsions made using low protein concentrations (0.5–2.0%, w/w), in comparison with their respective parent emulsions. However, the droplet size distribution was affected only very slightly by spray drying when the protein concentration was above 2.0% (w/w). The effects of maltodextrin concentration (1.0–25.0%, w/w) on the behaviour of WPI-stabilised emulsions (0.5–10.0%, w/w, WPI, 20.0%, w/w, soya oil) were also examined. Emulsions containing low levels of maltodextrin showed marked re-coalescence during spray drying and redispersion even at a WPI concentration of 10.0% (w/w).     

Characterization of Chemically and Thermally Treated Oil‐in‐Water Heteroaggregates and Comparison to Conventional Emulsions

Heteroaggregated oil‐in‐water (O/W) emulsions formed by targeted combination of oppositely charged emulsion droplets were proposed to be used for the modulation of physical properties of food systems, ideally achieving the formation of a particulate 3‐dimensional network at comparably low‐fat content. In this study, rheological properties of Quillaja saponins (QS), sugar beet pectin (SBP), and whey protein isolate (WPI) stabilized conventional and heteroaggregated O/W emulsions at oil contents of 10% to 60% (w/w) were investigated. Selected systems having an oil content of 30% (w/w) and different particle sizes (d₄₃ ≤ 1.1 or ≥16.7 μm) were additionally subjected to chemical (genipin or glutaraldehyde) and thermal treatments, aiming to increase network stability. Subsequently, their rheological properties and stability were assessed. Yield stresses (τ₀) of both conventional and heteroaggregated O/W emulsions were found to depend on emulsifier type, oil content, and initial droplet size. For conventional emulsions, high yield stresses were only observed for SBP‐based emulsions (τ₀,_SBP approximately 157 Pa). Highest yield stresses of heteroaggregates were observed when using small droplets stabilized by SBP/WPI (approximately 15.4 Pa), being higher than those of QS/WPI (approximately 1.6 Pa). Subsequent treatments led to significant alterations in rheological properties for SBP/WPI systems, with yield stresses increasing 29‐fold (glutaraldehyde) and 2‐fold (thermal treatment) compared to untreated heteroaggregates, thereby surpassing yield stresses of similarly treated conventional SBP emulsions. Genipin‐driven treatments proved to be ineffective. Results should be of interest to food manufacturers wishing to design viscoelastic food emulsion based systems at lower oil droplet contents.     

The interaction of milk sphingomyelin and proteins on stability and microstructure of dairy emulsions

The interaction between dairy proteins [micellar casein (MC) vs. whey protein isolate (WPI)] and phospholipids [PL; soy phosphatidylcholine (PC) vs. milk sphingomyelin (SM)] in an oil-in-water emulsion system was investigated. Sole PC–stabilized emulsion (1%, wt/vol) showed a significantly larger mean particle diameter (6.5 μm) than SM-stabilized emulsions (3.8 μm). The mean particle diameters of emulsions prepared by the combination of protein (1%, wt/vol) and PL (1%, wt/vol) did not significantly differ from the emulsions prepared with a single emulsifier (MC, WPI, and SM). Emulsion instability differed significantly among samples by a centrifugation-mediated accelerated stability test. Emulsion instability increased in the order of MC+SM < MC+PC, WPI+SM < WPI+PC < MC < SM < WPI < PC. Protein surface load determined by aqueous phase depletion was significantly decreased only in WPI+PC emulsion, whereas no significant difference was found between the MC+SM and WPI+SM emulsions. Topographic and phase images of emulsion surface by atomic force microscopy showed surface layers prepared by protein+PL combinations were composites with different mechanical properties, and PL formed a more compact domain than proteins. A smoother phase image was observed in MC+PL combinations than in WPI+PL counterparts. Based on the microstructure analysis using confocal laser scanning microscopy, combination and MC+SM formed a uniform and thick surface coating of fat droplets. More PC aggregates were observed in the emulsions containing PC (sole PC, MC+PC, and WPI+PC) compared with their SM counterparts. Based on these results, the appropriate selection of the PL matrix is important to modulate the emulsion stability of dairy emulsion products.     

Comparison of the Emulsifying Properties of Fish Gelatin and Commercial Milk Proteins

ABSTRACT: We have compared the flocculation, coalescence, and creaming properties of oil-in-water emulsions prepared with fish gelatin as sole emulsifying agent with those of emulsions prepared with sodium caseinate and whey protein. Two milk protein samples were selected from 9 commercial protein samples screened in a preliminary study. Emulsions of 20 vol% n -tetradecane or triglyceride oil were made at pH 6.8 and at different protein/oil ratios. Changes in droplet-size distribution were determined after storage and centrifugation and after treatment with excess surfactant. We have demonstrated the superior emulsifying properties of sodium caseinate, the susceptibility of whey protein emulsions to increasing flocculation on storage, and the coalescence of gelatin emulsions following centrifugation.     

Coalescence Index of Protein-Stabilized Emulsions

A simple method is proposed to estimate coalescence stability of protein-stabilized emulsions. Coalescence was accelerated through agitation and measured by change in emulsion turbidity over time. A coalescence index (CI) was determined and used to compare emulsions stabilized with casein, whey (WPI) and soy protein isolates (SPI). CI increased when stirring rate increased. Casein produced more stable emulsions, followed by WPI and SPI. High homogenization pressure increased coalescence stability of WPI and SPI-stabilized emulsions and decreased coalescence stability of casein-stabilized emulsions. Microscopic examination, showed agitation of the emulsion had clearly induced formation of large oil droplets which acted as coalescence nuclei.     

乳清分离蛋白聚集体乳化性能及其Pickering乳液稳定性

以乳清分离蛋白（whey protein isolate,WPI）为原料,分别在pH 2.0和pH 7.0条件下,85 ℃加热12 h制备2 种不同形态的蛋白质聚集体,研究2 种聚集体微观形貌的特征以及不同pH值和盐离子浓度下的乳化性能;采用透射电镜、动态光散射、光学显微镜等技术手段,探究WPI与2 种聚集体稳定的Pickering乳液微观结构、盐离子稳定性和热稳定性。结果表明：WPI分别在pH 2.0和pH 7.0且高温加热条件制得2 种微观形貌截然不同的蛋白聚集体（纤维状聚集体和球状聚集体）,并且2 种蛋白聚集体相较于WPI等电点均发生偏移,在不同pH值或盐离子浓度环境下,乳化性能均提高。2 种聚集体所稳定的Pickering乳液对不同pH值、盐离子浓度环境下有更好的稳定性,球状聚集体所稳定的Pickering乳液具有更好的热稳定性。这也为WPI聚集体稳定的Pickering乳液在乳饮料中的应用奠定基础。     

Effect of CMC Molecular Weight on Acid‐Induced Gelation of Heated WPI‐CMC Soluble Complex

Acid‐induced gelation properties of heated whey protein isolate (WPI) and carboxymethylcellulose (CMC) soluble complex were investigated as a function of CMC molecular weight (270, 680, and 750 kDa) and concentrations (0% to 0.125%). Heated WPI‐CMC soluble complex with 6% protein was made by heating biopolymers together at pH 7.0 and 85 °C for 30 min and diluted to 5% protein before acid‐induced gelation. Acid‐induced gel formed from heated WPI‐CMC complexes exhibited increased hardness and decreased water holding capacity with increasing CMC concentrations but gel strength decreased at higher CMC content. The highest gel strength was observed with CMC 750 k at 0.05%. Gels with low CMC concentration showed homogenous microstructure which was independent of CMC molecular weight, while increasing CMC concentration led to microphase separation with higher CMC molecular weight showing more extensive phase separation. When heated WPI‐CMC complexes were prepared at 9% protein the acid gels showed improved gel hardness and water holding capacity, which was supported by the more interconnected protein network with less porosity when compared to complexes heated at 6% protein. It is concluded that protein concentration and biopolymer ratio during complex formation are the major factors affecting gel properties while the effect of CMC molecular weight was less significant.     

Interfacial and emulsifying properties of lentil protein isolate

The dynamic interfacial tension (DIFT) at oil–water interface, diffusion coefficients, surface hydrophobicity, zeta potential and emulsifying properties, including emulsion activity index (EAI), emulsion stability index (ESI) and droplet size of lentil protein isolate (LPI), were measured at different pH and LPI concentration, in order to elucidate its emulsifying behaviour. Sodium caseinate (NaCas), whey protein isolate (WPI), bovine serum albumin (BSA) and lysozyme (Lys) were used as benchmark proteins and their emulsifying property was compared with that of LPI. The speed of diffusion-controlled migration of these proteins to the oil/water interface, was in the following order: NaCas > LPI > WPI > BSA > Lys, while their surface hydrophobicity was in the following order: BSA > LPI > NaCas > WPI > Lys. The EAI of emulsions stabilised by the above proteins ranged from 90.3 to 123.3 m²/g and it was 93.3 ± 0.2 m²/g in LPI-stabilised emulsion. However, the stability of LPI-stabilised emulsions was slightly lower compared to that of WPI and NaCas-stabilised emulsions at the same protein concentration at pH 7.0. The ESI of LPI emulsions improved substantially with decrease in droplet size when protein concentration was increased (20–30 mg/ml). Reduction of disulphide bonds enhanced both the EAI and ESI compared to untreated samples. Heat treatment of LPI dispersions resulted in poor emulsion stability due to molecular aggregation. The stability of LPI-stabilised emulsions was found to decrease in the presence of NaCl. This study showed that LPI can be as effective emulsifiers of oil-in-water emulsions as are WPI and NaCas at ?20 mg/ml concentrations both at low and neutral pH. The emulsifying property of LPI can be improved by reducing the intra and inter-disulphide bond by using appropriate reducing agents.