Whey Protein Isolate Solutions Research Articles

Pilot-scale formation of whey protein aggregates determine the stability of heat-treated whey protein solutions—Effect of pH and protein concentration

Denaturation and consequent aggregation in whey protein solutions is critical to product functionality during processing. Solutions of whey protein isolate (WPI) prepared at 1, 4, 8, and 12% (wt/wt) and pH 6.2, 6.7, or 7.2 were subjected to heat treatment (85°C × 30 s) using a pilot-scale heat exchanger. The effects of heat treatment on whey protein denaturation and aggregation were determined by chromatography, particle size, turbidity, and rheological analyses. The influence of pH and WPI concentration during heat treatment on the thermal stability of the resulting dispersions was also investigated. Whey protein isolate solutions heated at pH 6.2 were more extensively denatured, had a greater proportion of insoluble aggregates, higher particle size and turbidity, and were significantly less heat-stable than equivalent samples prepared at pH 6.7 and 7.2. The effects of WPI concentration on denaturation/aggregation behavior were more apparent at higher pH where the stabilizing effects of charge repulsion became increasingly influential. Solutions containing 12% (wt/wt) WPI had significantly higher apparent viscosities, at each pH, compared with lower protein concentrations, with solutions prepared at pH 6.2 forming a gel. Smaller average particle size and a higher proportion of soluble aggregates in WPI solutions, pre-heated at pH 6.7 and 7.2, resulted in improved thermal stability on subsequent heating. Higher pH during secondary heating also increased thermal stability. This study offers insight into the interactive effects of pH and whey protein concentration during pilot-scale processing and demonstrates how protein functionality can be controlled through manipulation of these factors.

Enhancing the aqueous solubility of curcumin at acidic condition through the complexation with whey protein nanofibrils

In the present study, whey protein nanofibrils (WPN) were used as carriers to improve the aqueous solubility of curcumin at acidic conditions (pH 3.2) for expanding its applications in functional beverages and drinks. Nanofibrils with nanometric diameter (less than 15 nm) and micrometric length were produced by 5 h heating (85 °C) of whey protein isolate (WPI) solution at pH 2.0. WPN showed a higher surface hydrophobicity compared to the native parental proteins which improved its ability to form a soluble complex with curcumin. The water solubility of curcumin was increased by about 1200-folds through the complexation with WPN, whereas the complexation with WPI increased its solubility by approximately 180-folds. Fluorescence measurements and Fourier transform infra-red spectroscopy indicated that the hydrogen bonding and hydrophobic interactions were mainly contributed to the formation of curcumin-WPN complexes. Circular dichroism spectroscopy revealed that the secondary structures of WPN were not significantly affected by binding to curcumin. The structural phase of nanocomplexes also was studied using X-ray diffraction analysis. Complexation of whey protein nanofibrils with curcumin improved their apparent viscosity and surface activity which makes them appropriate candidates to design new functional food emulsions and beverages. The results of antioxidant activity measurements (DPPH radical scavenging activity and reducing power) also showed that the antioxidant capacity of curcumin was drastically improved through the complexation with WPN. Generally, this study suggests that the whey protein nanofibril could be employed as a material to enhance the food applications of curcumin as a water-insoluble bioactive compound.

Multi-scale understanding of the effects of the solvent and process on whey protein emulsifying properties: Application to dairy emulsion

The combined effects of solvent and processing on whey protein isolate (WPI) emulsifying properties were investigated using a multi-scale approach in realistic conditions. WPI was solubilized in purified water or permeate then no treated, heat treated, mechanically treated (rotor/stator-sonication) or treated by dynamic pressure (16, 100, 350 MPa). Afterward, the treated WPI solutions were used to make emulsions. The approach revealed links between physico-chemical and interfacial properties of the WPI, and the final structure of the resulting emulsions. As expected, the heat treatment caused great changes, with the aggregation of the proteins and the increase in the exposure of hydrophobic zones of the aggregates. The high dynamic pressure of 350 MPa also led to changes in physico-chemical properties, but the mechanical treatment only caused few changes. The adsorption kinetics and reorganization of WPI at the interface depended on their aggregation state, hydrophobicity, ζ-potential and total free SH. All the non-aggregated WPI adsorbed as a thick film at the oil/water interface except the ones treated at 350 MPa, which adsorbed as a thinner film. The sample treated at 100 MPa might have the most close-packed interfacial film, with the highest amount of protein loaded. When aggregated, the whey proteins needed more time to adsorb at the interface and few reorganized once adsorbed. The final interfacial elasticity was higher in water than in permeate. Depending on the solvent, heating and pressure, the variety of physico-chemical properties obtained resulted in different emulsion structures: stable, more or less aggregated or slightly coalesced. A diagram based on the solvent and processing effects was proposed to link organizations at the three studied scales.

Exploiting Complex Formation between Polysaccharides and Protein Microgels To Influence Particle Stabilization of W/W Emulsions.

Protein particles were complexed with polysaccharides, and the effect on their capacity to stabilize water-in-water (W/W) emulsions was investigated. Protein microgels were formed by heating aqueous solutions of whey protein isolate. The microgels were subsequently mixed with anionic or cationic polysaccharides: κ-carrageenan (κ-car) or chitosan, respectively. The molar mass and radius of the complexes formed in dilute microgel suspensions (40 mg/L) were characterized by light scattering techniques as a function of the pH and the composition. The structure and stability of complexes formed at a higher microgel concentration (3 g/L) were studied by confocal laser scanning microscopy. It was found that small stable complexes can be formed with κ-car between pH 4.3 and pH 5.5 and with chitosan between pH 4.1 and pH 6.5, that is, both below and above the isoionic point of the microgels (pI = 5.0). Complexation with polysaccharides stabilized aqueous suspensions of microgels in the pH range where they flocculated in the absence of polysaccharides (4.3-5.5). W/W emulsions were produced by mixing dextran and poly(ethylene oxide) solutions. Microgels added to these emulsions spontaneously form a layer around the dispersed droplets, which inhibits coalescence to different extents depending on the conditions. The effect of complexation on the structure of the emulsions was investigated as a function of the pH. It is shown that stable liquid-like emulsions can be obtained in the pH range where emulsions containing only microgels flocculate.

Effect of pH, carbohydrates, and NaCl on functional properties of whey proteins

The effect of pH (7,5 and 3), and the addition of glucose, sucrose and Litesse II, as well as sodium chloride (1,2 and 3%) on the functional properties of whey protein isolate and whey protein concentrate was investigated. Foaming capacity and emulsifying activity index were significantly affected by the change of the pH in the prepared solutions. The addition of glucose to protein solutions had a slight impact (P>0.05) on the functional properties, while the foaming capacity of whey protein isolate solution significantly increased with the presence of sucrose. Litesse II significantly increased the foaming capacity of whey protein isolate and whey protein concentrate solutions, whereas the emulsifying activity index of whey protein isolate solution significantly increased with adding 1 and 2 % Litesse II. The addition of NaCl significantly increased the foaming properties of whey protein isolate and whey protein concentrate solutions and increased the emulsifying activity index of whey protein concentrate solution.

The effect of direct and indirect heat treatment on the attributes of whey protein beverages

Thermal processing of ready-to-drink high protein beverages can have a substantial impact on the physical and sensory properties of the final product for long-life milks such as extended shelf life and ultra high temperature processed products. Direct and indirect heat treatment technologies were applied to whey protein isolate (WPI) -based beverages containing 4, 6 or 8% (w/w) protein. Lower levels of protein denaturation (66–94%) were observed using direct heating compared with indirect heating (95–99%) across protein levels and heating temperatures (121 and 135 °C final heat). Direct heat treatment resulted in significantly lower viscosity and less extensive changes to the volatile profile, compared with indirect heat treatment. Overall, the application of direct and indirect heat treatment to WPI solutions resulted in significantly different final products in terms of appearance, physical characteristics and volatile profile, with direct heating resulting in many enhanced properties compared with conventional indirect heat treatment.

Whey protein isolate for the preparation of covalent immobilization beads

Whey protein isolate (WPI) was employed, for the first time, to activate carrageenan (Car) beads for the covalent immobilization of the Aspergillus oryzae β-D-galactosidase (β-gal). These Car beads were subjected to a WPI treatment step followed by a glutaraldehyde (GA) treatment step in order to enable such covalent immobilization. The WPI treatment was optimized via the Box-Behnken Design (BBD). The BBD anticipated that treating the Car beads with a 2.36% WPI solution of pH 5.25 for 7.04 h would allow for the attainment of an immobilized β-gal's activity recovery percent of 34.43%. A verification experiment was accomplished while employing the abovementioned conditions and an immobilized β-gal's activity recovery percent of 34.80 ± 1.11% was attained. It was also shown that the immobilization of β-gal onto the GA-WPI treated Car beads did not alter the enzyme's optimum temperature or optimum pH. Moreover, a reusability study was conducted and 93.84 ± 0.72% of the immobilized β-gal's initial observed activity was preserved during the 13th reusability cycle.

Controlling denaturation and aggregation of whey proteins during thermal processing by modifying temperature and calcium concentration

Whey protein isolate solutions (8.00 g protein/100 g; pH 6.8) were treated for 2 min at 72, 85 or 85 °C with 2.2 mM added calcium Ca to produce four whey protein systems: unheated control (WPI‐UH), heated at 72 °C (WPI‐H72), heated at 85 °C (WPI‐H85) or heated at 85 °C with added Ca (WPI‐H85Ca). Total levels of whey protein denaturation increased with increasing temperature, while the extent of aggregation increased with the addition of Ca, contributing to differences in viscosity. Significant changes in Ca ion concentration, turbidity and colour on heating of WPI‐H85Ca, compared to WPI‐UH, demonstrated the role of Ca in whey protein aggregation.

Acid-induced gelation of whey protein aggregates: Kinetics, gel structure and rheological properties

Acid-induced gelation of fractal aggregates with different sizes formed by preheating whey protein isolate solutions has been investigated in the protein concentration range 10–60 g/L as a function of the net protein charge density (α). Homogeneous gels could be formed in a range of α that depended on the heating time and temperature. Decrease of the negative charge density of the proteins increased the rate of gelation, but lowering α to less negative values than approximately −2.5 by adding HCl increased turbidity of the solutions and led to syneresis. Strong syneresis also occurred at more negative net charge densities (higher pH) if the heat treatment was too severe. The elastic modulus of homogeneous gels increased with increasing protein concentration. The heterogeneity of the gels decreased with increasing protein concentration. The temperature dependence of the gelation rate was characterized by an activation energy of 155 kJ/mol independent of the charge density, protein concentration and aggregate size. Increase of the temperature increased the gelation rate, but did not influence the elastic modulus of the gels. The present findings on acid-induced gelation are compared with gelation of the same WPI aggregates induced by adding CaCl2 reported elsewhere.

Influence of seeding and stirring on the structural properties and formation yield of whey protein microgels

The effect of seeding whey protein isolate (WPI) solution with pre-made whey protein soluble aggregates (WPSAs) and stirring on the yield and structural and chemical attributes of whey protein microgels (WPMs) was investigated. Fluorimetry indicated that the heat-exposed hydrophobic amino acids were buried again by WPM formation. Full substitution of WPI with WPSA under non-stirred conditions generated smaller WPMs of ∼200 nm diameter, imaged by atomic force microscopy. Furthermore, full substitution yielded WPMs of a lower percentage proportion of β-sheet structure and a higher proportion of random structures than those of the sample prepared from the 50/50 mix of WPI and WPSA. Stirring increased the hydrodynamic size of WPMs at every ratio of WPI/WPSA. Microgelation under stirring at a WPI/WPSA ratio of 25/75 gave the highest yield (80%) of WPM. Gel electrophoresis indicated that stirring and full substitution of WPI with WPSA caused formation of WPMs rich in β-lactoglobulin monomer.

A detailed investigation of whey protein isolate solutions and gels reveals a number of novel characteristics

• Anomalous structures and behaviour in WPI solutions and gels found at pH 6.2. • Maximum onset temperature of β-lactoglobulin denaturation occurred at pH 3.4. • Order of aggregate formation determined transition region gel microstructure. • Spherical aggregates gelled more rapidly; fine-stranded gels firmed more on cooling. • G′ correlated positively with fracture stress and negatively with fracture strain.

Extending protein functionality: Microfluidization of heat denatured whey protein fibrils

The functional attributes of globular proteins can be extended by controlling the nature of the aggregates they form. In this study, the effect of thermal treatment (85°C/20 min) and high pressure microfluidization (20,000 psi, 1 pass) on the physical properties of whey protein isolate solutions (5–9%; pH 2) was investigated. Heating solutions of native whey protein isolate (8 wt%) under these acidic conditions led to the formation of highly viscous solutions (η = 306 mPa s) with low turbidity (τ = 0.04 cm−1), which was attributed to the formation of protein fibrils (effective d = 310 nm). Microfluidization of these protein fibrils decreased their length (effective d = 97 nm) leading to a substantial reduction in solution viscosity (η = 3.8 mPa s), and a slight reduction in turbidity (τ < 0.03 cm−1). The impact of solution pH (2–7) on the appearance and rheology of native, heated, and heated-microfluidized whey protein isolate solutions was then examined. For all systems, highly turbid solutions (τ > 1 cm−1) were formed at pH values close to the isoelectric point of the whey proteins (pH 4.5) due to protein self-association caused by reduction of the electrostatic repulsion between the protein molecules. Highly viscous or gelled solutions were formed for the heated and heated-microfluidized proteins across a wide pH range, which was attributed to the presence of fibrils. The study showed that the functional attributes of whey proteins can be modulated by thermal and high-pressure homogenization treatment.

Formation and characterization of thiol-modified fibrillated whey protein isolate solution with enhanced functionalities

The effect of thiolation using propanethiol on the functionalities of fibrillated whey protein isolate (WPI) solution at different pH values was studied. Fibrillated WPI solutions were thiolated at different molar ratios of propanethiol:carboxyl group (0.5:1, 1:1, 2:1, 3:1, 4:1) and the highest esterification extent ratio was obtained at 4:1 (pH 9). We also found that the thiolation process improved the foaming capacity and foam stability. TEM micrographs evidenced aggregation of thiol-modified fibrillated WPI. A network of shortened fibrils attached to each other was formed upon thiolation, suggesting good physical interaction. This was coherent with the increment of zeta potential values, indicating a greater repulsion force to retard fibrils aggregation. Thiolation enhanced emulsifying stability index of thiol-modified fibrillated WPI solution (pH 8) and diminished its susceptibility to pH changes. This has broadened the potential application of fibrils as food ingredients.

Influence of pulsed light treatment on the aggregation of whey protein isolate

The effect of pulsed light (PL) on the aggregation of whey protein isolate (WPI) solutions was investigated. PL fluence values from 4 to 16J/cm2 were used to treat WPI (1% w/v) solutions in sodium phosphate buffer (pH=7.5). Whey protein structural modification and aggregation were assessed through the determination of free SH-groups and UV-absorption spectra. Additionally, covalent and non-covalently linked protein-protein interactions were identified through the measurement of turbidity, aggregation index, particle size distribution, and SDS-PAGE.WPI upon PL treatment showed structural changes as demonstrated by the immediate increase of free SH-group content (unfolding) and the subsequent formation of a small fraction of aggregation of unfolded proteins, due to both hydrophobic interactions and the formation of disulphide bonds. Turbidity, mean particle size, and aggregation index increased in samples treated at PL fluence from 4 to 16J/cm2. Furthermore, particle size distribution analysis of samples treated at higher fluence indicated that WPI dimer dissociation and formation of larger particles were likely to occur. The association of intermediate and larger protein molecules as well as the formation of soluble aggregates between β-lactoglobulin and α-lactalbumin were also observed in gel electrophoresis analysis. In conclusion, the results of this investigation demonstrated the potential of PL treatments to induce protein denaturation, with a minimal formation of soluble protein aggregates.

Effects of high intensity ultrasound on acid-induced gelation properties of whey protein gel

Pre-heated whey protein isolate (WPI) solution (10% w/v, 85°C for 30min) was prepared and subjected to high-intensity ultrasound (HUS, 20kHz) at different durations (5–40min) before acidification to determine the effects of HUS on glucono-δ-lactone (GDL)-induced gelation properties of whey protein gel. Results showed that HUS reduced the particle size and increased surface free sulfhydryl groups of pre-heated whey protein solution. Free sulfhydryl (-SH) content of the acid-induced WPI gels (GIWG) and protein solubility in presence of 8M urea were significantly reduced (P<0.05) by HUS (20 or 40min), indicating that HUS facilitated formation of more disulfide bonds during/after the gelation process. HUS significantly increased (P<0.05) the water holding capacity (WHC), gel strength, gel firmness (G′) as well as the frequency dependence of GIWG. The WHC, gel strength and gel firmness were positively correlated with surface free -SH content of pre-heated WPI and negatively correlated with particle size of pre-heated WPI and free -SH content of GIWG. The results indicated that high intensity ultrasound could be used for modifying whey protein to improve its gelling properties.

Formation of concentrated biopolymer particles composed of oppositely charged WPI and pectin for food applications

ABSTRACTThe application of protein–polysaccharide complexes as potential structure modifier, fat replacer, or emulsifying agents in food dispersions has gained increasing interest amongst scientists and manufacturers. Based on associative complexation, low biopolymer concentrations are typically used to generate particulated complexes. The current study, however, presents results that focused on the formation of concentrated biopolymer dispersions. A simple heat treatment was applied to tailor the overall water content of the biopolymer dispersion. For that purpose, whey protein isolate (WPI) and citrus pectin (DE 71%) solutions were mixed at different pH and biopolymer ratios to induce complex coacervation and subsequently heat-treated (ϑ = 90–95°C). Phase separation behavior, microstructural, rheological, and electrical properties of the complexes were investigated by surface charge, turbidity, particle size, rheometry, and light microscopy measurements. Results revealed that complexation was induced under acidic conditions, whereas high WPI:citrus pectin ratios led to positive surface charges, promoting the formation of large and dense particles. In addition, concentrated complex dispersions with water contents ≥80% could be manufactured and easily re-dispersed, whereas complexes maintained their particulate structures. Results are of importance for future studies where we intend to incorporate concentrated biopolymer particles as structuring agents in complex food matrices.

Characterisation of heat-induced protein aggregation in whey protein isolate and the influence of aggregation on the availability of amino groups as measured by the ortho-phthaldialdehyde (OPA) and trinitrobenzenesulfonic acid (TNBS) methods.

Whey protein isolate (WPI) solutions, with different levels of aggregated protein, were prepared by heating (5% protein, pH 7, 90°C for 30min) WPI solutions with either 20mM added NaCl (WPI+NaCl), 5mM N-ethylmaleimide (WPI+NEM) or 20mM added NaCl and 5mM NEM (WPI+NaCl+NEM). Gel electrophoresis demonstrated that the heated WPI and WPI+NaCl solutions had higher levels of aggregated protein, due to more covalent interactions between proteins, than the heated WPI+NEM and WPI+NaCl+NEM solutions. There were marked differences in the levels of amino groups between all heated WPI solutions when measured by the OPA and TNBS methods, with lower levels being measured by the TNBS method than by the OPA method. These results demonstrate that the measurement of available amino groups by the OPA method is less impacted than by the TNBS method after heat-induced structural changes, arising from disulfide or sulfhydryl-disulfide bond-mediated aggregation of whey protein molecules.

Ultrasound‐induced changes in physical and functional properties of whey proteins

SummaryUse of high‐intensity ultrasound to modify certain functional properties of whey proteins is an alternative to traditional method in food industry. Whey protein isolate (WPI) solutions were treated with an ultrasound probe (20 kHz) at different intensities (20% or 30% amplitude) and durations (10 or 20 min). Results showed that ultrasound treatment changed physical and several functional properties of whey proteins including decreased particle size (from 190.4 nm to 138.0 nm), increased surface hydrophobicity (from 5.13 × 105 to 5.77 × 105), free sulphydryl groups (from 52.64 μmol SH g−1 to 53.64–58.77 μmol SH g−1), solubility (from 74.95% to 89.70%), emulsion activity index (from 3.18 m2 g−1 to 3.59–5.32 m2 g−1) and emulsion stability index (from 62.26 min to 71.44–104.83 min), and changed viscosity (from 5.51 mPa.s to 4.81–5.64 mPa.s). Therefore, we conclude that high‐intensity ultrasound can be potentially applied to whey proteins to improve its specific functions during food processing.

Effect of hydrogen peroxide on improving the heat stability of whey protein isolate solutions

Whey protein isolate (WPI) solutions (12.8%w/w protein) were treated with varying concentrations of H2O2 in the range of 0-0.144 H2O2 to protein ratios (HTPR) by the addition of the required quantity of H2O2 and deionized water. The samples were analyzed for heat stability, rheological properties, denaturation level of β-lactoglobulin (β-LG) and α-lactalbumin (α-LA). The samples treated with H2O2 concentration >0.072 (HTPR) showed significant improvement in the heat stability, and decreased whey protein denaturation and aggregation. The WPI solution treated with H2O2 (>0.072 HTPR) remained in the liquid state after heat treatment at 120°C, whereas the control samples formed gel upon heat treatment. Detailed analysis of these samples suggested that the improvement in the heat stability of H2O2 treated WPI solution was attributed to the significant reduction in the sulfhydryl-disulfide interchange reaction during denaturation of β-LG and α-LA.

Whey protein isolate and flaxseed (Linum usitatissimum L.) gum electrostatic coacervates: Turbidity and rheology

Coacervates were prepared with flaxseed (Linum usitatissimum L.) gum (FG) and whey protein isolate (WPI) solutions in water. Structural transitions during coacervate formation were monitored by turbidimetry and zeta potentiometry. Biopolymer mixing ratio R (1:4–15:1, w/w) and pH (6.0–1.4) effects on pH-dependent phase transitions (pHc, pHϕ1, and pHϕ2) were examined. Where R = 1:1 (w/w), pHc, pHϕ1, and pHϕ2 were observed at pH 5.4, pH 5.0, and pH 1.8, respectively. The highest optical density (OD600 = 0.617 ± 0.009) occurred at pH 3.4, the pHmax. As R increased from 1:4 to 15:1, pHϕ1 and pHϕ2 also increased (pHϕ1 4.2 to 5.2 and pHϕ2 1.8 to 2.2). Accordingly, pHmax shifted from 3.0 to 4.8 while pHc was independent of R. The shift of pHmax was consistent with isoelectric point (IEP) of the WPI-FG mixture determined by electrophoretic mobility measurements. The maximum WPI-FG coacervate formation occurred at R = 2:1 (w/w) and pHmax = 3.8 with OD600 = 0.783 ± 0.018. Dynamic shear viscosity and viscoelasticity of WPI-FG coacervates were determined at a range of pH and R. WPI-FG coacervates exhibited shear-thinning behavior and gel-like properties. The highest dynamic viscosity and viscoelasticity of WPI-FG coacervates were observed at pH 3.8 with R = 2:1 (w/w). Electroneutrality of WPI-FG mixture favored coacervate formation with a more compact structure and improved rheological properties. Light microscopy observations revealed that uncharged WPI-FG mixtures formed coacervates that were more compact than charged coacervates and exhibited improved rheological properties.