Stability of oil: Water emulsions of amaranth proteins. Effect of hydrolysis and pH

In this contribution we have determined the effect of limited enzymatic hydrolysis on the emulsifying capacity of amaranth proteins. The action of enzyme (alcalase and trypsin) and the pH of the continuous phase of the oil/water emulsion (pH 2.0, 6.3 and 8.0) were the variables analyzed. The results obtained show that amaranth protein isolates, AI, contain proteins species capable of forming and stabilizing emulsions, mainly at acidic pH (2.0) and to a lesser extent at pH 8.0. While the emulsions obtained are sensitive to creaming and flocculation, they do not undergo destabilization by coalescence. The emulsions prepared from proteins subjected to low grade trypsin hydrolysis (TH2.2) are sensitive to creaming - flocculation, whereas alcalase-hydrolyzed proteins (AH1.7 and AH9.5) exhibited a significant destabilization by creaming, flocculation and coalescence, mainly at pH 6.3. The effect of the pH of the aqueous phase was determining on the emulsion stability beside the structural and physicochemical characteristics of protein species utilized as tensioactive. At acidic pH (pH 2.0) the unfolding and charge of polypeptides and the capacity of form a viscoelastic film at the interface were essential while at alkaline pH (pH 8.0) the balance among high and low molecular mass protein species and flexibility of the molecule fixed the emulsions properties.     

Pea protein exhibits a novel Pickering stabilization for oil-in-water emulsions at pH 3.0

In this paper we reported that pea protein isolate (PPI) at pH 3.0 exhibits a novel Pickering stabilization for oil-in-water emulsions. At pH 3.0, most of the proteins in PPI were present in the nanoparticle form, with the hydrodynamic diameter of 134–165 nm depending on the concentration (c; 0.25–3.0 g/100 mL). For the emulsions formed at a specific oil fraction of 0.2, increasing the c from 0.25 to 3.0 g/100 mL resulted in a considerable reduction in the emulsion size, while their creaming stability progressively increased, and especially at c values higher than 2 g/100 mL, no creaming occurred even after storage of 20 days. Confocal laser scanning microscopy observations showed that increasing the c resulted in a progressive increase in extent of droplet flocculation, and at higher c values, a network consisting of flocculated droplets could be formed. The emulsions formed at c values above 1.0 g/100 mL exhibited extraordinary stability against coalescence. The flocculated droplet network formation was closely associated with the increased amount of adsorbed proteins at the interface. The results suggest that pea proteins exhibit a good potential to act as a kind of Pickering stabilizers for oil-in-water emulsions at acidic pHs.     

Emulsifying properties of canola and flaxseed protein isolates produced by isoelectric precipitation and salt extraction

The emulsifying (emulsion capacity, EC; emulsion activity/stability indices, EAI–ESI and creaming stability, CS) and physicochemical properties (surface charge/hydrophobicity, protein solubility, interfacial tension, and droplet size) of chickpea (ChPI), faba bean (FbPI), lentil (LPI), and pea (PPI) protein isolates produced by isoelectric precipitation and salt extraction were investigated relative to each other and a soy protein isolate (SPI). Both the legume source and method of isolate production showed significant effects on the emulsifying and physicochemical properties of the proteins tested. All legume proteins carried a net negative charge at neutral pH, and had surface hydrophobicity values ranging between 53.0 and 84.8 (H₀-ANS), with PPI showing the highest value. Isoelectric precipitation resulted in isolates with higher surface charge and solubility compared to those produced via salt extraction. The EC values ranged between 476 and 542 g oil/g protein with LPI showing the highest capacity. Isoelectric-precipitated ChPI and LPI had relatively high surface charges (~−22.3 mV) and formed emulsions with smaller droplet sizes (~ 1.6 μm), they also displayed high EAI (~ 46.2 m²/g), ESI (~ 84.9 min) and CS (98.6%) results, which were comparable to the SPI.     

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.     

Emulsifying Properties of Legume Proteins Compared to β‐Lactoglobulin and Tween 20 and the Volatile Release from Oil‐in‐Water Emulsions

The emulsifying properties of plant legume protein isolates (soy, pea, and lupin) were compared to a milk whey protein, β‐lactoglobulin (β‐lg), and a nonionic surfactant (Tween 20). The protein fractional composition was characterized using sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. The following emulsion properties were measured: particle diameter, shear surface ζ‐potential, interfacial tension (IT), and creaming velocity. The effect of protein preheat treatment (90 °C for 10 min) on the emulsifying behavior and the release of selected volatile organic compounds (VOCs) from emulsions under oral conditions was also investigated in real time using proton transfer reaction‐mass spectrometry. The legume proteins showed comparable results to β‐lg and Tween 20, forming stable, negatively charged emulsions with particle diameter d_3,2 < 0.4 μm, and maintained stability over 50 d. The relatively lower stability of lupin emulsions was significantly correlated with the low protein surface hydrophobicity and IT of the emulsion. After heating the proteins, the droplet size of pea and lupin emulsions decreased. The VOC release profile was similar between the protein‐stabilized emulsions, and greater retention was observed for Tween 20‐stabilized emulsions. This study demonstrates the potential application of legume proteins as alternative emulsifiers to milk proteins in emulsion products.     

Structure and stability of heat-treated concentrated dairy-protein-stabilised oil-in-water emulsions: A stability map characterisation approach

Emulsion instabilities such as depletion flocculation, coalescence, aggregation and heat-induced protein aggregation may be detrimental to the production of sterilised food emulsions. The type and the amount of protein present in the continuous phase and at the oil–water interface are crucial in the design of emulsions with appropriate stability. In this study, four oil-in-water model emulsion systems (pH 6.8–7.0) were formulated, characterised and categorised according to the potential interactions between protein-coated or surfactant-coated emulsion droplets and non-adsorbed proteins present in the continuous phase. The heat stability, the creaming behaviour and the flow behaviour of the model emulsions were influenced by both the emulsifier type and the type of protein in the continuous phase. The results suggest that this stability map approach of predicting droplet–droplet, droplet–protein and protein–protein interactions will be useful for the future design of heat-stable emulsion-based beverages with good creaming stability at high protein concentrations.     

Emulsifying properties of pea protein/guar gum conjugates and mayonnaise application

Modified plant protein may be used as a healthy and more functional emulsifier in food products. The objective of this study was to evaluate the emulsifying properties of functionally enhanced pea protein (i.e. pea protein conjugated with guar gum, G-PPI) and its potential application to mayonnaise, compared with unmodified pea protein. Emulsions containing G-PPI were prepared at different pH, salt concentrations, protein concentrations and oil/water ratios. Mayonnaise samples were prepared using the pea proteins or egg yolk powder. Various characteristics of the emulsions, including droplet size, apparent viscosity, viscoelasticity and microstructure, were analysed. The emulsions with G-PPI had significantly increased stability of up to 89.4% and apparent viscosity of up to 48.62 mPa.s. The G-PPI emulsion had a smaller average droplet size of 934.4 nm at pH 7 compared with the PPI emulsion (stability 62.7%, apparent viscosity 22.8 mPa.s and droplet size 1664.8 nm). The pH, NaCl concentration, protein concentration and oil/water ratio greatly affected the emulsifying properties. The G-PPI mayonnaise at higher protein concentrations (6 or 8%) exhibited excellent emulsifying and rheological properties. The modified pea protein through the green modification process with natural polysaccharides could be used as a safe and functional emulsifier in different emulsified foods.     

Complexation of pea protein isolate with dextran sulphate and interfacial adsorption behaviour and O/W emulsion stability at acidic conditions

This study was aimed at improving the emulsifying property and physical stability of pea protein isolate (PPI) stabilised emulsions at acidic conditions by complexation with dextran sulphate (DS). Soluble and insoluble complexes with different charge and particle size were formed depending on the phase separation behaviour. The surface adsorption of PPI became slower after complexation with DS, but the percentage of adsorbed proteins at the oil–water interface was not affected. The formation of PPI–DS soluble complexes at high content of DS (≥0.4%) significantly improved the negative net charges of PPI, prevented the aggregation of protein, which further improved the emulsifying property of PPI at acidic conditions through the strong electrostatic repulsion and steric hindrance effects. Insoluble complexes with relatively weak net charge and large particles were formed at low DS content (≤0.2%), resulting in the bridging flocculation of oil droplets at pH 5 and 4. Thus, the emulsifying ability of PPI under acidic conditions could be significantly improved by formation of soluble complexes with DS.     

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.     

Emulsifying properties of the transglutaminase-treated crosslinked product between peanut protein and fish (Decapterus maruadsi) protein hydrolysates

BACKGROUND: Defatted peanut meal, a protein‐rich by‐product from the oil extraction industry, is underutilised owing to its inferior functional properties. In this study, transglutaminase (TGase) crosslinking and proteolysis were used to improve the emulsifying properties of peanut protein isolate (PPI) extracted from the meal. PPI and PPI hydrolysate (PPIH) were conjugated separately with fish (Decapterus maruadsi) protein hydrolysate (DPH), catalysed by TGase to obtain improvements in the emulsifying properties. RESULTS: Analyses by electrophoresis and high‐performance liquid chromatography indicated that polymers were formed in all TGase‐treated samples. In emulsions of PPIH, PPI‐DPH and PPIH‐DPH the volume/surface average particle diameter (d₃₂), creaming and instability phenomenon were decreased and the zeta‐potential was increased after TGase treatment, showing improved emulsifying activity and emulsion stability. In the case of PPI, TGase treatment had no effect on the emulsifying activity, but the emulsion stability of TGase‐treated PPI was improved. CONCLUSION: The study showed that TGase crosslinking and proteolysis could improve the emulsifying properties of PPI, while proteolysis followed by TGase crosslinking proved more efficient. The emulsifying properties of the heterologous protein systems of PPI‐DPH and PPIH‐DPH were also improved by TGase treatment. Copyright © 2010 Society of Chemical Industry     

Hydrocolloids as emulsifiers and emulsion stabilizers

We consider the essential molecular features of hydrocolloids having the ability to act as emulsifying agents and emulsion stabilizing agents. The criteria for effectiveness in protecting newly formed droplets against flocculation and coalescence are contrasted with the requirements to maintain long-term stability against aggregation, creaming and Ostwald ripening. To illustrate various aspects of stability behaviour, comparison is made between the physico-chemical characteristics of hydrocolloid emulsifying agents and those of other kinds of food emulsifying agents – surfactants, proteins and nanoparticles. Interfacial complexation between protein and polysaccharide may occur through covalent bonding or electrostatic bonding. For the case of electrostatic protein–polysaccharide complexes, the interfacial nanostructure and the stabilizing properties of the adsorbed layer are dependent, amongst other things, on the sequence of adsorption of the biopolymers to the emulsion droplet surface.     

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.     

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.     

Characterisation of fish oil emulsions stabilised by sodium caseinate

Fish oil emulsions varying in sodium caseinate concentration (25% w/w oil and 0.1–1.0% w/w protein, giving oil-to-protein ratios of 250–25) were investigated in terms of their creaming stability, rheological properties, the mobility of oil droplets and the oil/protein interaction at the interface. The presence of excessive protein in an emulsion (i.e., at 1% w/w) caused the aggregation of oil droplets through depletion flocculation, resulting in low creaming stability and high low-shear viscosity. At a lower protein concentration (0.1% w/w), when protein was limited, the emulsion droplets were stabilised by bridging flocculation and showed good stability to creaming. Shear-thinning behaviour was observed for both flocculated emulsions. A reduction in the low-shear viscosity and a Newtonian flow was obtained for the emulsion containing an intermediate concentration of protein (0.25% w/w). At this concentration, there was relatively little excess unadsorbed protein in the continuous phase; thus the emulsion was most stable to creaming. NMR was used to characterise these emulsion systems without dilution. Shorter T₂ values (by low-field ¹H NMR), for the emulsions containing both high (1% w/w) and low (0.1% w/w) amounts of protein, indicated increased restricted mobility of oils, caused by depletion or bridging flocculation. The line broadening in oil signals in the high-field NMR spectra (¹H, ¹³C) indicated increased interaction between oil molecules and proteins at the interface with increasing protein concentration in emulsions. In addition, ³¹P NMR spectra, which reflect the mobility of the casein component only, showed increased line broadening, with reduction in protein content due to the relatively higher proportion of the protein being adsorbed to the interface of the oil droplets, compared to that in the continuous phase (i.e., as the oil-to-protein ratio was increased). The T₂ values of resonances of the individual groups on oil molecules, obtained using high-field ¹H NMR, reflected their different environments within the oil droplet.     

Ability of flaxseed and soybean protein concentrates to stabilize oil-in-water emulsions

The ability of flaxseed protein concentrate (FPC) to stabilize soybean oil-in-water emulsion was compared with that of soybean protein concentrate (SPC). The stability of emulsions increased with increase in protein concentration. The FPC-stabilized emulsions had smaller droplet size and higher surface charge, but worse stability at the same protein concentration compared to SPC-stabilized emulsions. Oil-in-water emulsions stabilized by both proteins were diluted and compared at different pH values (3–7), ionic strength (0–200 mM NaCl) and thermal treatment regimes (25–95 °C for 20 min). Considerable emulsion droplet flocculation occurred around iso-electric point of both proteins: FPC (pH 4.2) and SPC (pH 4.5). FPC and SPC-stabilized emulsions remained relatively stable against droplet aggregation and creaming at NaCl concentration below 100 and 50 mM, respectively. The emulsions stabilized by both proteins were fairly stable within these thermal processing regimes. FPC appears to be less effective as an emulsifier compared to SPC due to its lower emulsion viscosity. Hence, FPC could be more effective in emulsions that are fairly viscous.     

Characterisation of emulsion properties and of interface composition in O/W emulsions prepared with hen egg yolk, plasma and granules

Comprehension of hen egg yolk emulsifying properties remains incomplete because competition between its various emulsifiers (proteins and lipoproteins containing phospholipids) has not been clearly elucidated and colloidal interactions between yolk-stabilised oil droplets have not been documented. Recent studies emphasised the interest of the fractionation of yolk into plasma and granules to improve this comprehension. In the present study, we characterised, concurrently, emulsion properties (oil droplet size and stability against creaming) and interface attributes (interfacial concentrations of proteins and phospholipids, SDS-PAGE profiles of adsorbed proteins and zeta potential) in oil-in-water (O/W) emulsions prepared with yolk, plasma and granules. We observed these features at four physicochemical conditions (pH 3.0 or 7.0 and at 0.15 or 0.55 M NaCl). Emulsion properties in emulsions made with yolk or plasma varied similarly as a function of pH and NaCl concentration whereas granules emulsions exhibited distinct properties. Therefore the main contributors to yolk emulsifying properties are to be sought for among plasma constituents (proteinaceous or phospholipids). Since, in plasma emulsions, variations of emulsion stability against creaming correlated exclusively to variations of protein interfacial concentration, a driving contribution of the proteinaceous part of plasma, namely apo-LDL, was hypothesised. In the pH and ionic strength ranges studied, zeta potentials of the interfaces were low, excluding extended electrostatic repulsion between oil droplets. We deduced that steric repulsion is the main interaction opposing to droplet aggregation in food emulsions made with yolk.     

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.     

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.     

Stability of Whey Protein Concentrate/Sunflower Oil Emulsions as Affected by Sucrose and Xanthan Gum

The effect of the addition of sucrose and xanthan gum, protein concentration, and processing method on the stability and destabilization mechanism type of emulsions formulated with two commercial whey protein concentrate powders was described and quantified following system changes with a Turbiscan TMA 2000, a light scattering equipment and a confocal laser scanning microscope. Two different processing methods that gave particle sizes with different orders of magnitude were compared: homogenization by ULTRA-TURRAX (UT) and by ultrasound (US). The addition of sucrose to the aqueous phase of emulsions significantly diminished volume-weighted mean diameter (D _4,3) and improved stability. When the aqueous phase contained xanthan gum, the main destabilization mechanism for UT emulsions changed from creaming to flocculation. For US emulsions, although some aggregation was detected by confocal laser scanning microscopy, it was not great enough to modify the backscattering average (BS_av) in the middle zone of the tube (20–50 mm). At low protein concentrations, the profiles corresponded to destabilization of small aggregates. In those conditions, creaming was markedly enhanced as evident from creaming rate values. Independently of aqueous phase composition, US emulsions stabilized by protein concentrations higher than 5 wt% were stable, indicating that whey proteins were good emulsion stabilizers at pH close to 7. This study shows the relevance of protein type on stability and describes for the first time a behavior for whey proteins different from the one reported for caseins in literature.     

植物球蛋白/姜黄素纳米复合物的制备及其对O/W型Pickering乳液氧化稳定性影响

本论文以两类植物球蛋白:豌豆分离蛋白(PPI)和大豆分离蛋白(SPI)为材料制备荷载姜黄素蛋白纳米复合物,并探究荷载前后蛋白所制备乳液的物理和氧化稳定性差异。结果表明:PPI和SPI在pH 3.0和pH 7.0下荷载前后蛋白纳米颗粒粒径没有明显变化。pH 7.0时两蛋白姜黄素荷载量均高于pH 3.0,各pH下SPI荷载量要高于PPI。表面疏水性的显著降低与荧光淬灭现象发生表明形成两种蛋白纳米复合物的主要作用力为疏水相互作用,同时在两pH下,PPI比SPI荧光蓝移趋势更明显且有效淬灭常数也更大,即更易形成复合物。与原蛋白相比,荷载后各蛋白颗粒所制备乳液乳化活性有少许降低,同时pH 3.0时各蛋白颗粒乳化活性要高于pH 7.0。各乳液生成初级氧化产物脂质氢过氧化物浓度的变化趋势与生成次级氧化产物TBARS相类似,均为荷载姜黄素后各乳液氧化水平加速,同时pH 3.0时各类型乳液油滴氧化程度均高于pH 7.0。