Comparison of rheological properties of acid gels made from heated casein combined with β-lactoglobulin or egg ovalbumin

Gelation of heat-treated milk protein solutions by acid fermentation was performed using a model system of micellar casein alone (systR), micellar casein and β-lactoglobulin (systB) or micellar casein and egg ovalbumin (systO) dissolved in a milk ultrafiltrate and heat-treated at 90°C for 24 min. Solubility of globular proteins at given pH values and their interactions with casein were determined. Particle size and ζ-potential of the protein systems were measured before and after heat treatment. Acid gelation of the heated systems was monitored using dynamic low-amplitude strain oscillation. Ovalbumin alone or with casein produced larger particles than casein alone or with β-lactoglobulin upon heat treatment. The systems gelled at different pH values, i.e. 4.88, 5.47 and 5.88 in systR, systB and systO, respectively. The latter two pH values were clearly related to the pH of the loss of solubility of the heated globular proteins. While the gelation pattern for systR resembled unheated milk, the pattern for systB and systO showed the usual maximum for tan δ of heated milk.     

乳铁蛋白与牛乳中其他蛋白质相互作用机制研究进展

牛乳蛋白分为酪蛋白和乳清蛋白,其中乳铁蛋白是乳清蛋白中的一种,具有多种生物学功能,被广泛应用于食品、医药、化妆品工业等领域。在牛乳中,乳铁蛋白带正电荷,会与一些带负电荷的蛋白质,如酪蛋白、骨桥蛋白、β-乳球蛋白、血清白蛋白、免疫球蛋白等发生相互作用。这种相互作用影响着这些蛋白的生物化学功能或者分离制备特性,尤其是后者更是工业化生产中需要关注的问题。本文阐述了乳铁蛋白与牛乳中其他蛋白质之间的相互作用机制及其应用现状,对于开发乳铁蛋白的工业化制备方法、以及系统理解牛乳中各种活性蛋白间的协同生物作用等方面都具有重要意义,为乳铁蛋白生物学特性的深入研究和工业化生产技术开发提供一定依据。     

Polymerization of β-lactoglobulin and bovine serum albumin at oil-water interfaces in emulsions by transglutaminase

Polymerization of β-lactoglobulin and bovine serum albumin at the oil—water interfaces in n-tetradecane-in-water emulsions induced by the transglutaminase reaction was studied. The emulsions were incubated with transglutaminase for various times, and adsorbed and unadsorbed protein fractions at the oil—water interfaces were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. While only monomers were detected in the unadsorbed fractions, polymers were observed in the adsorbed fractions of the both proteins. The sizes and amounts of the polymers increased with incubation time. The incubation with transglutaminase caused much flocculation of the emulsion stabilized by β-lactoglobulin. An increase in viscosity was also observed with the flocculation. The flocculation was probably initiated by the formation of ε-(γ-glutamyl)-lysyl isopeptide bonds between β-lactoglobulin molecules adsorbed on different oil droplets. In the case of the emulsion stabilized by bovine serum albumin, however, the flocculation and the increase in viscosity occurred to only limited extents by the transglutaminase reaction. This suggests that ε-(γ-glutamyl)-lysyl isopeptide bonds induced by the transglutaminase reaction were formed only between neighboring molecules of bovine serum albumin on the same droplet.     

Effect of β-Lactoglobulin Variant and Environmental Factors on Variation in the Detailed Composition of Bovine Milk Serum Proteins

Between November 1979 and November 1981, 17,086 test-day milk samples were collected from individual Holstein cows in 62 Quebec herds. Samples were analyzed for protein, fat, casein, serum protein, somatic cell count, and the relative percentages of β-lactoglobulin, α-lactalbumin, bovine serum albumin, and the immunoglobulins. Cows included in the study were phenotyped with respect to β-lactoglobulin. Unadjusted means ± standard error for the relative percentages of β-lactoglobulin, α-lactalbumin, bovine serum albumin, and immunoglobulins were 64.80 ± .07%, 21.54 ± .05%, 6.51 ± .02%, and 7.15 ± .04%. Least-square analyses showed that month of test, stage of lactation, age of cow, somatic cell count, and phenotype of the cow for β-lactoglobulin had significant effects on the relative percentages of β-lactoglobulin, α-lactalbumin, bovine serum albumin, and immunoglobulins. Test-day milk yield, fat percent, protein percent, casein percent, casein number, and serum protein percent, when included in a statistical model as covariates, had a significant effect on the relative percentage of Ig. Relative proportion of β-lactoglobulin and α-lactalbuminim were not significantly affected by serum protein and total protein contents. For bovine serum albumin, only the covariates fat percent and serum protein percent were not significant.     

Conditions for the Formation of Heat-Induced Gels of Some Globular Food Proteins

The quality of thermally induced aggregates of the globular proteins conalbumin, serum albumin, ß-lactoglobulin and lysozyme has been examined at various salt concentrations and pH values. The properties of the aggregates were characterized by their dry matter content. The results are given as simple phase diagrams. The following areas of dry matter content were found: solubility; transparent and opaque gels (dry matter content of 5–9%); precipitates (dry matter content above 9). Gels were formed only close to conditions of solubility. Only serum albumin was found to be a protein with good gelling properties. A small gelling area was registered for ß-lactoglobulin, while no gelling was observed for conalbumin or lysozyme under the conditions examined. No common simple physical characteristic of the proteins used could be correlated to good gelling behavior.     

Changes in protein-protein and protein-polysaccharide interactions induced by high pressure

β-Lactoglobulin and bovine serum albumin (BSA) protein solutions (0.1%, 0.2% and 2.5%), when subjected to high pressure treatment (800 MPa for 20 min) at neutral pH, were denatured and some aggregates formed. The total calorimetric enthalpy of 2.5% solutions of the pressure-treated proteins decreased to virtually zero for both β-lactoglobulin and BSA following pressure treatment. Isoelectric focussing patterns (IEF) indicated that aggregation occurred in both proteins and there was a concomitant loss of sulphydryl groups (42% for β-lactoglobulin and 55% for BSA), suggesting that protein aggregation after high pressure processing was caused, at least in part, by the formation of -S-S- bridges. The surface hydrophobicity of the two proteins was modified, increasing (40%) with β-lactoglobulin and decreasing (41%) with BSA. Pressure treatment of 1:1 mixtures of BSA and dextran sulphate (DS) yielded structures with a significant enthalpy. However, addition of DS to β-lactoglobulin had little effect on the thermograms, suggesting that the DS either protects the protein against pressure induced unfolding or enables the pressure-denatured protein to regain some secondary structure.     

Rapid Separation and Quantification of Major Caseins and Whey Proteins of Bovine Milk by Polyacrylamide Gel Electrophoresis

A rapid polyacrylamide gel electrophoretic method was developed for separating and quantifying major proteins in casein and whey protein fractions of bovine milk. For casein separation, best results were achieved by an 8% polyacrylamide gel containing 4 M urea and a top layer of large pore sample gel; for whey protein the most satisfactory separation was with 12% polyacrylamide gel in the absence of urea and a large pore gel. Electrophoretic conditions for separation of whey protein were also applicable for identifying genetic variants of β-lactoglobulin. Densitometric tracings were used to resolve and quantify protein peaks from stained bands in the gels. Casein was resolved into three major fractions with relative proportions of 50:30:15 for α_s1- and α_s2-casein, β-casein, and κ-casein. Whey protein was resolved into four major fractions with relative proportions of approximately 60:20:7:13 for β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulin.     

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.     

Proteins recovered from acid whey/skim milk mixtures heated at alkaline pH values

Skim milk and mixtures prepared by combining acid whey with skim milk at volume ratios of 2:1, 1:1, 1:2, 1:3 and 1:4 were adjusted to pH 7.5 and heated at 90°C × 15 min. Protein was isolated from these heated samples by precipitation at pH 4.6 and it was found that 65% of the whey protein was recovered in each case. Non-recovered proteins included the proteose peptones and small quantities of β-lactoglobulin, α-lactalbumin and bovine serum albumin. The solubility of these isolates, which contained from 10–25% whey protein, decreased to > 95% when the whey protein exceeded ˜16%. Further characterization of the isolate, prepared from the 1:1 volume ratio of acid whey and skim milk, showed that ˜50% of the whey protein was insoluble, bound to casein and non-functional while the other ˜50% was complexed with casein and was soluble. The addition of a reducing agent suggests that sulphydryl bonding alone is not responsible for complex formation.     

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.     

牛奶过敏原表位研究进展

详细地综述了牛乳酪蛋白、α-乳白蛋白、β-乳球蛋白、牛血清白蛋白的过敏原B细胞表位以及T细胞表位的研究进展,介绍了牛乳过敏原之间构象性表位的相似形以及线性表位序列位置的特异性,同时也简述了牛乳过敏原表位定位的方法及表位定位的应用。     

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.     

On the effect of high-pressure treatment on the surface activity of β-casein

High-pressure treatment (up to 800 MPa) of the milk protein β-casein has an insignificant effect on the time-dependent surface tension of a dilute solution of this disordered protein at pH 7. This is in contrast to a solution of globular protein, bovine serum albumin (BSA), which is substantially affected at 200 MPa and above.     

Effect of hydrocolloids on the thermal denaturation of proteins

The thermal denaturation of bovine serum albumin (BSA), lysozyme and whey protein isolate (WPI) in the presence of hydrocolloids (pectin, guar gum, ι-carrageenan) was investigated. A decrease in the thermal stability of lysozyme was observed in the mixture of protein with ι-carrageenan. The increase in the enthalpy of denaturation (ΔH) of BSA and lysozyme in the presence of hydrocolloids was attributed to the protection of globular proteins against aggregation through blockage of their hydrophobic binding sites by the bulky polysaccharide moeity. Biopolymers had a stabilizing effect on WPI. The thermal stability was the highest in the presence of pectin, whereas the lowest transition temperature was observed in the presence of guar gum. A single transition peak was observed for pure WPI. However, WPI exhibited two transition temperatures when together with pectin and i-carrageenan. WPI was stable against heat denaturation at acidic pH values (pH 4.0), while it was denatured at a low temperature at an alkaline pH (pH 9.0) in the presence of pectin. This was attributed to the formation of extra hydrogen bonding. The increase in the concentration of pectin has little affect on the heat stability of WPI; however, it reduces the cooperativity of transition.     

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.     

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

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

Heat-Induced Gelation of Individual Whey Proteins A Dynamic Rheological Study

Heat-induced gelation of the bovine whey proteins [serum albumin (BSA), β-lactoglobulin (β-Lg) and α-lactalbumin (α-La)] has been studied individually and in mixture at different conditions by a dynamic rheological method. Values in the shear stiffness modulus (/G*/) appeared on heating at low protein concentration for BSA (～2%) and at intermediate concentration for β-Lg (～ 5%). α-La did not form a heat-induced gel of concentrations up to 20% (w/v). The ratio of viscous to elastic properties (loss factor) at maximum possible measuring temperature was below 0.07 for the BSA gels and 0.1–0.3 for the β-Lg gels. The temperature of gelation was highly dependent on pH. In mixture one protein could not be exchange for another without changing the gelation behavior of the mixture.     

Changes in the antioxidant properties of protein solutions in the presence of epigallocatechin gallate

β-Casein and α-casein showed radical-scavenging activities in aqueous solution, whereas bovine serum albumin (BSA), α-lactalbumin and β-lactoglobulin showed much weaker antioxidant activity, when assessed by the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical-scavenging assay. However, β-casein and α-casein showed reduced antioxidant activity after storage at 30 °C. An increase in radical-scavenging activity and a fall in fluorescence of the protein component were evident after 6 h, when BSA, β-lactoglobulin or casein were mixed with EGCG, and excess EGCG was removed, indicating the formation of a complex with this protein on mixing. Storage of all the proteins with EGCG at 30 °C caused an increase in the antioxidant activity of the isolated protein component after separation from excess EGCG. This showed that EGCG was reacting with the proteins and that the protein-bound catechin had antioxidant properties. The reaction of EGCG with BSA, casein and β-lactoglobulin was confirmed by the loss of fluorescence of the protein on storage, and the increase in UV absorbance between 250 and 400 nm. The increase in antioxidant activity of BSA after storage with EGCG was confirmed by the ferric reducing antioxidant potential (FRAP) and the oxygen radical antioxidant capacity (ORAC) assays.     

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.     

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.