Depletion flocculation and thermodynamic incompatibility in whey protein stabilised O/W emulsions

The stability of whey protein stabilised emulsions, containing methylcellulose added after emulsification in their bulk phase, was investigated. The phase diagram of the ternary system whey proteins/methylcellulose/water was first established and used to identify the conditions permitting polymer phase separation within the emulsion bulk phase. Emulsions containing a whey protein and methylcellulose concentration in the bulk phase below and above the phase separation threshold could therefore be prepared. Below the phase separation threshold, the creaming rate of the oil droplets was faster than the one predicted by the Stokes equation, due to methylcellulose-induced depletion flocculation. Above the phase separation threshold, the destabilisation of the emulsion involved different mechanisms, depending on the emulsifier adsorbed at the O/W interface. In the case of Tween 40 stabilised droplets, depletion flocculation led to a complete creaming of the fat globules while phase separation led to the formation of two polymer-rich phases, namely a protein-rich phase at the bottom of the tube and a methylcellulose-rich phase above. In the case of whey protein stabilised droplets, phase separation between bulk whey proteins and methylcellulose occurred, and the fat globules were entrapped in the protein-rich phase. These results permitted to describe the destabilisation mechanisms of both Tween 40 and whey protein stabilised emulsions in the presence of unadsorbed polysaccharide. They could be used to better understand the destabilisation processes arising in food emulsions, especially in those emulsions containing whey proteins, small surfactant molecules and polysaccharides.     

Characterization and acid-induced gelation of butter oil emulsions produced from heated whey protein dispersions

Whey protein isolate was dispersed at 4% or 8% (w/v) and heated at neutral pH to produce protein polymers. Butter oil, up to 20%, was homogenized in heated whey protein dispersions at pressure ranging from 10 to 120 MPa. Emulsion gelation was induced by acidification with glucono-δ-lactone. Whey protein polymers produced finely dispersed emulsions with fat droplet diameter ranging from 340 to 900 nm. Homogenization pressure was the main factor influencing droplet size. At low fat volume fraction, the emulsions exhibited Newtonian behaviour. As fat content increased, shear thinning behaviour developed as a result of depletion flocculation. Emulsion consistency index increased with protein and fat concentrations. Increasing homogenization pressure had no effect on Newtonian emulsions but promoted flocculation and significantly increased the consistency of high fat emulsions. Protein concentration was the main factor explaining emulsion gel hardness and syneresis. Syneresis decreased with increasing fat content in the gel.     

Manipulating oral behaviour of food emulsions using different emulsifiers

This study aims to investigate the assumption that the presence of α-amylase in the human saliva will interact instantly with starch and will lead to very different oral behaviour and enhanced flavour release. Hence, orange oil flavoured emulsion was prepared with whey protein isolates (WPI) and modified starch (MS). The stability and flavour release of emulsions were examined through in vitro and in vivo studies. MS emulsion mixed with artificial saliva containing α-amylase resulted in more pronounced changes in mean particle size from 0.185 to 2.35 μm and a significant increase in viscosity. Morphology and turbidity revealed strong flocculation, coalescence and creaming. However, WPI emulsion exhibited very little changes in stability and behaviour. Similar results were observed during oral digestion (in vivo) for both emulsion systems. Moreover, a higher intensity of flavour release (37%) was observed in MS emulsion than WPI. This work demonstrated that a starch-stabilised emulsion has a very different oral behaviour to that of a protein-stabilised emulsion.     

Modifications in stability and structure of whey protein-coated o/w emulsions by interacting chitosan and gum arabic mixed dispersions

The influence of chitosan and gum arabic mixtures on the behaviour of o/w emulsions has been investigated at pH = 3.0. The emulsion behaviour, properties and microstructure were found to be greatly dependent on the precise gum arabic to chitosan ratio. Mixing of gum arabic with chitosan leads to the formation of coacervates of a size dependent on their ratio. Incorporation of low gum arabic to chitosan weight ratios into whey protein-coated emulsions causes depletion flocculation and gravity-induced phase separation. Increasing the polysaccharide weight ratio further, a droplet network with a rather high viscosity (at low shear stress) is generated, which prevents or even inhibits phase separation. At even higher gum arabic to chitosan ratios, the emulsion droplets were immobilised into clusters of an insoluble ternary matrix. Although the emulsion droplet charge had the same sign as that of the coacervates, clusters of oil droplets in a ternary matrix were generated. A mechanism to explain the behaviour of the whey protein-stabilised o/w emulsions is described on the basis of confocal and phase contrast microscopic observations, rheological data, zeta potential measurements, particle size analysis and visual assessment of the macroscopic phase separation events.     

Factors that influence the coalescence stability of protein-stabilised emulsions,estimated from the proportion of oil extracted by hexane

The coalescence stability of protein-stabilised emulsions was estimated by measuring the degree to which the oil content could be extracted by hexane. The hexane extraction method is an empirical one but it correlates well with both an absolute method, such as increase in droplet size, and with another ‘accelerating’ technique, oil separation by centrifugation. Moreover, the hexane extraction method is capable of measuring coalescence stability over a wide range of instability, whereas the centrifugation method only provides information about the final stages of emulsion instability. Among the proteins studied, caseinates were generally the best stabilisers, especially at pH 6. Soya proteins gave rise to emulsions of minimal stability, whereas whey protein concentrate and blood plasma resulted in emulsions of medium stability. The coalescence instability of the protein-stabilised emulsions, viewed overall, was significantly and positively related to the droplet size, the degree of flocculation and the amount of protein in the membrane. High values of these emulsion parameters were due mainly to frequent recoalescence and bridging during emulsification. To minimise these effects emulsifying conditions creating high protein: surface area ratios should be used, as well as proteins that quickly change their conformation at an interface and have low aggregation numbers in solution.     

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.     

Stability of oil‐in‐water emulsions as influenced by thermal treatment of whey protein dispersions or emulsions

Properties of whey protein concentrate stabilised emulsions were modified by protein and emulsion heat treatment (60–90 °C). All liquid emulsions were flocculated and the particle sizes showed bimodal size distributions. The state and surface properties of proteins and coexisting protein/aggregates in the system strongly determined the stability of heat‐modified whey protein concentrate stabilised emulsions. The whey protein particles of 122–342 nm that formed on protein heating enhanced the stability of highly concentrated emulsions. These particles stabilised protein‐heated emulsions in the way that is typical for Pickering emulsions. The emulsions heated at 80 and 90 °C gelled due to the aggregation of the protein‐coated oil droplets.     

Whey protein–carboxymethylcellulose interaction in solution and in oil-in-water emulsion systems. Effect on emulsion stability

Carboxymethylcellulose (CMC) was used as coagulation aid to precipitate the whey proteins from defatted milk serum and the ability of the resulting whey protein concentrate (WPC, protein content: 63.69%) to aid in the physicochemical stabilization of oil-in-water emulsions, during ageing or following the application of heat or freeze–thaw treatment, was investigated, along with the stability of emulsion systems prepared with a commercial whey protein isolate. The stability of WPC emulsions against droplet flocculation and creaming, and to a lesser extent against droplet coalescence, depended on the presence of the CMC molecules in the emulsion continuous phase and the extent of adsorbed protein–polysaccharide interactions as affected by the emulsion pH. Studies on whey protein–CMC interaction were conducted, both in biopolymer mixture solutions and emulsion systems, by applying zeta potential measurement and viscometry techniques. These results were combined with data on protein surface hydrophobicity and on methylene blue-binding ability of CMC molecules and indicated that whey protein–CMC interaction may take place in solution, both at neutral as well as at acidic environments, leading, depending on pH, to the formation of soluble or non-soluble amphiphilic conjugates. In emulsion systems, however, conjugate formation is observed only at relatively acidic pH environments, probably because at a neutral or at a slightly acidic pH whey protein adsorption to the emulsion droplet surface and molecular unfolding does not favour protein–polysaccharide interaction.     

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.     

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.     

Spray-dried whey protein/lactose/soybean oil emulsions. 2. Redispersability, wettability and particle structure

In the present paper redispersion and wettability experiments of spray-dried whey protein-stabilized emulsions are presented. Emulsion droplet size after redispersion gives information about eventual coalescence between emulsion droplets in the powder matrix during drying or storage, resulting in an increase in emulsion droplet size after redispersion. Results from redispersion experiments are combined with previously presented knowledge about powder surface composition and particle structure to elucidate internal processes in the powder matrix and external processes on the powder surface during drying and storage of whey protein powder. The results show that with addition of lactose to whey protein-stabilized emulsions, emulsion droplet structure remains intact in the powder matrix during drying since the emulsion droplet size in the redispersed spray dried emulsion is unchanged. In the absence of lactose there is a growth in emulsion droplet size after redispersion of the spray-dried whey protein-stabilized emulsion, showing that a coalescense of emulsion droplets occurs during the drying or redispersion process. Storage of the whey protein-stabilized powders in a humid atmosphere (relative humidity 75%, 4 days) induces changes in some powders. When the powder contains a critical amount of lactose there is a remarkable increase in emulsion droplet size after redispersion of humid stored powders compared with the emulsion before drying and with the redispersed dry stored powder. In addition, there is a release of encapsulated fat after humid storage of lactose-containing powders detected by electron spectroscopy for chemical analysis. For powders which do not contain any lactose there is no increase in emulsion droplet size after storage in a humid atmosphere compared with the redispersed dry stored emulsion. Addition of only a small amount of lactose prevents coalescence of emulsion droplets and the subsequent increase in droplet size during drying. If the lactose content is kept rather low neither an effect on the droplet size after storage under humid conditions nor a release of fat onto powder surfaces is detected. Furthermore, wettability of the spray-dried whey protein-stabilized emulsions by water is presented. It is concluded that it is beneficial to wettability in water to have as high a coverage of lactose on the powder surface as possible. In addition, a review of particle structure for powders of various composition is presented.     

Prebiotic emulsions stabilised by whey protein and kefiran

The goal of this work has been to assess the influence of a health-promoting hydrocolloid, such as kefiran, in oil-in-water emulsions (O/W: 50/50) containing whey protein isolate (1.0% wt.). Different kefiran concentration levels (0%, 0.25%, 0.5%, 1% wt.) were studied, observing a shift from a fluid-like to a solid-like behaviour and higher viscosities when kefiran content increased. A pseudoplastic behaviour was detected for all systems studied. The observed evolution is attributed to the thickening effect exerted by kefiran, which promoted stability, in spite of observing a certain flocculation degree within the emulsion systems, demonstrated through the addition of sodium dodecyl sulphate (1% wt.). Furthermore, short-term stability of these pseudoplastic emulsions has been verified by laser-scattering techniques. Thus, a Sauter diameter around 1 µm remained unaltered up to 7 days since preparation. The addition of kefiran in the formulation would benefit from their well-known health-promoting effects.     

Intracellular fate of retinyl acetate-loaded submicron delivery systems by in vitro intestinal epithelial cells: A comparison between whey protein-stabilised submicron droplets and micelles stabilised with polysorbate 80

Submicron peanut oil-in-water emulsion stabilised with whey proteins, was processed by ultra-high-pressure homogenisation at 200 MPa (first-stage, 2-pass homogenisation) and an initial emulsion temperature (T_in) of 24 °C. Retinyl acetate (RAC) was selected as a model of a lipophilic biomolecule to be carried by submicron droplets. For comparison, micelles of retinyl acetate (RAC-micelles) were prepared at atmospheric pressure using polysorbate 80 (Tween® 80) as emulsifier. Both types of bio-particles displayed submicron diameters. Their behaviour was investigated on TC7-cell monolayers to characterise their influence on monolayer integrity (TER measurements), cell metabolic activity (MTT-assay) and cell membrane integrity (LDH-leakage). TC7-cell exposure to submicron droplets UHPH-elaborated as shuttles for RAC, and stabilised with proteins, did not impair TC7-cell viability compared with control cells (without deposit) or cells exposed to RAC-micelles. Such results demonstrated UHPH-processing innocuity in terms of Novel Food Regulation and implementation of UHPH as a novel technology. TC7-cell internalisation of whey proteins coating submicron oil droplets was observed using confocal microscopy and fluorescent probes. Cellular uptake of RAC was determined for both systems, after extraction from TC7 cells and HPLC quantitation. Results revealed RAC incorporation into TC7 cells and cellular turn-over of RAC into retinol for both systems without previous in vitro digestion. RAC bioaccessibility appeared yet better in the form of RAC-micelles than after entrapment into oily submicron droplets. Nevertheless, submicron droplets stabilised by proteins displayed a better physical stability than did RAC-micelles, which is the main feature for further developments of carrier systems.     

Effects of ultra-high pressure homogenization on the properties and structure of interfacial protein layer in whey protein-stabilized emulsion

The effect of high pressure homogenization on the properties of whey protein adsorbed on emulsion interface was determined by competitive adsorption with Tween 20, Fourier transform infrared (FT-IR) spectroscopy, and RP-HPLC. The amount of whey proteins desorbed by Tween 20 increased at higher pressure. The results of FT-IR spectroscopy showed that higher homogenization pressures led to decrease of α-helix and increase of β-sheet indicating the formation of fewer interactions with the lipid phase and more interaction between adsorbed whey proteins, respectively. In RP-HPLC profile, the retention time of whey protein desorbed from high pressured emulsion was shorter than that from low-pressured emulsion. From these results, we have shown that higher pressures cause the formation of a more compact interfacial protein layer. Finally, “high pressure” emulsions are more stable than “low-pressure” ones, because of the protein layer formed by protein-protein interactions as well as the decrease of droplet size.     

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.     

Interaction between Emulsion Droplets and Escherichia coli Cells

ABSTRACT Oil‐in‐water emulsions (20% n‐hexadecane, v/v) were stabilized by dodecyltrimethylammonium bromide (DTAB), Tween 20, or sodium dodecyl sulfate (SDS). Particle size distribution and creaming stability were measured before and after adding Escherichia coli cells to emulsions. Both E. coli strains promoted droplet flocculation, coalescence, and creaming in DTAB emulsions, although JM109 cells (surface charge = ‐35 mV) caused faster creaming than E21 cells (surface charge = ‐5 mV). Addition of bacterial cells to SDS emulsions promoted some flocculation and coalescence, but creaming stability was unaffected. Droplet aggregation and accelerated creaming were not observed in emulsions prepared with Tween 20. Surface charges of bacterial cells and emulsion droplets played a key role in emulsion stability.     

Stability of oil–water primary emulsions stabilised with varying levels of casein and whey proteins affected by high-intensity ultrasound

This study evaluates physical and chemical stability of ultrasound-assisted grape seed oil primary emulsions stabilised by varying compositions of caseins to whey proteins (80:20, 60:40, 50:50 and 40:60) at different sono-operating conditions (81.9 and 117.0 J mL⁻¹). Physical and chemical stabilities were influenced by both sonication energy densities and milk protein compositions. Emulsions prepared at 81.9 J mL⁻¹ energy density with ≥40% whey protein fraction (60:40, 50:50, 40:60 and WPI) showed greater physical stability than the emulsions sonicated at 117.0 J mL⁻¹ which exhibited physical instability due to the depletion flocculation mechanism at the critical casein concentration (≥40%). The emulsion oxidative stability was found to be affected by sonication conditions as 117.0 J mL⁻¹ induced the oxidation reactions once the whey concentration exceeds 40%. Therefore, ultrasound prepared emulsions with casein to whey ratios of 60:40, 50:50, 40:60 and WPI at 81.9 J mL⁻¹ energy density was found to be stable for 10 days at 4 °C.     

Emulsification mechanisms and characterizations of cold, gel-like emulsions produced from texturized whey protein concentrate

A novel supercritical fluid extrusion (SCFX) process was used to successfully texturize whey protein concentrate (WPC) into a product with cold-setting gel characteristics that was stable over a wide range of temperature. It was further hypothesized that incorporation of texturized WPC (tWPC) within an aqueous phase could improve emulsion stability and enhance the rheological properties of cold, gel-like emulsions. The emulsifying activity and emulsion stability indices of tWPC and its ability to prevent coalescence of oil-in-water (o/w) emulsions were evaluated and compared with the commercial WPC80. The cold, gel-like emulsions were prepared at different oil fractions (φ = 0.20–0.80) by mixing oil with the 20% (w/w) tWPC dispersion at 25 °C and evaluated using a range of rheological techniques. Microscopic structure of cold, gel-like emulsions was also observed by Confocal Laser Scanning Microscope (CLSM). The results revealed that the tWPC showed excellent emulsifying properties compared to the commercial WPC in slowing down emulsion breaking mechanisms such as creaming and coalescence. Very stable with finely dispersed fat droplets, and homogeneous o/w gel-like emulsions could be produced. Steady shear viscosity and complex viscosity were well correlated using the generalized Cox–Merz rule. Emulsions with higher viscosity and elasticity were obtained by raising the oil fraction. Only 4% (w/w) tWPC was needed to emulsify 80% (w/w) oil with long-term storage stability. The emulsion products showed a higher thermal stability upon heating to 85 °C and could be used as an alternative to concentrated o/w emulsions and in food formulations containing heat-sensitive ingredients.     

Impact of whey protein isolates and concentrates on the formation of protein nanoparticles‐stabilised Pickering emulsions

Whey protein nanoparticles (NPs) were prepared by heat‐induced method. The influences of whey protein isolates (WPIs) and concentrates (WPCs) on the formation of NPs were first investigated. Then Pickering emulsions were produced by protein NPs and their properties were evaluated. After heat treatment, WPC NPs showed larger particle size, higher stability against NaCl, lower negative charge and contact angle between air and water. Dispersions of WPC NPs appeared as higher turbidity and viscosity than those of WPI NPs. The interfacial tension of WPC NPs (~7.9 mN/m at 3 wt% NPs) was greatly lower than that of WPI NPs (~12.1 mN/m at 3 wt% NPs). WPC NPs‐stabilised emulsions had smaller particle size and were more homogeneous than WPI NPs‐stabilised emulsions. WPC NPs‐stabilised emulsions had higher stability against NaCl, pH and coalescence during storage.     

Interfacial composition and stability of emulsions made with mixtures of commercial sodium caseinate and whey protein concentrate

The interfacial composition and the stability of oil-in-water emulsion droplets (30% soya oil, pH 7.0) made with mixtures of sodium caseinate and whey protein concentrate (WPC) (1:1 by protein weight) at various total protein concentrations were examined. The average volume-surface diameter (d₃₂) and the total surface protein concentration of emulsion droplets were similar to those of emulsions made with both sodium caseinate alone and WPC alone. Whey proteins were adsorbed in preference to caseins at low protein concentrations (<3%), whereas caseins were adsorbed in preference to whey proteins at high protein concentrations. The creaming stability of the emulsions decreased markedly as the total protein concentration of the system was increased above 2% (sodium caseinate >1%). This was attributed to depletion flocculation caused by the sodium caseinate in these emulsions. Whey proteins did not retard this instability in the emulsions made with mixtures of sodium caseinate and WPC.