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.     

EGCG与乳清蛋白相互作用的光谱分析

探究表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)与乳清分离蛋白(whey protein isolate,WPI)间的相互作用。测定不同pH值与不同离子浓度条件下WPI的紫外吸收光谱、导数光谱和荧光光谱,利用SternVolmer方程判断荧光猝灭机制,采用位点结合模型公式计算结合常数和结合位点数。结果表明,紫外吸收光谱和导数光谱结果显示EGCG改变了WPI中酪氨酸和色氨酸残基所处的微环境,使WPI的分子构象发生了变化。荧光光谱结果显示EGCG可以有规律地猝灭WPI的内源荧光,猝灭机理为静态猝灭,EGCG与WPI结合常数在pH 2.0~9.0范围内随pH的增大先减小后增大,在离子浓度0.10 mol/L^0.20 mol/L范围内随离子浓度的增大而增大,结合位点数约为1。EGCG与WPI间存在静电相互作用。     

Stability of citral in protein- and gum arabic-stabilized oil-in-water emulsions

Citral is a major flavor component of citrus oils that can undergo chemical degradation leading to loss of aroma and formation of off-flavors. Engineering the interface of emulsion droplets with emulsifiers that inhibit chemical reactions could provide a novel technique to stabilize citral. The objective of this study was to determine if citral was more stable in emulsions stabilized with whey protein isolate (WPI) than gum arabic (GA). Degradation of citral was equal to or less in GA- than WPI-stabilized emulsion at pH 3.0 and 7.0. However, formation of the citral oxidation product, p-cymene was greater in the GA- than WPI-stabilized emulsion at pH 3.0 and 7.0. Emulsions stabilized by WPI had a better creaming stability than those stabilized by GA because the protein emulsifier was able to produce smaller lipid droplets during homogenization. These data suggest that WPI was able to inhibit the oxidative deterioration of citral in oil-in-water emulsions. The ability of WPI to decrease oxidative reactions could be due to the formation of a cationic emulsion droplet interface at pH 3.0 which can repel prooxidative metals and/or the ability of amino acids in WPI to scavenge free radical and chelate prooxidative metals.     

Ionic strength and buffering capacity of emulsifying salts determine denaturation and gelation temperatures of whey proteins

Ionic conditions affect the denaturation and gelling of whey proteins, affecting the physical properties of foods in which proteins are used as ingredients. We comprehensively investigated the effect of the presence of commonly used emulsifying salts on the denaturation and gelling properties of concentrated solutions of β-lactoglobulin (β-LG) and whey protein isolate (WPI). The denaturation temperature in water was 73.5°C [coefficient of variation (CV) 0.49%], 71.8°C (CV 0.38%), and 69.9°C (CV 0.41%) for β-LG (14% wt/wt), β-LG (30% wt/wt), and WPI (30% wt/wt), respectively. Increasing the concentration of salts, except for sodium hexametaphosphate, resulted in a linear increase in the denaturation temperature of WPI (kosmotropic behavior) and an acceleration in its gelling rate. Sodium chloride and tartrate salts exhibited the strongest effect in protecting WPI against thermal denaturation. Despite the constant initial pH of all solutions, salts having buffering capacity (e.g., phosphate and citrate salts) prevented a decrease in pH as the temperature increased above 70°C, resulting in a decline in denaturation temperature at low salt concentrations (≤0.2 mol/g). When pH was kept constant at denaturation temperature, all salts except sodium hexametaphosphate, which exhibited chaotropic behavior, exhibited similar effects on denaturation temperature. At low salt concentration, gelation was the controlling step, occurring up to 10°C above denaturation temperature. At high salt concentration (>3% wt/wt), thermal denaturation was the controlling step, with gelation occurring immediately after. These results indicate that the ionic and buffering properties of salts added to milk will determine the native versus denatured state and gelation of whey proteins in systems subjected to high temperature, short time processing (72°C for 15 s).     

pH and Heat Treatment Effects on Foaming of Whey Protein isolate

The overrun obtained by whipping whey protein isolate (WPI) was significantly (p<0.05) affected by changing pH. Heating WPI at pH 4.0 reduced rate and amount of overrun. The highest overrun values for unheated WPI were observed at pH 5.0 and 7.0 after heating at 55°C for 10 min. The maximum foam stability for unheated WPI was obtained at pH 5.0. Heat treatment had little effect on stability at pH 4.0 or 7.0 but at pH 5.0, 80°C for 10 min improved stability by 65%. Based on surface pressure data, the rate of adsorption of β-lactoglobulin interfacial films and the work of compression correlated with overrun, maximum overrun, overrun development and foam stability.     

Enhancement of the functional properties of whey protein by conjugation with maltodextrin under dry‐heating conditions

Conjugation of whey protein isolate (WPI) and maltodextrin (MD, dextrose equivalent of 6) was achieved by dry‐heating at an initial pH of 7.0, at 60 °C and 79% relative humidity, with WPI: MD6 ratio of 1:1, for up to 24 h. Conjugation was achieved with limited development of colour and advanced Maillard products on 24 h of heating. Conjugation increased the protein solubility at pH 4.5, by 7.1–8.5%, compared to the unheated and heated WPI controls. Conjugation of WPI with MD6 enhanced the stability and retention of clarity in protein solutions heated at 85 °C for 10 min with 50 mM added NaCl.     

Modification of rheological properties of whey protein isolates by limited proteolysis

Whey protein isolate (WPI) was subjected to limited tryptic hydrolysis and the effect of the limited hydrolysis on the rheological properties of WPI was examined and compared with those of untreated WPI. At 10% concentration (w/v in 50 mM TES buffer, pH 7.0, containing 50 mM NaCl), both WPI and the enzyme-treated WPI (EWPI) formed heat-induced viscoelastic gels. However, EWPI formed weaker gels (lower storage modulus) than WPI gels. Moreover, a lower gelation point (77 °C) was obtained for EWPI as compared with that of WPI which gelled at 80 °C only after holding 1.4 min. Thermal analysis and aggregation studies indicated that limited proteolysis resulted in changes in the denaturation and aggregation properties. As a consequenece, EWPI formed particulated gels, while WPI formed fine-stranded gels. In keeping with the formation of a particulate gel, Texture Profile Analysis (TPA) of the heat-induced gels (at 80 °C for 30 min) revealed that EWPI gels possessed significantly higher (p < 0.05) cohesiveness, hardness, gumminess, and chewiness but did not fracture at 75% deformation. The results suggest that the domain peptides, especially β-lactoglobulin domains released by the limited proteolysis, were responsible for the altered gelation properties.     

Encapsulation of eugenol using Maillard-type conjugates to form transparent and heat stable nanoscale dispersions

Maillard-type protein-carbohydrate conjugates are known for their excellent emulsifying properties and have been used to encapsulate volatile oils and flavor compounds. In the present study, eugenol was used as a model compound for encapsulation in conjugates of whey protein isolate (WPI) and maltodextrins (MD) made using different WPI:MD mass ratios and MD chain lengths. The encapsulation involved two steps, emulsifying an oil phase of eugenol dissolved in hexane into an aqueous phase with dissolved conjugates and spray drying the emulsion. Mass yield up to 82.7 g/100 g and encapsulation efficiency as high as 35.7 g/100 g eugenol were observed. After hydrating spray-dried powders, several samples with an eugenol content above its solubility limit demonstrated transparent dispersions at pH 3.0 and 7.0 after heating at 80 °C for 15 min, corresponding to mean diameters smaller than ca. 100 nm. One treatment also showed transparent dispersions after heating at pH 5.0, which is near the isoelectric point of whey proteins, in contrast to gel formation for a control prepared with a mixture of non-conjugated WPI and MD. The present study demonstrates potential to produce food grade nanoscale systems for delivering lipophilic bioactives in functional beverages, without adversely affecting their visual appearance.     

Thermal Stability Improvement of Rice Bran Albumin Protein Incorporated with Epigallocatechin Gallate

Rice bran albumin protein (RAP) is sensitive to thermal changes and tends to degrade when exposed to high‐temperature processing. In this work, RAP–epigallocatechin‐3‐gallate (EGCG) complex (RAPE) was prepared and the thermal stability was evaluated. Fluorescence results showed that EGCG could interact with RAP with a binding number n of 0.0885:1 (EGCG:RAP, w/w) and a binding constant K of 1.02 (± 0.002) ×10⁴/M, suggesting both hydrogen bonding and van der Waals forces played an important role. FTIR analysis demonstrated that EGCG could induce secondary structural changes in RAP above a ratio of 1.6:1 (EGCG:RAP, w/w). Interestingly, the secondary structure changes of RAPE at different temperatures (25, 50, 60, 70, and 80 °C) were inhibited compared with that for RAP, suggesting RAPE was more resistant and stable to the heat treatment. In addition, a dense porous structure of RAPE was achieved due to the EGCG binding after thermal treatment. Furthermore, the T_peak temperature of RAPE increased significantly from 64.58 to 74.16 °C and the enthalpy also increased from 85.53 to 138.52 J/g with a mass ratio increasing from 0 to 3.2 (EGCG:RAP, w/w), demonstrating the thermal stability of RAPE. In addition, the valine, methionine, and lysine content in RAPE were significantly higher than RAP following 80 °C treatment for 20 min (P < 0.05), exhibiting enhanced amino acid profiles, which might be due to EGCG–RAP interactions and microenvironment changes around relevant amino acids. These findings demonstrate that EGCG has the potential to improve the thermal stability of sensitive proteins and is beneficial for usage in the food industry.     

Heat-Induced Aggregation Properties of Whey Proteins as Affected by Storage Conditions of Whey Protein Isolate Powders

The control of storage as any other manufacturing steps of dairy powders is essential to preserve protein functional properties. This study aimed to determine the effects of different storage conditions on both protein denaturation and protein lactosylation in whey protein isolate (WPI) powder, and also on heat-induced aggregation. Two different storage temperature conditions (20 and 40 °C) were studied over 15 months. Our results showed that protein lactosylation progressively increased in WPI powders over 15 months at 20 °C, but heat-induced aggregation properties did not significantly differ from non-aged WPI. On the other hand, powders presented a high level of denaturation and aggregation at 40 °C from the first 2 weeks of storage, involving first protein lactosylation and then aggregation in the dry state. This was correlated with an increasing Browning Index from 15 days of storage. These changes occurred with a decrease in aggregate size after heat treatment at 5.8?≤?pH?≤?6.6 and modification of heat-induced aggregate shapes.     

Isoelectric protein precipitation from mild-acidic extracts of de-oiled sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) press cake

Sunflower protein extraction using sodium chloride solutions allows high extraction yields at pH ~6 while preventing oxidation and covalent binding of phenolic compounds to the proteins, which usually occurs under alkaline conditions. Since protein solubility is enhanced at high NaCl concentrations, isoelectric precipitation had to be adapted in the present study besides developing subsequent desalting steps. Precipitation losses decreased from 62 to 31% with increasing NaCl (0.6–2.0 mol/L) and protein concentrations of the solution (6–14 mg/mL) and decreasing pH of precipitation (pH 4.5–3.0). Maximum yields were achieved at low temperature (8 °C) and upon instant acid addition. After adsorptive removal of co-extracted phenolics from the protein extracts, overall protein yields were considerably higher after precipitation at pH 3.5 compared with 4.5, but only slightly higher after washing of the precipitates. The physico-chemical properties of the protein isolates did not differ significantly except for the marked protein denaturation upon precipitation at pH 3.5. From the proposed process, light-coloured protein isolates of high purity (>98%) are obtained, which are suitable for food use.     

Effects of Polymerized Whey Proteins on Consistency and Water-holding Properties of Goat's Milk Yogurt

ABSTRACT The effects of polymerized whey proteins (PWP) on functional properties of goat's milk yogurt were investigated. PWP were prepared by heating whey protein isolate (WPI) dispersion (8.0% protein, pH 7.0) at 90 °C for 30 min. Three reconstituted goat milk (RGM) (12% total solids [TS] as control; RGM with 2.4% unheated WPI; and RGM with 2.4% PWP) and 1 RGM with 16.7% TS were prepared and inoculated with 0.04% yogurt starter culture. Inoculated milk was incubated at 43 °C for 5 h, cooled to 4 °C in an ice‐water bath, and then placed at refrigerator (4 °C) overnight before testing. Incorporation of PWP significantly (P < 0.001) increased the viscosity (by 80%) and decreased the syneresis (by 25%) of the yogurt samples, whereas addition of unheated WPI did not significantly affect the viscosity and syneresis compared with the control. There were no changes in pH, TS, ash, fat, protein, and lactose contents among yogurt samples except the solids fortified control. Yogurt with 16.7% TS had the lowest syneresis but did not improve in viscosity. Transmission electron microscopy micrographs demonstrated that the microstructure of the goat's milk yogurt gel with PWP was denser than the control. Results of this study indicate that polymerized whey proteins may be a novel protein‐based thickening agent for improving the functional properties of goat's milk yogurt and other similar products.     

Effects of heating rate and pH on fracture and water-holding properties of globular protein gels as explained by micro-phase separation

The effect of heating rate and pH on fracture properties and held water (HW) of globular protein gels was investigated. The study was divided into 2 experiments. In the 1st experiment, whey protein isolate (WPI) and egg white protein (EWP) gels were formed at pH 4.5 and 7.0 using heating rates ranging from 0.1 to 35 °C/min and holding times at 80 °C up to 240 min. The 2nd experiment used one heating condition (80 °C for 60 min) and probed in detail the pH range of 4.5 to 7.0 for EWP gels. Fracture properties of gels were measured by torsional deformation and HW was measured as the amount of fluid retained after a mild centrifugation. Single or micro-phase separated conditions were determined by confocal laser scanning microscopy. The effect of heating rate on fracture properties and HW of globular protein gels can be explained by phase stability of the protein dispersion and total thermal input. Minimal difference in fracture properties and HW of EWP gels at pH 4.5 compared with pH 7.0 were observed while WPI gels were stronger and had higher HW at pH 7.0 as compared to 4.5. This was due to a mild degree of micro-phase separation of EWP gels across the pH range whereas WPI gels only showed an extreme micro-phase separation in a narrow pH range. In summary, gel formation and physical properties of globular protein gels can be explained by micro-phase separation. PRACTICAL APPLICATION: The effect of heating conditions on hardness and water-holding properties of protein gels is explained by the relative percentage of micro-phase separated proteins. Heating rates that are too rapid require additional holding time at the end-point temperature to allow for full network development. Increase in degree of micro-phase separation decreases the ability for protein gels to hold water.     

Release of Single-Cell Protein by Induced Cell Lysis

A temperature sensitive lysis mutant of Saccharomyces cerevisiae JD 109, which grows normally at 27°C, stops growing and partially lyses at a nonpermissive temperature (37°C). In studies in a 14L fermentor with temperature shifted (from 27°C to 37°C) at 3.0 g/L cells, the effect of pH was investigated with the result that the rate of lysis is faster at pH 8 than at pH 5.6; the protein in the cells decreases from 45 to 35%. When both pH (from 5.6 to 8.0) and temperature are shifted, the culture showed a more noticeable inhibition of growth and lysis than when only pH was changed and the temperature was maintained at 27°C.     

ATP and Creatine Phosphate Breakdown in Spiked Plaice Muscle during Storage, and Activities of Some Enzymes Involved

The onset of rigor-mortis of plaice Puralichthys olivaceus spiked at the brain started much faster at 0°C than at 10°C. Correspondingly, ATP and creatine phosphate breakdown and lactic acid accumulation in the muscle were faster at 0°C. ATP concentration was constant at 5 μmol/g until creatine phosphate decreased from the initial conccntration at around 20 μmol/g to 5 μmol/g, irrespective of storage temperature. Simultaneously, lactic acid accumulated, slowly at first, until ATP concentration started decreasing, and then quickly accompanying the full-rigor state. Activities of the enzymes involved in ATP and creatine phosphate consumption-myofibrillar and sarcoplasmic AT-Pases and creatine kinase-were temperature-dependent, and decreased with decrease in temperature.     

Microcoagulation of a Whey Protein Isolate by Extrusion Cooking at Acid pH

A whey protein isolate (WPI) was coagulated by thermomechanical processing in a twin screw extruder. Nonaggregated semi-solid spreads were obtained only in the pH range 3.5–3.9, at ca 20% protein (77% water), a barrel temperature of 90–100°C and a screw speed of 100–200 rpm. WPI extrusion-coagulated at pH 3.9 displayed a high nitrogen solubility (NSI) (43–47%). Electrophoresis indicated that the β-lactoglobulin constituent was entirely soluble in 1% SDS, while scanning calorimetry revealed about 82% protein unfolding. WPI extrusion-coagulated at pH 4.5–6.8 displayed lower NSI (25%), were less soluble in 1% SDS, were 88% unfolded and had grainy texture. Light microscopy, centrifugation in glycerol solutions, and laser diffractometry indicated the acid spread (pH 3.9) was composed of small coagulated particles, mean diameter 11.5 μm (volume basis).     

Chemical and physical stability of protein and gum arabic-stabilized oil-in-water emulsions containing limonene

ABSTRACT:  An important flavor component of citrus oils is limonene. Since limonene is lipid soluble, it is often added to foods as an oil-in-water emulsion. However, limonene-containing oil-in-water emulsions are susceptible to both physical instability and oxidative degradation, leading to loss of aroma and formation of off-flavors. Proteins have been found to produce both oxidatively and physically stable emulsions containing triacylglycerols. The objective of this research was to determine if whey protein isolate (WPI) could protect limonene in oil-in-water emulsion droplets more effectively than gum arabic (GA). Limonene degradation and formation of the limonene oxidation products, limonene oxide and carvone, were less in the WPI- than GA-stabilized emulsions at both pHs 3.0 and 7.0. These data suggest that WPI was able to inhibit the oxidative deterioration of limonene in oil-in-water emulsions. The ability of WPI to decrease oxidative reactions could be due to the formation of a cationic emulsion droplet interface at pH 3.0, which can repel prooxidative metals, and/or the ability of amino acids in WPI to scavenge free radical and chelate prooxidative metals.     

Influence of sucrose on cold gelation of heat‐denatured whey protein isolate

A solution of heat‐denatured whey proteins was prepared by heating 100 g kg⁻¹ whey protein isolate (WPI) at pH 7.0 to 75 °C for 15 min in the absence of salt. Heat treatment caused the globular protein molecules to unfold, but electrostatic repulsion opposed strong protein–protein aggregation and so prevented gel formation. When the heat‐denatured whey protein solution was cooled to room temperature and mixed with 15 mM CaCl₂, it formed a gel. We investigated the influence of the presence of sucrose in the protein solutions prior to CaCl₂ addition on the gelation rate. At relatively low concentrations (0–100 g kg⁻¹), sucrose decreased the gelation rate, presumably because sucrose increased the aqueous phase viscosity. At higher concentrations (100–300 g kg⁻¹), sucrose decreased the gelation rate, probably because sugar competes for the water of hydration and therefore increases the attraction between proteins. These data have important implications for the application of cold‐setting WPI ingredients in sweetened food products such as desserts. © 2000 Society of Chemical Industry     

鹰嘴豆分离蛋白的胶凝性

研究了蛋白质浓度、pH、NaCl及CaCl2对鹰嘴豆分离蛋白胶凝性的影响。pH3.0、无盐加入时,蛋白质分散液以溶液状存在;pH3.0、0.1mol/LNaCl与pH7.0、高离子强度(NaCl:0.5～1.0mol/L)条件下,蛋白质分散液表现出半溶液状性质。pH3.0、高离子强度(NaCl:0.5～1.0mol/L)与pH7.0、低离子强度(NaCl:0～0.1mol/L)条件下,蛋白质分散液以凝胶状存在。pH3.0、NaCl浓度0.5～1.0mol/L与pH7.0、NaCl浓度0～0.1mol/L时具有相似的胶凝动力学。CaCl2对蛋白质的胶凝性影响与NaCl基本相同。pH3.0时,CaCl2的浓度为0.1和0.3mol/L时的凝胶强度分别为24和26.4g;NaCl浓度为0.1、0.5、1.0mol/L时的凝胶强度分别为7.6、8.4和9.3g。     

Gelation of single heated vs. double heated whey protein isolate

Gelation of single and double heated whey protein dispersions was investigated using Ca²⁺ as inducing agents. Whey protein isolate (WPI) dispersions (10% w/w) were single heated (30 min, 80 °C at pH 7.0) or double heated (30 min, 80 °C at pH 8.0 and 30 min, 80 °C at pH 7.0) and diluted to obtain the desired protein and/or calcium ions concentration (4–9% and 5–30 mm, respectively). Calcium ions were added directly or by using a dialysis method. Double-heated dispersions gelled faster at lower protein and calcium ion concentrations than single-heated dispersions. Gels obtained from double-heated dispersions had lower values of shear strain and shear stress at fracture than gels obtained from single-heated dispersions. Double heating caused a significant complex modulus (G*) increase at 4% WPI and 15 mm calcium ions in comparison with gels obtained from single-heated dispersion. Less significant differences between gels made from double and single-heated dispersions were observed at 6% WPI, however a higher value of complex modulus was obtained for 8% protein gels from the single-heated solution. Native and non-reduced SDS–PAGE did not show clearly the effect of different procedures of heating on the quantities of polymerised proteins. Proteins in double-heated dispersions had higher hydrophobicity. Increased calcium concentration caused decreased protein hydrophobicity for both single and double-heated solutions.