Formation of biopolymer particles by thermal treatment of β-lactoglobulin–pectin complexes

The purpose of this study was to prepare and characterize biopolymer particles based on thermal treatment of protein–polysaccharide electrostatic complexes formed from a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin). Initially, the optimum pH and pectin concentration for forming protein–polysaccharide complexes were established by mixing 0.5 wt% β-lactoglobulin solutions with beet pectin (0–0.5 wt%) at different pH values (3–7). Biopolymer complexes in the sub-micron size range (d = 100–300 nm) were formed at pH 5.0 and 0.1 wt% pectin. These particles were then subjected to a thermal treatment (30–90 °C at 0.8 °C min⁻¹). The presence of pectin increased the thermal aggregation temperature of the protein, although aggregate formation was still observed when the protein–polysaccharide systems were heated above about 70 °C. The impact of pH (3–7) on the properties of heat-treated biopolymer particles (83 °C, 15 min, pH 5) was then established. The biopolymer particles were stable to aggregation over a range of pH values, which increased as the amount of pectin was increased. The biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components, or as substitutes for lipid droplets.     

Complex coacervation between β-lactoglobulin and acacia gum in aqueous medium

The compatibility of β-lactoglobulin (β-lg) and acacia gum in aqueous medium was investigated as a function of the pH (3.6–5.0), the protein to polysaccharide weight ratio (50:1–1:20) and the total biopolymer concentration (0.1–5 wt%). The ternary phase diagrams obtained at low ionic strengths (0.005–10.7 mM) typically accounted for phase separation through complex coacervation. Thus a drop-shaped two-phase region was anchored in the water-rich corner. The electrostatic nature of the interactions between the two biopolymers was pointed out according to the pH dependence of the two-phase region's breadth. Following the absorbance of the mixtures at 650 nm, the influence of the protein to polysaccharide ratio was also demonstrated. Electrophoretic mobility (μ_E) measurements and chemical analyses of separated phases revealed the formation of soluble and insoluble coacervates and complexes. A remarkable value of the protein to polysaccharide weight ratio (2:1) at pH 4.2 gave the same protein to polysaccharide (Pr:Ps) ratio in the two phases after 2 days, implying that electrostatic interactions are maximum between β-lg and acacia gum. The increase of the total biopolymer concentration reduced the influence of pH and protein to polysaccharide ratio. Also, the increase of the pH close to the β-lg IEP reduced the influence of the total biopolymer concentration and Pr:Ps ratio. As the biopolymer content was increased at pH 3.6 and 4.2, the relative β-lg solubility increased probably because of the self-suppression of complex coacervation.     

Effect of polysaccharide charge on formation and properties of biopolymer nanoparticles created by heat treatment of β-lactoglobulin–pectin complexes

Biopolymer nanoparticles can be formed by heating globular protein/polysaccharide mixtures above the thermal denaturation temperature of the protein under pH conditions where the two biopolymers are weakly electrically attracted to each other. In this study, the influence of polysaccharide linear charge density on the formation and properties of these biopolymer nanoparticles was examined. Mixed solutions of globular proteins (β-lactoglobulin) and anionic polysaccharides (high and low methoxyl pectin) were prepared. Micro-electrophoresis, dynamic light scattering, turbidity and atomic force microscopy (AFM) measurements were used to determine the influence of protein-to-polysaccharide mass ratio (r), solution pH, and heat treatment on biopolymer particle formation. Biopolymer nanoparticles (d < 500 nm) could be formed by heating protein–polysaccharide complexes at 83 °C for 15 min at pH 4.75 and r = 2:1 in the absence of added salt. The biopolymer particles formed were then subjected to pH and salt adjustment to determine their stability. The pH stability was greater for β-lactoglobulin-HMP complexes than for β-lactoglobulin-LMP complexes. The addition of 200 mM sodium chloride to heated complexes greatly improved the pH stability of HMP complexes, but decreased the pH stability of LMP complexes. The biopolymer particles formed consisted primarily of β-lactoglobulin, which was probably surrounded by a pectin coating at low pH values. AFM measurements indicated that the biopolymer nanoparticles formed were spheroid in shape. These biopolymer particles may be useful as delivery systems or fat mimetics.     

Thermal analysis of β-lactoglobulin complexes with pectins or carrageenan for production of stable biopolymer particles

Biopolymer nanoparticles can be formed by thermal treatment of electrostatic complexes of globular proteins and anionic polysaccharides. The purpose of this study was to provide insights into the physicochemical origin of biopolymer particle formation using differential scanning calorimetry (DSC) and temperature-scanning turbidity measurements. DSC measurements indicated that high methoxyl pectin (HMP), low methoxyl pectin (LMP) and carrageenan (C) had little impact on the thermal denaturation temperature of β-lactoglobulin (T_m ～ 78 °C) at pH 4.75, where electrostatic complexes are formed. Temperature scanning turbidity measurements indicated that extensive biopolymer aggregation occurred above T_m for β-lactoglobulin-pectin systems, but not for β-lactoglobulin-carrageenan systems. This difference was attributed to the greater strength of the attractive electrostatic interactions between the protein and carrageenan molecules, compared to the protein and pectin molecules. The biopolymer particles formed by heating β-lactoglobulin-pectin complexes were relatively stable to association/dissociation from pH 3 to 7 for HMP and from pH 4 to 7 for LMP, whereas the β-lactoglobulin-C complexes were highly unstable to pH changes. The β-lactoglobulin-pectin nanoparticles (d = 200–300 nm) may therefore be useful as natural delivery systems or fat replacers in the food, pharmaceutical, cosmetic and other industries.     

Impact of cosolvents (polyols) on globular protein functionality: Ultrasonic velocity, density, surface tension and solubility study

The objective of this study was to understand how cosolvents influence the molecular and functional properties of globular proteins in aqueous solutions. The ultrasonic velocity, density and adiabatic compressibility of cosolvent solutions (0–50 wt% sorbitol or glycerol) were measured in the absence and presence of a globular protein (1 wt% β-lactoglobulin) at 30 °C. These measurements were used to calculate the partial specific apparent volume and adiabatic compressibility of the protein. The protein's volume decreased and its compressibility increased in the presence of high cosolvent concentrations, which were attributed to changes in the properties of the protein interior and solvation layer. Sorbitol was more effective than glycerol at decreasing the protein volume at high cosolvent concentrations, which may be because glycerol has some surface activity and may therefore accumulate around hydrophobic regions on the protein surface. Our data were used to account for the observation that sorbitol is more effective than glycerol at increasing the thermal stability and self-association of the β-lactoglobulin. A better understanding of the influence of protein–cosolvent–solvent interactions on the functionality of globular proteins may facilitate the design of protein-based products.     

Cold-set thickening mechanism of β-lactoglobulin at low pH: Concentration effects

There is an interest in developing protein based thickening agents for nutritional considerations. A procedure to convert whey protein concentrates or isolates into a pH modified cold-thickening ingredient was developed. Concentration effects on thickening mechanism of this whey protein ingredient were studied with a β-lactoglobulin model system at the pH of the modification procedure, 3.35. In this study, concentration effects on thermal aggregation of β-lactoglobulin were studied at low pH using capillary and rotational viscometry, transmission electron microscopy (TEM), and high performance liquid chromatography coupled with multi-angle laser light scattering (HPLC-MALS). From the results of capillary viscometry, a critical concentration (C_c  6.9% w/w) was identified below which no significant thickening functionality could be achieved. Microscopy revealed formation of flexible fibrillar network at pH 3.35 during heating at all concentrations. These flexible fibrils had a diameter of about 5 nm and persistence length of about 35 nm as compared to more linear and stiff fibrils formed at pH 2 and low ionic strength conditions. Under similar heating conditions at concentration above C_c, larger aggregates similar to microgels were observed compared to the concentration below C_c, where isolated fibrils with an average contour length of about 130 nm were observed. These microgels and apparently stronger interactions between aggregates at concentrations above C_c were seemingly responsible for thickening functionality of heated β-lactoglobulin solutions and subsequently modified powders. Further investigation of β-lactoglobulin aggregation at this pH may provide capability to mechanistically tailor the functional attributes of modified ingredients.     

Biopolymer Nanoparticles from Heat-Treated Electrostatic Protein–Polysaccharide Complexes: Factors Affecting Particle Characteristics

ABSTRACT: Biopolymer nanoparticles can be formed by heating globular protein–ionic polysaccharide electrostatic complexes above the thermal denaturation temperature of the protein. This study examined how the size and concentration of biopolymer particles formed by heating β-lactoglobulin–pectin complexes could be manipulated by controlling preparation conditions: pH, ionic strength, protein concentration, holding time, and holding temperature. Biopolymer particle size and concentration increased with increasing holding time (0 to 30 min), decreasing holding temperature (90 to 70 °C), increasing protein concentration (0 to 2 wt/wt%), increasing pH (4.5 to 5), and increasing salt concentration (0 to 50 mol/kg). The influence of these factors on biopolymer particle size was attributed to their impact on protein–polysaccharide interactions, and on the kinetics of nucleation and particle growth. The knowledge gained from this study will facilitate the rational design of biopolymer particles with specific physicochemical and functional attributes.     

Influence of complexing carboxymethylcellulose on the thermostability and gelation of α-lactalbumin and β-lactoglobulin

Thermostability and gelation of the main proteins of whey, α-lactalbumin (α-lac) and β-lactoglobulin (β-lg) recovered by selective complexation with carboxymethylcellulose (CMC) was studied to evaluate its functionality in food systems. Their behavior was compared to the non-complexed proteins. Both complexes showed a maximum stability at pH 4, that is close to the pH of obtention of β-lg/CMC coacervate (pH 4) and α-lac/CMC coacervate (pH 3.2). Protein complexation increased the thermostability of β-lg by approximately 6–8 °C and that of α-lac by approximately 26 °C due to immobilization of protein molecules in a complex, mainly by electrostatic interactions and because of different amounts of bound polysaccharide. The denaturation enthalpy of complexed proteins markedly decreased as compared to free proteins. Storage modulus (G′) and loss modulus (G″) were recorded to reflect the structure development during heating β-lg/CMC and α-lac/CMC complexes at different pH values. β-lg/CMC complex at 20 wt% was a viscoelastic liquid at pH values within 2 and 8 but upon heating turned to a particulate viscoelastic gel. However, α-lac/CMC complex formed before heating opaque, large visible white particulate aggregates that sticked together to give a solid viscoelastic structure that was not further modified by thermal processing.     

Microstructural characterization of β-lactoglobulin–konjac glucomannan systems: Effect of NaCl concentration and heating conditions

In the initial part of this study, the high temperature (85 °C) microscopic phase behaviour of β-lactoglobulin (0.4–6%, w/w)–konjac (0.05–0.75%, w/w) mixtures containing 50 mM NaCl was established using confocal laser scanning microscopy (CLSM). Also, the effects of heating time (heating temperature: 78 °C) and NaCl concentration (0–75 mM) on protein denaturation kinetics and the phase behaviour in 2%, w/w, β-lactoglobulin–0.4%, w/w, konjac mixtures were investigated using turbidimetry, protein denaturation measurement, CLSM and image analysis techniques. Segregative phase separation occurred in heat-treated β-lactoglobulin–konjac mixtures containing biopolymer and NaCl concentrations exceeding certain critical levels, due to heat and NaCl induced β-lactoglobulin denaturation/aggregation. The microstructural properties of selected heated (to 85 °C for 30 min) and cooled (to 25 °C) β-lactoglobulin–konjac mixtures containing different NaCl levels were studied using CLSM and rheological measurements and the results showed that the microstructure can be distinguished as miscible, phase separated or phase separated containing stable protein inclusions dependent on NaCl concentration. Response surface methodology was used to determine the minimum NaCl concentrations required for phase separation and for formation of phase separated systems containing stable inclusions in a wide concentration range of heated and cooled β-lactoglobulin (0.8–2%, w/w)–konjac (0.2–0.75%, w/w) mixtures. The results show that the microstructural and rheological properties of β-lactoglobulin–konjac mixtures can be controlled by selecting appropriate mixture biopolymer and NaCl concentrations and heating conditions.     

Core-Shell Biopolymer Nanoparticles Produced by Electrostatic Deposition of Beet Pectin onto Heat-Denatured β-Lactoglobulin Aggregates

ABSTRACT:  The purpose of this study was to produce and characterize core-shell biopolymer particles based on electrostatic deposition of an anionic polysaccharide (beet pectin) onto amphoteric protein aggregates (heat-denatured β-lactoglobulin [β-lg]). Initially, the optimum conditions for forming stable protein particles were established by thermal treatment (80 °C for 15 min) of 0.5 wt%β-lg solutions at different pH values (3 to 7). After heating, stable submicron-sized ( d = 100 to 300 nm) protein aggregates could be formed in the pH range from 5.6 to 6. Core-shell biopolymer particles were formed by mixing a suspension of protein aggregates (formed by heating at pH 5.8) with a beet pectin solution at pH 7 and then adjusting the pH to values where the beet pectin is adsorbed (< pH 6). The impact of pH (3 to 7) and salt concentration (0 to 250 mM NaCl) on the properties of the core-shell biopolymer particles formed was then established. The biopolymer particles were stable to aggregation from pH 4 to 6, but aggregated at lower pH values because they had a relatively small ζ-potential. The biopolymer particles remained intact and stable to aggregation up to 250 mM NaCl at pH 4, indicating that they had good salt stability. The core-shell biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components or as substitutes for lipid droplets.     

Combined effect of an oxygen absorber and oregano essential oil on shelf life extension of rainbow trout fillets stored at 4 °C

In the present study the combined effect of an O₂ absorber and oregano essential oil (0.4% v/w) on shelf life extension of rainbow trout fillets (Onchorynchus mykiss) stored under refrigeration (4 °C) was investigated. The study was based on microbiological [TVC, Pseudomonas spp., Lactic Acid Bacteria, H₂S-producing bacteria including Shewanella putrefaciens, Enterobacteriaceae and Clostridium spp.), physicochemical (pH, PV, TBA, TVBN and Drip loss) and sensory (odor, taste) changes occurring in the product as a function of treatment and storage time. Aerobically-packaged rainbow trout fillets stored at 4 °C were taken as control samples. Results showed that TVC exceeded 7 log cfu/g on day 4 of storage for control samples, day 7–8 for samples containing oregano oil, day 9 for samples containing the O₂ absorber and day 12–13 for samples containing the O₂ absorber and oregano oil. Pseudomonas spp., Enterobacteriaceae and LAB were only partially inhibited by the O₂ absorber and/or the oregano oil. In all cases the inhibition effect was more pronounced when the combination of O₂ absorber with oregano essential oil was used. pH decreased from an initial value of 6.65–6.09 and subsequently increased to 6.86 due to formation of protein decomposition products. % Drip loss ranged between 7% and 11–12% at the end of the product shelf life. PV values ranged between 11.4 and 27.0 meq O₂/kg oil while malondialdehyde (MDA) ranged between 9.6 and 24.5 mg/kg. TVBN ranged between 10.6 and 54.6 mg/kg at the time of sensory rejection. Sensory shelf life was 4 days for the control samples, 7–8 days for samples containing oregano oil, 13–14 days for samples containing the O₂ absorber and 17 days for samples containing the O₂ absorber plus oregano oil.     

Maturation effects in fish gelatin and HPMC composite gels

This study assessed the effectiveness of using hydroxylpropylmethylcellulose (HPMC) to enhance mechanical strength and thermal stability in fish skin gelatin (FG). The significant increase in absorbance (A₄₀₀) observed after HPMC had been added to FG and then matured indicated successful formation of a composite gel. Increased gel strength and storage modulus (G′) indicated the enhanced gelation ability of the matured composite gel, while increased melting temperature (T_m) and enthalpy (ΔH) indicated its improved thermal stability. Maturation-related rheological property improvements were more noticeable at 4 °C than 10 °C, but no apparent differences in T_m improvement were observed between 4 °C and 10 °C maturation. Nevertheless, the composite gel exhibited reversible cold and thermal gelation properties.     

Ferrous bisglycinate content and release in W1/O/W2 multiple emulsions stabilized by protein–polysaccharide complexes

Ferrous bisglycinate aqueous solution was entrapped in the inner phase (W₁) of water-in-oil-in-water (W₁/O/W₂) multiple emulsions. The primary ferrous bisglycinate aqueous solution-in-mineral oil (W₁/O) emulsion contained 15% (w/w) ferrous bisglycinate, had a dispersed phase mass fraction of 0.5, and was stabilized with a mixture of Grindsted PGPR 90:Panodan SDK (6:4 ratio) with a total emulsifiers concentration of 5% (w/w). This primary emulsion was re-emulsified in order to prepare W₁/O/W₂ multiple emulsions, with a dispersed mass fraction of 0.2, and stabilized using protein (whey protein concentrate (WPC)):polysaccharide (gum arabic (GA) or mesquite gum (MG) or low methoxyl pectin (LMP)) complexes (2:1 ratio) in the W₂ aqueous phase. The W₁/O/W₂ multiple emulsion stabilized with WPC:MG (5% w/w total biopolymers concentration) provided smaller droplet sizes (2.05 μm), lower rate of droplet coalescence (7.09 × 10⁻⁷ s⁻¹), better protection against ferrous bisglycinate oxidation (29.75% Fe³⁺) and slower rate of ferrous bisglycinate release from W₁ to W₂ (K_H = 0.69 mg mL⁻¹ min^−0.5 in the first 24 h and 0.07 mg mL⁻¹ min^−0.5 for the next 19 days of storage time). Better encapsulation efficiencies, enhanced protection against oxidation and slower release rates of ferrous bisglycinate were achieved as the molecular weight of the polysaccharide making up protein:polysaccharide complex was higher. Thus, the factor that probably affected most the overall functionality of multiple emulsions was the thickness of the complex adsorbed around the multiple emulsion oil droplets. These thicknesses determined indirectly by measuring the z-average diameter of the complexes, and that of the WPC:MG (529.4 nm) was the largest.     

Factors influencing the production of o/w emulsions stabilized by β-lactoglobulin–pectin membranes

A primary emulsion was prepared by homogenizing 10 wt% corn oil with 90 wt% aqueous β-lactoglobulin solution (0.5 wt% β-lg, pH 3 or 7) using a two-stage high-pressure valve homogenizer. This emulsion was mixed with aqueous pectin (citrus, 59% DE) stock solution (2 wt%, pH 3 or 7) and NaCl solution to yield secondary emulsions with 5 wt% corn oil, 0.225 wt% β-lactoglobulin, 0.2 wt% pectin and 0 or 100 mM NaCl. The final pH of the emulsions was then adjusted (3–8). Primary and secondary emulsions were ultrasonically treated (30 s, 20 kHz, 40% amplitude) to disrupt any flocculated droplets. Secondary emulsions were more stable than primary emulsions at intermediate pHs. Secondary emulsions prepared at pH 7 had smaller particle diameters (0.35 to 6 μm) than those prepared at pH 3 (0.42 to 18 μm) across the whole pH range studied, and also had smaller diameters than the primary emulsions (0.35 to 14 μm). Ultrasound treatment reduced the particle diameter of both primary and secondary emulsions and lowered the rate of creaming. The presence of NaCl screened the charges and thus the electrostatic interaction between biopolymer molecules and primary emulsion droplets. Secondary emulsions were more stable to the presence of 100 mM NaCl at low pHs (3–4) than primary emulsions. This study shows that stable emulsions can be prepared by engineering their interfacial membranes using the electrostatic interaction of natural biopolymers, especially at intermediate pHs where proteins normally fail to function.     

Modulation of thermal stability and heat-induced gelation of β-lactoglobulin by high glycerol and sorbitol levels

The influence of glycerol and sorbitol on the thermal stability and heat-induced gelation of β-lactoglobulin (β-lg) in aqueous solutions was investigated. The thermal stability of β-lg was characterized by measuring the thermal denaturation temperature (T_m) using differential scanning calorimetry, while its gelation properties were characterized by measuring the gelation temperature (T_gel) and final gel rigidity (G^∗) using dynamic shear rheology. All experiments were carried out using aqueous solutions containing 10% (w/w) β-lg, glycerol (0–70% w/w) or sorbitol (0–55% w/w), and 5 mM phosphate buffer (pH 7.0). No salt was added to these solutions so that there was a relatively strong electrostatic repulsion between the protein molecules, which usually prevents gelation. When the cosolvent concentration was increased from 0% to 50%, T_m increased from 74 to 86 °C for sorbitol, but only from 74 to 76 °C for glycerol, which indicated that sorbitol was much more effective at stabilizing the native state of the globular protein than glycerol. Protein solutions containing sorbitol (0–55%) did not form a gel after heating, but those containing glycerol formed gels when the cosolvent concentration exceeded about 10%, with G^∗ increasing with increasing glycerol concentration. We attribute these effects to differences in the preferential interactions of polyols and water with the surfaces of native and heat-denatured proteins, and their influence on the protein–protein collision frequency.     

Interactions of polysaccharides with β-lactoglobulin spread monolayers at the air–water interface

In the present work we have studied the static (film structure and elasticity) and dynamic characteristics (surface dilatational properties) of β-lactoglobulin (βLG) monolayers spread at the air–water interface in the presence of polysaccharides in the aqueous phase, at 20 °C and at pH 7. The measurements were performed on a fully automated Wilhelmy-type film balance. As polysaccharides with interfacial activity we have used propylene glycol alginates (PGA). To evaluate the effect of the degree of PGA esterification and viscosity, different commercial samples were studied-kelcoloid O (KO), kelcoloid LVF (KLVF) and manucol ester (MAN). Xanthan gum (XG) and λ-carrageenan (λC) were studied as non-surface active polysaccharides. The results reveal a significant effect of surface active and non-surface active polysaccharides on static—when the polysaccharide was added in the subphase the π-A isotherms shifted to higher surface pressure values as the time increased-and dynamic—the presence of polysaccharide in the aqueous phase decreased the surface dilatational modulus of a pure β-lactoglobulin monolayer-characteristics of β-lactoglobulin monolayers. To explain the observed effects three phenomena were taken into account: (i) the ability of the polysaccharide to adsorb at the interface by it-self and to increase the surface pressure, (ii) the interfacial complexation of the polysaccharide with the adsorbed protein and (iii) the existence of a limited thermodynamic compatibility between the protein and polysaccharide, depending on the protein-polysaccharide system.     

Study of the formation of complexes between DNA and esterified dairy proteins

Like with many naturally occurring basic proteins such as histones and lysozymes, plasmid DNA can interact with methylated α-lactalbumin (ALA) and methylated β-lactoglobulin (BLG) forming complexes. The stabilities of these complexes were tested at different pH, temperatures and salt concentrations, and after enzyme digestion with DNase I and pepsin. Incubation at 37°C for long periods (up to 24 h) allowed the interaction of DNA with low concentrations of esterified proteins to take place. High temperature treatment (100°C) for short periods of time enhanced complex formation after 5 min of heating in case of both DNA/methylated ALA and DNA/methylated BLG. The complex of DNA with methylated BLG was more stable than that of DNA and methylated ALA, when the thermal treatment at 100°C was extended to 10 min. Both complexes were formed in larger amounts and were more stable at acid pH (3–6). Generally, at acid pH, the concentration of the stable complex of DNA and methylated BLG was larger than that of the complex between DNA and methylated ALA. These complexes were still quite stable at very acid pH (1–2) but not at all at very basic pH (10–11). Formation and stability of studied complexes of DNA with esterified proteins were generally dependent on salt concentration. Magnesium chloride had the greatest inhibitory effect on the formation and the stability of these complexes while potassium acetate had the least. The inhibitory effect of KCl on both complex formation and stability was observed in the range 0.4–1.0 . The complexes between DNA and esterified milk proteins or lysozyme were more resistant to hydrolysis with DNase I than free non-complexed DNA. Surprisingly, the DNA/methylated ALA complex was more resistant to DNase I digestion than the DNA/methylated BLG or DNA/lysozyme complexes. All the studied proteins were resistant to pepsin when complexed with DNA.     

Protein–polysaccharide interactions: The determination of the osmotic second virial coefficients in aqueous solutions of β-lactoglobulin and dextran

Solutions containing dextran and solutions containing mixtures of dextran +β-lactoglobulin are studied by membrane osmometry. The low concentration range of these solutions is considered. From the measured osmotic pressures the virial coefficients are obtained. These are analyzed using the osmotic virial coefficient of β-lactoglobulin solutions published earlier by us [Schaink, H.M., & Smit, J.A.M. (2000). Determination of the osmotic second virial coefficient and the dimerization of beta-lactoglobulin in aqueous solutions with added salt at the isoelectric point. PCCP, 2, 1537–1541]. The second cross-virial coefficient A₁₂ is found to be positive indicating a repulsive and probably mainly steric interaction between neutral in nature dextran and and practically uncharged β-lactoglobulin (pH=5.18). The measurements show that the β-lactoglobulin has only a small tendency to form multimers in the presence of dextran. The phase diagram of solutions of dextran+Whey Protein Isolate (appr. 60% β-lactoglobulin) is also presented. The McMillan–Mayer equation of state that considers only the second virial coefficients is found to be unreliable for the extrapolation up to the concentrations at which phase separation is expected.     

Influence of glycerol and sorbitol on thermally induced droplet aggregation in oil-in-water emulsions stabilized by β-lactoglobulin

The influence of polyol cosolvents (glycerol and sorbitol) on the flocculation stability of hydrocarbon oil-in-water emulsions stabilized by a globular protein was examined. Salt (150 mM NaCl) and polyols (0–40 wt%) were added to n-hexadecane oil-in-water emulsions stabilized by β-lactoglobulin (β-Lg, pH 7.0) either before or after isothermal heat treatments (30–90 °C for 20 min). When salt was added to emulsions before heat treatment, appreciable droplet flocculation was observed below the thermal-denaturation temperature of the adsorbed β-Lg (T_m∼70 °C), and more extensive flocculation was observed above T_m. On the other hand, when salt was added after heat treatment, appreciable droplet flocculation still occurred below T_m, but little flocculation was observed above T_m. Addition of cosolvents to the emulsions increased the temperature where extensive droplet flocculation was first observed when they were heated in the presence of salt, which was attributed to their ability to increase T_m and to reduce the droplet collision frequency, with sorbitol being more effective than glycerol. Our results are interpreted in terms of the influence of the cosolvents on protein conformational stability, protein-protein interactions and the physiochemical properties of aqueous solutions. This study has important implications for the formulation and production of protein stabilized oil-in-water emulsions for industrial applications, such as foods, pharmaceuticals and cosmetics.     

Effect of microbial transglutaminase treatment on thermal stability and pH-solubility of heat-shocked whey protein isolate

Whey protein isolate (WPI) dispersions (5% protein, pH 7.0) were subjected to heat-shock at 70 °C for 1, 5 and 10 min. The heat-shocked WPI dispersions were treated with microbial transglutaminase (MTGase) enzyme, and thermal properties and pH-solubility of the treated proteins were investigated. Heat-shocking of WPI for 10 min at 70 °C increased the thermal denaturation temperature (T_d) of β-lactoglobulin in WPI by about 1.5 °C. MTGase treatment (30 h, 37 °C) of the heat-shocked WPI significantly increased the T_d of β-lactoglobulin by about 6.3–7.3 °C when compared with heat-shocked only WPI at pH 7.0. The T_d increased by about 13–15 °C following pH adjustment to 2.5; however, the T_d of heat-shocked WPI was not substantially different from heat-shocked and MTGase-treated WPI at pH 2.5. Both the heat-shocked and the heat-shocked-MTGase-treated WPI exhibited U-shaped pH-solubility profiles with minimum solubility at pH 4.0–5.0. However, the extent of precipitation of MTGase-treated WPI samples at pH 4.0–5.0 was much greater than all heat-shocked and native WPI samples. The study revealed that while MTGase cross-linking significantly enhanced the thermal stability of β-lactoglobulin in heat-shocked WPI, it caused pronounced precipitation at pH 4.0–5.0 via decreasing the hydrophilic/hydrophobic ratio of the water-accessible protein surface.