Protein-stabilized Nanoemulsions Research Articles

Changes in Emulsifying Properties, Droplet Size, Turbidity and Lipid Oxidation of Pea Protein Nanoemulsions Exposed to High-Intensity Ultrasound and High-Pressure Homogenization during Storage

The current work was conducted to explore the influence of two non-thermal technologies (high pressure homogenization (HPH) and high-intensity ultrasound (HIU)) on emulsifying properties, droplet size, turbidity and lipid oxidation of pea protein-stabilized nanoemulsions (PPNs) during seven days of storage. The smallest droplet size (198.3 nm) was obtained for the samples exposed to 10 min HIU at 100% amplitude among all treatments. The same sample also showed the least lipid oxidation (98 mmol/kg) during storage. There was a positive relationship between droplet size and turbidity values. All HIU-treated PPNs exhibited less turbidity and smaller droplet size where the control PPN samples demonstrated the most turbid structure (4.05) with the biggest droplet size (413.9 nm). Similar positive relationship was also found between the variables of droplet size and lipid oxidation. All HIU-treated PPNs exhibited less lipid oxidation and smaller droplet size where the control PPNs demonstrated the most lipid oxidation with the biggest droplet size. Last but not least, among the treatments, the PPNs exposed to 10 min HIU at 100% amplitude showed the highest emulsifying activity index (EAI, 212 m2g-1) and emulsifying stability index (ESI, 59 min), whereas the PPNs with no treatment showed the lowest EAI (69 m2g-1) and ESI (21 min).

Nanoemulsion stabilized by β-lactoglobulin: A promising strategy to encapsulate curcumin for topical delivery

Curcumin has many interesting biological properties. However, it is poorly soluble and stable in aqueous media and its formulation is complex. Protein-stabilized nanoemulsions (NEs) are proposed as encapsulating systems to increase curcumin solubility while protecting it from degradations. Results showed that the incorporation of curcumin within the oily phase of NEs did not modify the size and the zeta potential of nanodroplets as well as NEs stability over time. A creaming phenomenon was observed in NEs over an 80-day storage period. However, droplet stabilization against coalescence was ensured by the formation of an elastic oil/water interface following the adsorption of β-lactoglobulin and/or curcumin. An in vitro study with Franz cells showed that curcumin diffused slowly through a synthetic membrane mimicking the skin. Thus, curcumin encapsulation in the dispersed phase of NEs seemed to be a promising strategy for the development of a topical formulation as NEs increased curcumin solubility and allowed its controlled diffusion through a synthetic skin model.

Sea buckthorn pulp oil nanoemulsions fabricated by ultra-high pressure homogenization process: A promising carrier for nutraceutical

Food protein-stabilized with nanoemulsions were prepared by ultra-high pressure homogenization under appropriate homogenization condition, so as to improve the poor water solubility and instability of sea buckthorn pulp oil (SBPO). SBPO nanoemulsions stabilized by sodium caseinate (SC) and whey protein isolates (WPI) suggested good microstructures and rheological properties. SC-stabilized SBPO nanoemulsions system showed smaller mean particle size (<260 nm), lower Ke value (6.3–20.4%) and higher absolute value of zeta potential (55.70–67.20mV) over various pH (3–9), salinity (0–200 mM), temperature (20–80 °C). The SC-stabilized SBPO nanoemulsions showed no significantly increase in droplet size over 30 days at 4 °C and 25 °C, which proved it has good stability. The antioxidant activity of SC-stabilized SBPO nanoemulsions was evaluated by employing cellular antioxidant activity (CAA) model and the results showed good antioxidant activity. This study implied that protein-stabilized nanoemulsions are a promising carrier for extending the applications of SBPO as nutraceutical or functional dairy beverage.

Pea protein based vitamin D nanoemulsions: Fabrication, stability and in vitro study using Caco-2 cells

Pea protein-stabilized nanoemulsions were prepared to encapsulate vitamin D with the aim to develop novel non-dairy functional foods for vitamin D fortifications. Homogenization conditions of 20 kpsi and two homogenization cycles were identified as optimal conditions for producing stable nanoemulsions. The nanoemulsions exhibited controllable sizes (170–350 nm), good stability with zeta-potential of –25 mV, and high vitamin encapsulation efficiency (94–96%). Cellular uptake efficiency of small sized nanoemulsions (233 nm) was ~2.5 times higher than large sized nanoemulsions (350 nm). Interestingly, protein-based nanoemulsions exhibited significantly higher cellular uptake than emulsions prepared using a combination of protein and lecithin. The vitamin D transport efficiency across Caco-2 cells for small sized nanoemulsions (233 nm) was ~5.3 times greater than free vitamin D suspension. This research demonstrated that pea protein can be used as an effective emulsifier for preparing food nanoemulsions, which may enhance vitamin D bioavailability and improve vitamin deficiency status in aged population.

Salt and pH-Induced Attractive Interactions on the Rheology of Food Protein-Stabilized Nanoemulsions.

This research aimed to investigate the possibility of forming gelled nanoemulsions (NEs) by inducing attractive interactions among the nanodroplets. The effect of salt concentration and changes in pH on the stability and gelation behavior of 2, 4, and 5% sodium caseinate (SC) and whey protein isolate (WPI)-stabilized 40% canola oil-in-water NEs were investigated. For the effect of salt, sodium chloride was added in a concentration of 0.1, 0.5, and 1 M in the continuous phase of the NEs at neutral pH, whereas to study the effect of acidification, the pH of the NEs was adjusted to the isoelectric point (pI) of the proteins. The addition of salt led to attractive gelation in WPI NEs because of a screening of charge. In contrast, the gel strength of SC-stabilized NEs was reduced with salt, which was attributed to the loss of close packing of droplets and their surrounding repulsive barriers because of charge screening and to the steric barrier of interfacial SC preventing droplet aggregation. All the NEs with pH at the pI of proteins transformed into strong attractive gels made of droplet aggregates irrespective of the type or concentration of protein because of the complete charge neutralization. The strength of the acidified NE gels increased with a decrease in droplet size and the type of protein used. Overall, research on the effect of different environmental factors on the stability and gelation behavior of protein-stabilized NEs could be useful for possible applications of these nanoscale materials in various food systems.

Impact of an indigestible oil phase (mineral oil) on the bioaccessibility of vitamin D3 encapsulated in whey protein-stabilized nanoemulsions

These is some interest in replacing digestible fats with indigestible ones to decrease the energy-density of foods. The utilization of indigestible oils, however, may have unforeseen nutritional consequences, such as the reduction of vitamin bioavailability. In this study, the impact of an indigestible oil on the bioaccessibility of emulsified vitamin D3 (VD) was examined. We prepared four kinds of nanoemulsions using different combinations of a digestible oil (DO) and an indigestible oil (IO): DO only; IO only; an oil mixture (OM) consisting of 1:1 DO:IO mixed before homogenization; and, an emulsion mixture (EM) consisting of 1:1 DO:IO nanoemulsions mixed after homogenization. A gastrointestinal tract (GIT) model was employed to elucidate the kinetics of VD bioaccessibility from the nanoemulsions. Both the lipid digestion rate and vitamin bioaccessibility decreased in the same order: DO > OM ≈ EM > IO. The change in vitamin bioaccessibility over time under simulated small intestine conditions was also measured. With the exception of the IO nanoemulsions, the vitamin bioaccessibility increased to a maximum value after around 30 min, but then decreased during the following 24 h. This effect was attributed to an initial solubilization of the vitamin within the mixed micelles, followed by their precipitation during prolonged incubation. Our results show that lipid digestion, micelle solubilization, and micelle aggregation impact the in vitro bioaccessibility of vitamin D. This knowledge may be helpful for designing more efficacious nanoemulsion-based delivery systems for fat-soluble vitamins.

Physicochemical Properties and Storage Stability of Food Protein-Stabilized Nanoemulsions.

This study investigated the preparation and properties of corn oil nanoemulsions stabilized by peanut protein isolate (PPI), rice bran protein isolate (RBPI), soybean protein isolate (SPI), and whey protein isolate (WPI). The mean droplet diameter of four protein-stabilized nanoemulsions prepared via ultrasound method was less than 245 nm. PPI-stabilized nanoemulsions showed better stability for 4 weeks, while the mean droplet diameter of RBPI-stabilized nanoemulsions had exceeded 1000 nm during the third week of storage. Fourier transform infrared and interfacial tension (IT) analysis showed that the higher level of disordered structure and lower IT of proteins made the stability of nanoemulsions better. Moreover, bivariate correlation analysis discovered that α-helix (p < 0.01) and β-turn (p < 0.05) of proteins were related to the mean droplet diameter of nanoemulsions, the random coil (p < 0.05) was related to the zeta potential of nanoemulsions. This study provided new idea for the relationship between the structure of protein and properties of protein-stabilized nanoemulsions.

Enhancement of physicochemical properties of whey protein-stabilized nanoemulsions by interfacial cross-linking using cinnamaldehyde

The impact of incorporating different levels of cinnamaldehyde (CA) into the oil phase used to prepare whey protein isolate (WPI)-stabilized nanoemulsions was investigated. Stable nanoemulsions could not be formed by pure CA, which was attributed to gravitational separation, but they could be formed from mixtures of CA and triglycerides. The mean particle diameter produced by homogenization decreased with increasing CA, which was attributed to the reduction of oil phase interfacial tension and viscosity. CA enhanced the physical stability of the nanoemulsions to salt addition (0–500 mM) and thermal processing (30–90 °C), but could not prevent droplet flocculation near the protein's isoelectric point (around pH 5). SDS-PAGE and surface load measurements indicated that more protein accumulated at the droplet surfaces in the presence of CA, which was attributed to interfacial crosslinking. CA may therefore be a useful means of modulating the physicochemical properties of protein-stabilized nanoemulsions.

Physicochemical properties and storage stability of soybean protein nanoemulsions prepared by ultra-high pressure homogenization

This study investigated the effects of the ultrahigh pressure homogenization (pressure, protein concentration, oil phase fraction, pH, temperature, and ionic strength) and storage on the properties of nanoemulsions (100–500nm range), which were stabilized by laboratory-prepared soybean protein isolate (SPI), β-conglycinin (7S) and glycinin (11S). The nanoemulsions made with SPI, 7S and 11S proteins exhibited considerable stability over various ionic strengths (0–500mM NaCl), pH (<4 or >7), thermal treatments (30–60°C) and storage (0–45days). The far-UV spectra of SPI, 7S, 11S dispersions, and SPI-, 7S-, 11S protein-stabilized nanoemulsions were analyzed for the protein structural changes following lipid removal. The ultra-high pressure homogenization changed the secondary structure of SPI, 7S, 11S proteins in the nanoemulsions, and enhanced their stability. This study demonstrated that SPI, 7S, and 11S proteins can be used as effective emulsifiers in nanoemulsions prepared by ultra-high pressure homogenization.

Improved stabilization of nanoemulsions by partial replacement of sodium caseinate with pea protein isolate

In this research, pea protein isolate (PPI) was used to partially replace sodium caseinate (SC) in the formation and long-term stabilization of 5 wt% oil-in-water nanoemulsions. Both SC and PPI alone and their 1:1 mixture, at various total protein concentrations, were used to prepare the nanoemulsions using a high-pressure homogenizer. The presence of both the proteins at the oil droplet interface was confirmed by SDS-PAGE analysis of interfacial protein from the mixed-protein-stabilized nanoemulsions. All SC-stabilized nanoemulsions (d32 < 200 nm) displayed creaming due to depletion flocculation by excess proteins. PPI alone also failed to produce stable flowable nanoemulsions (d32 > 200 nm) due to excessive droplet and protein aggregation. Interestingly, nanoemulsions stabilized by 1:1 mixture of SC and PPI did not display any creaming or aggregation and remained stable for more than 6 months (d32 < 200 nm). It was proposed that under high-pressure homogenization disrupted pea proteins aggregate interacted with SC in the continuous phase of the nanoemulsions, which prevented depletion flocculation effect of SC by increasing the size and decreasing the number-density of the depletants and interfered with PPI aggregation thereby blocking both the destabilization mechanisms seen in individual protein-stabilized nanoemulsions. The interaction between the SC and PPI in the aqueous phase were confirmed by the change in surface hydrophobicity and intrinsic fluorescence of the homogenized mixed-protein solutions. The proposed method could be a novel way of utilizing plant proteins in the long-term stabilization of nanoemulsions in the food and beverage industry.

Reprint of: Impact of alcohols on the formation and stability of protein-stabilized nanoemulsions

Nanoemulsions are increasingly being used for encapsulation, protection, and delivery of bioactive lipids, however, their formation from natural emulsifiers is still challenging. We investigated the impact of alcohol on the formation and stability of protein-stabilized oil-in-water nanoemulsions prepared by high-pressure homogenization. The influence of different alcohols (ethanol, 1-propanol, and 1-butanol) at various concentrations (0–25% w/w) on the formation and stability of emulsions stabilized by sodium caseinate, whey protein isolate, and fish gelatin was investigated. The mean particle diameter decreased with increasing alcohol concentrations from 0 to 10%w/w, but extensive droplet aggregation occurred at higher levels. This phenomenon was attributed to enhanced protein–protein interactions between the adsorbed emulsifier molecules in the presence of alcohol leading to droplet flocculation. The smallest droplets (d<100nm) were obtained when 10%w/w 1-butanol was added to sodium caseinate-stabilized nanoemulsions, but relatively small droplets (d<150nm) could also be obtained in the presence of a food-grade alcohol (ethanol). This study demonstrated that alcohol addition might be a useful tool for producing protein-stabilized nanoemulsions suitable for use as delivery systems of lipophilic bioactive agents.

Impact of alcohols on the formation and stability of protein-stabilized nanoemulsions

Nanoemulsions are increasingly being used for encapsulation, protection, and delivery of bioactive lipids, however, their formation from natural emulsifiers is still challenging. We investigated the impact of alcohol on the formation and stability of protein-stabilized oil-in-water nanoemulsions prepared by high-pressure homogenization. The influence of different alcohols (ethanol, 1-propanol, and 1-butanol) at various concentrations (0–25% w/w) on the formation and stability of emulsions stabilized by sodium caseinate, whey protein isolate, and fish gelatin was investigated. The mean particle diameter decreased with increasing alcohol concentrations from 0 to 10%w/w, but extensive droplet aggregation occurred at higher levels. This phenomenon was attributed to enhanced protein–protein interactions between the adsorbed emulsifier molecules in the presence of alcohol leading to droplet flocculation. The smallest droplets (d<100nm) were obtained when 10%w/w 1-butanol was added to sodium caseinate-stabilized nanoemulsions, but relatively small droplets (d<150nm) could also be obtained in the presence of a food-grade alcohol (ethanol). This study demonstrated that alcohol addition might be a useful tool for producing protein-stabilized nanoemulsions suitable for use as delivery systems of lipophilic bioactive agents.

Food protein-stabilized nanoemulsions as potential delivery systems for poorly water-soluble drugs: preparation, in vitro characterization, and pharmacokinetics in rats

Nanoemulsions stabilized by traditional emulsifiers raise toxicological concerns for long-term treatment. The present work investigates the potential of food proteins as safer stabilizers for nanoemulsions to deliver hydrophobic drugs. Nanoemulsions stabilized by food proteins (soybean protein isolate, whey protein isolate, β-lactoglobulin) were prepared by high-pressure homogenization. The toxicity of the nanoemulsions was tested in Caco-2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide viability assay. In vivo absorption in rats was also evaluated. Food protein-stabilized nanoemulsions, with small particle size and good size distribution, exhibited better stability and biocompatibility compared with nanoemulsions stabilized by traditional emulsifiers. Moreover, β-lactoglobulin had a better emulsifying capacity and biocompatibility than the other two food proteins. The pancreatic degradation of the proteins accelerated drug release. It is concluded that an oil/water nanoemulsion system with good biocompatibility can be prepared by using food proteins as emulsifiers, allowing better and more rapid absorption of lipophilic drugs.

Protein-Stabilized Nanoemulsions and Emulsions: Comparison of Physicochemical Stability, Lipid Oxidation, and Lipase Digestibility

The properties of whey protein isolate (WPI) stabilized oil-in-water (O/W) nanoemulsions (d(43) ≈ 66 nm; 0.5% oil, 0.9% WPI) and emulsions (d(43) ≈ 325 nm; 0.5% oil, 0.045% WPI) were compared. Emulsions were prepared by high-pressure homogenization, while nanoemulsions were prepared by high-pressure homogenization and solvent (ethyl acetate) evaporation. The effects of pH, ionic strength (0-500 mM NaCl), thermal treatment (30-90 °C), and freezing/thawing on the stability and properties of the nanoemulsions and emulsions were compared. In general, nanoemulsions had better stability to droplet aggregation and creaming than emulsions. The nanoemulsions were unstable to droplet flocculation near the isoelectric point of WPI but remained stable at higher or lower pH values. In addition, the nanoemulsions were stable to salt addition, thermal treatment, and freezing/thawing (pH 7). Lipid oxidation was faster in nanoemulsions than emulsions, which was attributed to the increased surface area. Lipase digestibility of lipids was slower in nanoemulsions than emulsions, which was attributed to changes in interfacial structure and protein content. These results have important consequences for the design and utilization of food-grade nanoemulsions.

Fabrication of protein-stabilized nanoemulsions using a combined homogenization and amphiphilic solvent dissolution/evaporation approach

Oil-in-water nanoemulsions containing small lipid droplets ( d < 100 nm) are finding increasing applications within the food industry as delivery systems in transparent foods and beverages, and to increase the bioavailability of lipophilic active agents. In this study, we show that nanoemulsions can be fabricated from food-grade ingredients (corn oil, whey protein, and water) using simple processing operations (homogenization, dilution and solvent evaporation). Nanoemulsions were formed by homogenizing 10 wt% organic phase (corn oil and ethyl acetate) with 90 wt% aqueous phase (water and whey protein isolate). The mean particle diameter of the emulsions decreased with increasing ethyl acetate concentration in the organic phase, which was attributed to its ability to alter the size of the droplets produced during homogenization, as well as its ability to be removed from the droplets by dissolution and/or evaporation after homogenization. The particle size also decreased with increasing emulsifier concentration. These nanoemulsions may be useful as delivery systems in the food, pharmaceutical and cosmetics industries.

Structural Behaviour of Lipid Droplets in Protein-stabilized Nano-emulsions and Stability of α-Tocopherol

A stearin-rich milk fraction was used, alone or in combination with α-tocopherol, for the preparation of oil-in-water sodium caseinate-stabilized nano-emulsions. Fat droplets in these two emulsions were characterized for their size distribution and physical stability against aggregation–coalescence, for their heat-induced structural behaviour, and for their ability to protect α-tocopherol during oxidation. Inclusion of α-tocopherol led to changes in the particle size distributions of fat droplets: in the presence of α-tocopherol, approximately 75% of fat droplets were lower than 1 μm, instead of 100% in the absence of α-tocopherol. On the other hand, thermal transitions observed by differential scanning calorimetry showed that supercooling (the increase in differences between temperature of initial crystallization and melting completion) was higher in the emulsified milk fat samples containing α-tocopherol. In addition to a decrease in the temperature of fat crystallization leading this change in the supercooling effect of α-tocopherol, small- and wide-angle X-ray diffraction patterns (SAXS and WAXS, respectively) observed under cooling and re-heating cycles showed that heat-induced polymorphic transitions from\({\text{2L}}_{\text{ $ \alpha $ }} \to {\text{2L}}_{{\text{ $ \beta $ }}\prime } \) forms were more impeded in the emulsion containing α-tocopherol. Immobilisation of α-tocopherol in fat droplets composed by high melting temperature milk fat triglycerides seemed to lead to its protection against degradation.