Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-casein produced by thermolysin-catalysed hydrolysis

The thermolysin catalysed hydrolysates of α-lactalbumin and β-casein were fractionated by size-exclusion chromatography (SEC) and reversed-phase high performance liquid chromatography (RP-HPLC) in order to identify the peptides responsible for the high ACE-inhibitory activity of these hydrolysates. The SEC fractionation separated many co-eluting peptides into different fractions allowing individual peptides to be isolated in one or two subsequent semi-preparative RP-HPLC fractionation steps. Five potent ACE-inhibitory peptides from α-lactalbumin were isolated. They all contained the C-terminal sequence -PEW, corresponding to amino acid residues 24–26 in α-lactalbumin, and had IC₅₀ values of 1–5 μm. From one SEC fraction of the β-casein hydrolysate two potent ACE-inhibitory peptides were isolated and identified as f58-76 and f59-76 of β-casein A2. They both contained IPP as the C-terminal sequence and had IC₅₀ values of 4 and 5 μm. From another SEC fraction a new but less ACE-inhibitory peptide from β-casein was identified (f192–196; LYQQP).     

Influence of other whey proteins on the heat-induced aggregation of α-lactalbumin

The thermally induced aggregation of α-lactalbumin in solution and in the presence of serum protein-free casein micelles has been studied using gel permeation chromatography. No aggregation of α-lactalbumin was detected after heating at 90°C for 24 min. However, addition of β-lactoglobulin or serum albumin to the serum protein-free casein micelles + α-lactalbumin system caused aggregation of the α-lactalbumin, the rate and extent of this aggregation being dependent upon the concentration of free sulphydryl groups present in the other whey protein. It is therefore the sulphydryl group which is important and which appears to function by inducing cleavage of intramolecular disulphide bonds in the α-lactalbumin. This leads to the formation of intermolecular disulphide bridges and hence to aggregation of the α-lactalbumin.     

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.     

Heat-induced phase behavior of β-lactoglobulin/polysaccharide mixtures

The influence of polysaccharides on the thermal stability of β-lactoglobulin at pH 6.8 was investigated regarding polysaccharide type, concentration and size. Two kinds of polysaccharides, sulfate-containing polysaccharides (carrageenans and dextran sulfate with different molecular mass) and neutral polysaccharides (dextran with different molecular mass), were investigated. At low ratios of sulfate-containing polysaccharide to β-lactoglobulin, heat-induced aggregation was decreased as shown by lower turbidity. Increasing the ratio induced a significant increase in turbidity, leading to segregative phase separation. Phase diagrams were established by centrifugation, chemical assays and visual observation for β-lactoglobulin/kappa-carrageenan and β-lactoglobulin/dextran sulfates. Significant phases (stable, separated and gel) were found, indicating the varieties of phase behavior and a strong competition between phase separation and gelation caused by thermal treatment. Moreover, gelation was reversible in β-lactoglobulin/dextran sulfate systems depending on the polysaccharide concentration.     

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.     

Characterization of α-carrageenan solution behavior by field-flow fractionation and multiangle light scattering

The salt mediated molecular conformation change of alpha (α)-carrageenan was studied in 0.1 M solutions of NaCl, NaI, and KCl. Asymmetric Field-Flow Fractionation with multiangle laser light scattering (AFFF/MALLS) detection was used to determine the average molecular weight, radius of gyration, and hydrodynamic radius which were in turn used to calculate the molecular density. In the presence of 0.1 M NaCl, an inert salt that does not promote gelation, α-carrageenan has a denser structure compared to κ-carrageenan of a similar molecular weight. A distinct and dramatic increase in the molecular weight (factor of 2) was observed for α-carrageenan in 0.1 M NaI compared to 0.1 M NaCl. This combined with only a slight change in the radius of gyration, suggests intermolecular interaction to a more compact structure (e.g., coaxial helices). A similar increase in molecular weight is observed in 0.1 M KCl, accompanied with an approximate 50% increase in the radius of gyration as well as an increase in polydispersity. This may also be attributed to intermolecular interaction with helix formation (coaxial or lateral) or may be due to K⁺ cations interacting with naturally occurring residual ι-carrageenan in the sample. As previously reported for other carrageenans the random coil to helix transition of α-carrageenan appears to be stabilized by K⁺ cation or I⁻ anion in an aqueous environment.     

Effects of γ-irradiation on molar mass and properties of Konjac mannan

Konjac mannan (KM) is a water-soluble glucomannan with high molar mass. Here, the effects of γ-irradiation on the structure of KM, its viscosity, molar mass distribution and the state of sorbed water were studied after irradiation at reduced pressure. These changes were investigated using ESR, FT-IR, UV, viscometer, SEC–MALS and DSC. Free radical yields increased with absorbed dose. Irradiation led to chain scission, but introduced no significant new chemical groups into the structure, apart from a small increase in content of carbonyl groups. The intrinsic viscosity, molar mass and radius of gyration decreased rapidly with increasing dose up to 10 kGy and then at a slower rate. The Mark–Houwink–Sakurada equation for the KM gave [η]=5.30×10⁻⁴ M^0.78. The α value showed that KM molecules are solvated in the form of random coils in water. There are three types of sorbed water in irradiated KM as in the original KM. There is no significant change in water binding ability for KM with M_W greater than 2×10⁵.     

Susceptibility of an industrial α-lactalbumin concentrate to cross-linking by microbial transglutaminase

The susceptibility of an industrial α-lactalbumin concentrate to cross-linking with a microbial transglutaminase from Streptoverticillium mobaraense was investigated. At a protein concentration of 0.5% w v⁻¹, the maximum cross-linking was observed at 50°C, pH 5 and at 5 h of incubation time. Results from sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis showed that most of the monomeric form of α-lactalbumin was converted to polymers too large to move into the gel matrix. Addition of ethylenediamine tetraacetic acid or SDS prior to the incubation of protein–enzyme mixture, further enhanced the transglutaminase reaction with the industrial α-lactalbumin. Results from reverse phase chromatography indicated that cross-linking caused a broadening of the α-lactalbumin peak with little change in the average hydrophobicity of the protein. In contrast to the reported results on pure α-lactalbumin, the industrial α-lactalbumin concentrate showed considerable cross-linking with transglutaminase even without the reduction of the disulphide bonds. This difference was attributed to the partially unfolded secondary structures in the industrial α-lactalbumin concentrate.     

Competitive adsorption of α-lactalbumin in the molten globule state

The molten globule state of α-lactalbumin is a partially denatured form with native-like secondary structure and disordered tertiary structure. Using circular dichroism measurements, it was demonstrated that the molten globule state was produced by decreasing the pH to 2.0 at 25°C or by removing bound Ca²⁺ by treatment with ethylenediamine—tetraacetic acid (EDTA) at pH 7.5 and 40°C. Tension measurements showed that α-lactalbumin in the molten globule state is more easily unfolded at liquid interfaces than is the native protein. Results of competitive adsorption experiments involving α-lactalbumin and β-lactoglobulin at the oil droplet surface in emulsions are consistent with preferential adsorption of α-lactalbumin during emulsification when it is in the molten globule state. In contrast to the difficulty of exchange between α-lactalbumin and β-lactoglobulin at the oil-water interface in emulsions at 25°C, it has been found that the two whey proteins are able partially to displace one another from the oil—water interface at 40°C. While native α-lactalbumin was found to be readily displaced from the oil—water interface by β-lactoglobulin at 40°C, it was found that α-lactalbumin in the molten globule state in the presence of EDTA at 40°C had itself the capacity for displacing β-lactoglobulin from the interface.     

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.     

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.     

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.     

Comparative behaviour of goat β and αs1-caseins at the air–water interface and in solution

Here we present a comparative study of caprine β- and α_s1-caseins behaviours at the air–water interface and in solution. Both caseins were purified from the milk of a single goat homozygous at the α_s1- and β-Cn loci, with a high degree of purity (98%). Physical measurements (ellipsometry, surface pressure and surface rheology) were performed at the air–water interface, whereas SAXS measurements were performed on casein solutions. Our results clearly show that self-organizations, both at the air–water interface and in solution are different for β- and α_s1-caseins. β-casein is unfolded in solution and forms a network at the interface, while α_s1-casein forms compact objects in solution and is organised in fluid domains at the interface. We also show that the presence of Ca²⁺ in the subphase strongly disturbs the interfacial layer formed by the caseins. It is elsewhere worth noting that in solution, the aggregation of α_s1-casein induced by calcium ions is associated with a pronounced change in the molecular structural organisation of the protein, which seems to adopt, in these conditions, an unfolded structure.     

On solid-like rheological behaviors of globular protein solutions

Dynamic viscoelastic and steady flow properties of β-lactoglobulin, bovine serum albumin, ovalbumin, and α-lactalbumin aqueous solutions were investigated at 20°C. When a sinusoidal strain in the linear viscoelastic region was applied, the solutions of the globular proteins except for α-lactalbumin showed typical solid-like rheological behavior: the storage modulus G′ was always larger than the loss modulus G″ in the entire frequency range examined (0.1–100 rad/s). Under a steady shear flow, strong shear thinning behavior was observed with increasing shear rate from 0.001 to 800 s⁻¹, for the globular proteins except for α-lactalbumin. The values of the steady shear viscosity η were lower than those of the dynamic shear viscosity η* at a comparable time scale of observation, violating the Cox–Merz rule, and thus suggesting that a solid-like structure in a globular protein solution was susceptible to a steady shear strain. During isothermal gelation of the protein colloids at 70°C, no crossover between G′ and G″ was observed so that the gelation point was judged by an abrupt increase in the modulus or a sudden decrease in tanδ.     

Release of β-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine β-casein variants and milk-based infant formulas

The release of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated during simulated gastro-intestinal digestion (SGID) of bovine β-casein variants (n = 3), commercial milk-based infant formulas (n = 6) and experimental infant formulas (n = 3). SGID included pepsin digestion at pH 2.0, 3.0 and 4.0 and further hydrolysis with Corolase PP™. β-Casein (β-CN) variants were extracted from raw milks coming from cows of Holstein-Friesian and Jersey breeds. Genomic DNA was isolated from milk and the β-CN genotype was determined by a PCR-based method. Phenotype at protein level was determined by capillary zone electrophoresis in order to ascertain the level of gene expression. Recognition and quantification of BCMs involved HPLC coupled to tandem MS. Regardless of the pH, BCM7 generated from variants A1 and B of β-CN (5–176 mmol/mol casein) the highest amount being released during SGID of form B. As expected, the peptide was not released from variant A2 at any steps of SGID. BCM5 was not formed in hydrolysates irrespective of either the genetic variant or the pH value during SGID. Variants A1, A2 and B of β-CN were present in all the commercial infant formulae (IFs) submitted to SGID. Accordingly, 16–297 nmol BCM7 were released from 800 ml IF, i.e. the daily recommended intake for infant. Industrial indirect-UHT treatments (156 °C × 6–9 s) did not modify release of BCM7 and, during SGID, comparable peptide amounts formed in raw formulation and final heat-treated IFs.     

Effects of different food commodities on larval development and α-amylase activity of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae)

The present work was undertaken to study the influence of four commodities (wheat flour, dates, sorghum and barley) on Plodia interpunctella post-embryonic development. Larval weight, larval mortality, pupation and adult emergence were recorded. The study also aimed to find out the effect of these commodities on protein and glycogen production as well as on α-amylase activity. Results indicated that the weight of fourth instar larvae placed on dates increased gradually. Percentage mortality was low. Pupation and adult emergence were delayed. In contrast, the weight of larvae placed on wheat flour, sorghum or barley remained low. Pupation and adult emergence occurred sooner than among those placed on dates and the percentage mortality was highest for larvae placed on barley. Results also showed that protein content and α-amylase activity were lower for larvae placed on dates than for those placed on other commodities. The biochemical composition of different commodities showed that dates are a rich source of glucose, while their protein and starch contents were very low as compared to the other commodities. In contrast, wheat flour, sorghum and barley contained large amounts of starch and protein and low amounts of glucose. Thus, the reduction in α-amylase activity was probably due to the high levels of glucose in dates.     

Influence of high pressure on interactions of 11S globulin Vicia faba with ι-carrageenan in bulk solution and at interfaces

The influence of ι-carrageenan (ι-CAR) on the solution, interfacial and emulsifying properties of 11S globulin Vicia faba at low ionic strength and pH 8 has been investigated before and after high-pressure processing at 200 MPa for 20 min. The total calorimetric enthalpy (ΔH) and size exclusion chromatography studies for the pure 11S indicate that there is subunit dissociation and extensive aggregation of the protein during or following treatment. Under the same treatment conditions, 1-anilinonaphthalene-8-sulphonate (ANS) data has shown increased protein surface hydrophobicity. Pressure treatment of 11S gives much lower values of the surface tension, and apparent surface shear rheology experiments show that the molecules in the film adsorbed from the pressurised 11S are much more strongly interacting than those adsorbed from the native 11S. However, emulsions prepared with pressure processed 11S give substantially bigger droplets than those made with the untreated pure protein. Addition of ι-CAR to 11S reduces the denaturation temperature (T_m), the ΔH value, and protein surface hydrophobicity. Size exclusion chromatography at low ionic strength is indicative of complex formation. Tension measurements at the air–water interface are also consistent with the presence of a complex. Emulsions made with the simple 1:0.33 mixture of 11S+ι-CAR give emulsions with smaller droplets and pressure processing of the biopolymer mixture leads to emulsions with even smaller droplets. The presence of ι-CAR at low ionic strength appears to protect the globulin against pressure-induced aggregation.     

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.     

Polyelectrolyte screening effects on the dissolution of whey protein gels at high pH conditions

The literature reports an optimum NaOH concentration for the alkaline cleaning of whey deposits or gels; at NaOH concentrations higher than this optimum, cleaning proceeds much more slowly. Although this phenomenon is of great importance in the cleaning of dairy equipment, no conclusive physical explanation has yet been presented. In this study, we present strong evidence that the dissolution rate is affected by the equilibrium-swelling ratio in β-lactoglobulin (βLg) gels. The swelling ratio is greatly reduced in the presence of salts due to the polyelectrolyte screening effect of the cations. This has been observed in free-swelling βLg gels using gravimetrical analysis and in the uniaxial swelling of WPC gel deposits using fluid dynamic gauging. At high dissolution pH (>13.3), the high Na⁺ concentration reduces swelling in spite of the high surface charge of the protein. It is proposed that the reduction of the free volume inside the gel impedes the transport of the protein aggregates out of the NaOH penetration zone. We have also observed that the final dissolution rate of gels pre-soaked in 1 M NaOH or NaCl is similar, despite the difference in pH, and much lower than for untreated gels: the high Na⁺ concentration in the soaked gels hinders swelling, inhibiting the disentanglement of the protein clusters regardless of the high pH.     

Impact of cosolvents on formation and properties of biopolymer nanoparticles formed by heat treatment of β-lactoglobulin–Pectin complexes

The purpose of this study was to determine the influence of neutral cosolvents on the formation and properties of biopolymer nanoparticles formed by thermal treatment of protein–polysaccharide electrostatic complexes. Biopolymer particles were formed by heating (85 °C, 20 min) an aqueous solution containing a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin) above the thermal denaturation temperature (T_m) of the protein under pH conditions where the biopolymers formed electrostatic complexes (pH 5). The impact of two neutral cosolvents (glycerol and sorbitol) on the self-association of β-lactoglobulin and on the formation of β-lactoglobulin–pectin complexes was examined as a function of solution pH (3–7) and temperature (30–95 °C). Glycerol had little impact on the pH-induced self-association or aggregation of the biopolymers, but it did increase the thermal aggregation temperature (T_a) of the protein–polysaccharide complexes, which was attributed to its ability to increase aqueous phase viscosity. Sorbitol decreased the pH where insoluble protein–polysaccharide complexes were formed, and greatly increased their T_a, which was attributed to its ability to increase T_m, alter biopolymer–biopolymer interactions, and increase aqueous phase viscosity. This study shows that neutral cosolvents can be used to modulate the properties of biopolymer nanoparticles prepared by thermal treatment of protein–polysaccharide electrostatic complexes.