Effect of Whey Pretreatment on Composition and Functional Properties of Whey Protein Concentrate

The effect of pretreatment upon the composition and physicochemical and functional properties of whey, ultrafiltration (UF) retentate and freeze-dried and spray-dried whey protein concentrates (WPC) was investigated. Pretreatment was by cooling cheese whey to 0-5°C, adding calcium chloride, adjusting to pH 7.3, warming to 50°C, and removing the insoluble precipitate that formed by centrifugation or decantation. UF permeation flux rate of pretreated whey was about double that for control whey. Pretreated whey was essentially turbidity free, contained 85% less milkfat, 37% more calcium and 40% less phosphorus than whey. Pretreated whey WPC proteins were slightly more soluble at pH 3, but less functional for emulsification than whey WPC proteins. Neither whey WPC proteins nor pretreated whey WPC proteins was functional for foaming at 6% protein concentration.     

Elimination of β-Lactoglobulin from Whey to Simulate Human Milk Protein

When the pH of cottage cheese whey was adjusted to 4.5 in the presence of 6.7 mM FeCI³, β-lactoglobulin was eliminated from the whey as a precipitate. However, the majority of immunoglobuhns were also coprecipitated. To recover immunoglobulins together with α-lactalbumin, the whey pH was adjusted to 3.0 in the presence of 4.0 mM FeCI³. After centrifugation of the whey, the supernatant contained exclusively β-lactoglobulin; other whey proteins were found in the precipitate. Excess Fe₊₊₊ in the precipitate was removed by ion exchange or by ultrafiltration. This protein concentrate had a protein composition much more similar to that of human milk whey than that of ultrafiltered whey protein concentrate.     

Structural consequences of dry heating on Beta-Lactoglobulin under controlled pH

Heating in dry state has gained a lot of interest in pharmaceutical and food industries for viral and microbial decontamination of thermo sensible products. Controlled dry heating has now become a common industrial process for improving the functional properties of food proteins. Besides this improvement, chemical modifications in protein structures involving degradation of amino acids, new intra or inter-molecular disulfide bonds, isopeptide bonds, and some other links may occur. These chemical modifications are favored by severe heating under neutral or alkaline conditions, and except disulfide interchanges, are usually not predominant during heat treatments in solution and their occurrence is not well understood. Understanding chemical modifications in protein structure that occur during dry heating of protein powders is a prerequisite for reproducible properties of final products at industrial scale. In the present work, we focused on how dry heating under acidic pH conditions affects the chemical modifications and denaturation/aggregation reactions of whey proteins. Model whey protein (β-lactoglobulin) powders obtained from freeze drying of protein concentrates adjusted to pHs 2.5 & 6.5 were adjusted to a fixed water activity (Aw 0.23). The samples were dry heated at 100 °C for up to 24 hours and structural modifications induced during dry heating were followed. We showed that whatever the pH value, proteins were characterized by irreversible mass losses of 18 Da. Such mass losses were increased at lower pH value. Dry heating mainly generated small aggregates (dimers and oligomers). Strikingly, at pH 2.5 intermolecular disulfide bonds were the only crosslinks between proteins in the aggregated forms while covalent crosslinks other than disulfide bonds also participated at pH 6.5. β-Lactoglobulin hydrolysis was also detected at pH 2.5 and some of the peptides were incorporated in the oligomers. These results underline that chemical modifications in protein structures induced by dry heating are highly pH dependent. Hence, strict control of pH conditions for dry heating is indispensable to give reproducible functionality in products where such modified proteins are incorporated as ingredient.     

Extraction of Whey Proteins with Magnesium and Zinc Salts

A method was developed for the extraction of protein from cottage cheese whey with magnesium and zinc salts. After neutralization of the whey, both magnesium and zinc salts (at 4 and 2% wt/vol, respectively) precipitated most of the protein nitrogen plus some nonprotein nitrogen and lactose. Calcium at these concentrations was only partially effective. Yields of dried precipitate obtained from cottage cheese whey containing approximately 7% solids ranged from 25 to 34 g/L. Precipitates contained 16 to 21% protein and from 11 to 24% lactose; the remainder was ash. Zinc was the preferred reagent because it was effective at neutral pH; magnesium, alone or with calcium, required alkaline pH for thorough precipitation of protein. All reagents tested were effective precipitants of whey protein in a one-step procedure at room temperature.     

Biochemical studies on proteins from cheese whey and blood plasma by-products

Efforts have been done to recover proteins from waste liquors rich in protein in a soluble form. Cheese whey and animal bloods are byproducts from the manufacture of cheese and meat. It contains a variety of proteins which can be reclaimed. The efficiency of protein precipitation from the sweet-cheese whey by the use of hydroxyethyl cellulose (HEC) was similar to that precipitated by the use of carboxymethyl cellulose (CMC). Both are greater than that precipitated by trichloro acetic acid. The same results of the efficiency of precipitation were attained when the plasma protein was precipitated. It was found that cheesewhey protein-HEC-complex and plasms protein-HEC-complex contain a large amount of essential amino acids. Electrophoretic separation of whey protein complex showed that β-Lactoglobulin forms the major fraction while in case of plasma protein complex albumin forms the major fraction. The fractionation patterns of different complexes with HEC, CMC or TCA gave the same components and about the same ratio. It appears from these results that HEC-protein complexes are preferable than CMC-protein complexes or proteins precipitated by TCA. Chemical analysis of whey protein complexes revealed that lactose content of whey protein-HEC-complex was higher than that of CMC-complex or protein precipitated by TCA. Elemental analysis of protein complexes showed that the level of sodium, phosphorus, and potassium was increased while that of copper or zinc decreased. Cellulose derivative protein complexes showed no significant effects on the liver or kindney function of albino rat and these results indicated that no toxic effect was observed from the uses of these protein complexes in feeding.     

Thermal modifications of structure and co-denaturation of alpha-lactalbumin and beta-lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (beta-lactoglobulin or alpha-lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other codenaturation partner, and of existence of a previous thermal pretreatment (industrial whey). alpha-Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at > or = 85 degrees C. After its denaturation, alpha-lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys-X and Arg-X bonds). Heating over 85 degrees C of beta-lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co-denaturation of alpha-lactalbumin with beta-lactoglobulin increased their aggregation and resulted in complete exposure of beta-lactoglobulin peptic cleavage sites and partial unveiling of alpha-lactalbumin tryptic cleavage sites. The exposure of alpha-lactalbumin tryptic cleavage sites was slightly enhanced when the alpha-lactalbumin/beta-lactoglobulin mixture was heated at pH 7.5. Co-denaturation of fresh whey by heating at 95 degrees C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by beta-mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.     

Effect of Processing Temperature and Storage Time on Nonfat Dry Milk Proteins

Changes in physicochemical properties of pooled nonfat milk preheated to 63°C (I), 74°C (II), and 85°C (III), before spray-drying were examined. Insoluble material from III contained more protein (particularly at reduced pH) and more coagulated protein-lactose aggregates than either I or II. Soluble material from III was practically depleted of whey proteins which were utilized to form complexes stabilized through disulfide bonds. Milk protein micelles from III were heavier (ca 1 × 10¹¹ g/mole) than either I or II. An unsweetened milk-orange juice blend, which was pasteurized at 63°C for 30 min and stored at 4°C, developed a precipitate which contained more protein and pectin, but less sucrose than the supernatant.     

Precipitation of proteins from potato juice with bentonite

The recovery of proteins from potato juice by treatment with bentonite has been investigated. All proteins can be precipitated from potato juice by acidification and addition of bentonite. The acid-coagulatable protein fraction is adsorbed less by bentonite than the acid-soluble protein fraction. Maximum adsorption of the acid-soluble fraction occurs at pH 5.0. The working conditions recommended for obtaining a protein-free potato juice are acidification to pH 4.5 and addition of bentonite to obtain a weight ratio of soluble protein: bentonite of 0.9. At the natural pH of potato juice (pH 5.8-6.0), adsorption of potato proteins on bentonite is irreversible. About 62% of the adsorbed protein can be recovered by alkali treatment at pH 13.     

Thermal modifications of structure and codenaturation of α‐lactalbumin and β‐lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (β‐lactoglobulin or α‐lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other co‐denaturation partner, and of existence of a previous thermal pretreatment (industrial whey). α‐Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at ⪈85°C. After its denaturation, α‐lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys‐X and Arg‐X bonds). Heating over 85°C of β‐lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co‐denaturation of α‐lactalbumin with β‐lactoglobulin increased their aggregation and resulted in complete exposure of β‐lactoglobulin peptic cleavage sites and partial unveiling of α‐lactalbumin tryptic cleavage sites. The exposure of α‐lactalbumin tryptic cleavage sites was slightly enhanced when the α‐lactalbumin/β‐lactoglobulin mixture was heated at pH 7.5. Co‐denaturation of fresh whey by heating at 95°C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by β‐mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.     

Proteins recovered from milks heated at alkaline pH values

Approximately 95% of available nitrogen can be precipitated from milk on adjustment to pH 4.6 after heating at 90°C × 15 minutes at its natural pH and pH 7.5, while 89% can be precipitated after heating at pH 10.0 at 60°C × 3 minutes. Non-recovered protein includes some serum albumin, β-lactoglobulin, α-lactalbumin and proteose peptones. Protein isolates precipitated from milk heated at pH >7.0 are more soluble in the pH range 6.0–7.0 than those precipitated from milk heated at its natural pH. Whey proteins complex onto the casein micelles after heating milk at its natural pH, while on heating at pH >7.0 whey proteins appear to interact with k-casein in the serum phase. When N-ethylmaleimide is present in milk during heating the percentage protein recovered on pH 4.6 precipitation is decreased, confirming that disulphide linkage is involved in complex formation. However, addition of β-mercaptoethanol to recovered isolates did not result in dissociation of the casein/whey protein complex, suggesting that forces other than disulphide bonding are also involved in maintaining the complex.     

Gelation of casein-whey protein mixtures

Heated milk consists of a mixture of whey protein-coated casein micelles and soluble whey protein aggregates. The acid-induced gelation properties of heated milk are consistently different from those of unheated milk—i.e., a shift in gelation pH, stronger gels, and a different microstructure of the gels. In this study we investigated the role of the different fractions of denatured whey proteins on the acid-induced gelation, the gel hardness, and the microstructure. Both whey protein fractions contribute to the observed shift in gelation pH, although by a different mechanism. Obtaining gels with high gel hardness occurs most effectively when all denatured whey proteins are present as whey protein aggregates. It was observed that disulfide bridge exchange reactions during the acid-induced gelation at ambient temperature play an important role for both whey protein fractions. Additionally, disulfide interactions seem to occur between the aggregates and the casein micelles during the gel state. In this study, we show the development of a new approach for confocal scanning laser microscopy measurements—i.e., separate staining of the proteins in milk. By using this method, we were able to determine that, although whey protein aggregates are not linked to the casein micelles, they nevertheless gel at the same moment. This work adds to a better understanding of the role of denatured whey proteins during acid-induced gelation and could improve the effective use of whey proteins.     

Effects of gamma radiation on microbial,physicochemical, and structural properties of whey protein model system

Gamma radiation has been used in food processing for many years, though it has certain effects on food components. Whey protein solutions (10%/30%, wt/vol) were treated with gamma radiation at various dosages (10–25 kGy) and evaluated for microbial changes in the solutions and physicochemical and structural changes of whey proteins. Whey protein solutions after gamma radiation showed substantially lower populations of all viable microorganisms than those of controls. The 10% whey protein solution treated at radiation of 20 or 25 kGy remained sterile for up to 4 wk at room temperature. Gamma radiation increased viscosity and turbidity and decreased soluble nitrogen of whey protein solutions compared to nonradiated control samples regardless of radiation dosage. Nonreducing sodium dodecyl sulfate-PAGE suggested that whey proteins under gamma radiation treatment formed aggregates with high molecular weights. Reducing sodium dodecyl sulfate-PAGE showed that disulfide bonds played a role in gamma radiation-induced whey protein cross-linking. Scanning and transmission electron microscopy micrographs exhibited large aggregates of whey proteins after gamma radiation treatment. Results suggested that gamma radiation could be applied to whey protein solution for purposes of reducing microbial counts and cross-linking protein molecules.     

Disulfide Bond Formation Affects Stability of Whey Protein Isolate Emulsions

The viscosity and degree of flocculation of 20 wt% n-hexadecane oil-in-water emulsions stabilized by whey protein isolate (1 wt% WPI in 0.05M phosphate buffer, pH 7.0) increased as the emulsions aged. These effects were reduced when N-ethylmaleimide, a sulfhydryl blocking agent, was added to the emulsions immediately after homogenization, but were not completely eliminated. Gel electrophoresis (SDS-PAGE) showed an increase in the extent of intermolecular disulfide bond formation between proteins absorbed at the droplet interface with time. Floes were probably formed initially by noncovalent bonding or bridging flocculation, and then stabilized by disulfide bonds between proteins absorbed to different droplets.     

L-组氨酸修饰乳清蛋白结构及其热诱导凝胶特性

采用L-组氨酸(L-His)作为蛋白凝胶功能性的增强剂,将其加入乳清分离蛋白溶液中制备热诱导凝胶,研究L-His对乳清蛋白结构及其凝胶特性的影响.结果 表明:在乳清蛋白等电点(pI 5.2)时蛋白形成尺度约1 700 nm、具有极小比表面积且几乎不带电的蛋白聚集体,远离蛋白等电点时则所形成的聚集体大小约为400 nm;...     

Roles of charge interactions on astringency of whey proteins at low pH

Whey proteins are a major ingredient in sports drink and functional beverages. At low pH, whey proteins are astringent, which may be undesirable in some applications. Understanding the astringency mechanism of whey proteins at low pH could lead to developing ways to minimize the astringency. This study compared the astringency of β-lactoglobulin (β-LG) at low pH with phosphate buffer controls having the same amount of phosphate and at similar pH. Results showed that β-LG samples were more astringent than phosphate buffers, indicating that astringency was not caused by acid alone and that proteins contribute to astringency. When comparing among various whey protein isolates (WPI) and lactoferrin at pH 3.5, 4.5, and 7.0, lactoferrin was astringent at pH 7.0 where no acid was added. In contrast, astringency of all WPI decreased at pH 7.0. This can be explained by lactoferrin remaining positively charged at pH 7.0 and able to interact with negatively charged saliva proteins, whereas the negatively charged WPI would not interact. Charge interactions were further supported by β-LG or lactoferrin and salivary proteins precipitating when mixed at conditions where β-LG, lactoferrin, or saliva themselves did not precipitate. It can be concluded that interactions between positively charged whey proteins and salivary proteins play a role in astringency of proteins at low pH.     

Factors regulating astringency of whey protein beverages

A rapidly growing area of whey protein use is in beverages. There are 2 types of whey protein-containing beverages: those at neutral pH and those at low pH. Astringency is very pronounced at low pH. Astringency is thought to be caused by compounds in foods that bind with and precipitate salivary proteins; however, the mechanism of astringency of whey proteins is not understood. The effect of viscosity and pH on the astringency of a model beverage containing whey protein isolate was investigated. Trained sensory panelists (n = 8) evaluated the viscosity and pH effects on astringency and basic tastes of whey protein beverages containing 6% wt/vol protein. Unlike what has been shown for alum and polyphenols, increasing viscosity (1.6 to 7.7 mPa·s) did not decrease the perception of astringency. In contrast, the pH of the whey protein solution had a major effect on astringency. A pH 6.8 whey protein beverage had a maximum astringency intensity of 1.2 (15-point scale), whereas that of a pH 3.4 beverage was 8.8 (15-point scale). Astringency decreased between pH 3.4 and 2.6, coinciding with an increase in sourness. Decreases in astringency corresponded to decreases in protein aggregation as observed by turbidity. We propose that astringency is related to interactions between positively charged whey proteins and negatively charged saliva proteins. As the pH decreased between 3.4 and 2.6, the negative charge on the saliva proteins decreased, causing the interactions with whey proteins to decrease.     

Isothermal gelation of proteins. 1. Urea-induced gelation of whey proteins and their gelling mechanism

The changes in dynamic elastic moduli of whey proteins [whey protein isolates, β-lactoglobulin (B-Lg), α-lactalbumin (A-La) and bovine serum albumin (BSA)] at various concentrations in the presence of 8 molldm³ urea with time were measured at 25°C, because whey protein-urea systems set to gels automatically at room temperature without heating. From the time dependence behavior of elastic moduli for the proteins, the individual proteins were characterized as BSA having good, B-Lg intermediate and A-La poor urea-induced gelation. The disulfide bonds and hydrogen bonds played important roles in the formation the urea-induced gels.     

Distinction of different heat-treated bovine milks by native-PAGE fingerprinting of their whey proteins

Native-PAGE (polyacrylamide gel electrophoresis) was used for the simultaneous qualitative and quantitative analysis of whey proteins of raw, commercial and laboratory heat-treated bovine milks. Four whey protein bands, including β-lactoglobulin variants (β-LG A and B), could be distinctively separated in the gel. The results showed that levels of the major whey proteins were reduced by less than 23% in the pasteurized milks and by more than 85% in the UHT milks as compared with raw milk. The α-lactalbumin (α-LA) exhibited the strongest heat-tolerance: about 32% of it remained in its native state after the milk was heated at 100 °C for 10 min. About 42% of β-LG A and 53% of β-LG B were lost after the milk was heated at 75 °C for 30 min. Blood serum albumin (BSA) was lost almost completely when the milk at pH 5.0 was heated at a temperature of 75 °C or higher. The β-LGA and β-LGB were much more stable at low pH than in neutral conditions.     

Influence of pH,Casein, and Whey Protein Denaturation on the Composition,Crystal Size,and Yield of Lactose from Condensed Whey

Lactose was crystallized from condensed whey under varying conditions of pH (3.76, 4.84, 5.59, and 6.43), degree of protein denaturation (4.2, 32.2, 46.9, and 78.4%), and casein addition (.2, .3, and .45%). Increasing the amount of whey protein denaturation in the condensed whey resulted in crude lactose containing more protein and ash, particularly when near the isoelectric point for whey proteins. Excessive whey protein denaturation increased viscosity of the mother liquor, decreased lactose crystal size, and made washing of the crude lactose more difficult. Casein had no adverse effects on lactose quality, yield, or crystal size.Generally, the yield of washed/dried lactose increased as whey protein denaturation increased from 4.2 to 46.9%; however, this was due mainly to inclusion of protein and mineral in the crystallized lactose, particularly at pH 4.84. Moderate protein denaturation (up to 32%) had no adverse affect on lactose quality. Washed/dried lactose prepared from condensed whey (pH 4.84 and 78.4% whey protein denaturation) contained excessive protein (5.0%) and ash (2.0%). Lactose quality improved with lower amounts of whey protein denaturation and crystallizing at pH on either side of the isoelectric point.     

Production of an exopolysaccharide-containing whey protein concentrate by fermentation of whey

Using whey as a fermentation medium presents the opportunity to create value-added products. Conditions were developed to partially hydrolyze whey proteins and then ferment partially hydrolyzed whey with Lactobacillus delbrueckii ssp. bulgaricus RR (RR; an EPS-producing bacterium). In preliminary experiments, pasteurized Cheddar cheese whey was treated with Flavourzyme to partially hydrolyze the protein (2 to 13% hydrolyzed). Fermentation (2 L, 38 degrees C, pH 5.0) with RR resulted in EPS levels ranging from 95 to 110 mg of EPS per liter of hydrolyzed whey. There were no significant differences in the amount of EPS produced during fermentations of whey hydrolyzed to varying degrees. Since a high level of hydrolysis was not necessary for increased EPS production, a low level of hydrolysis (2 to 4%) was selected for future work. In scale up experiments, whey was separated and pasteurized, then treated with Flavourzyme to hydrolyze 2 to 4% of the protein. Following protease inactivation, 60 L of partially hydrolyzed whey was fermented at 38 degrees C and pH 5.0. After fermentation, the broth was pasteurized, and bacterial cells were removed using a Sharples continuous centrifuge. The whey was then ultrafiltered and diafiltered to remove lactose and salts, freeze-dried, and milled to a powder. Unfermented hydrolyzed and unhydrolyzed whey controls were processed in the same manner. The EPS-WPC ingredients contained approximately 72% protein and 6% EPS, but they exhibited low protein solubility (65%, pH 7.0; 58%, pH 3.0).