Casein Composition of Cow's Milk of Different Chymosin Coagulation Properties

Five good and four poor chymosincoagulating individual cow milk samples were analyzed for casein composition using hydroxyapatite chromatography and polyacrylamide gel electrophoresis to establish possible relationships between casein fractions and differences in coagulation properties.Samples exhibited wide variation in casein composition. Poor chymosincoagulating milk had higher content of γ- and degraded caseins and lower κ- and β-caseins than the good-coagulating milk. One milk sample that did not coagulate 30 min after chymosin addition had low α_S-casein concentration and an additional major casein fragment (not identified). A substantial peak representing unidentified minor caseins was apparent in a poorcoagulating milk sample, which coagulated early but whose coagulum did not become firm in 30 min. Excluding the nonclotting sample, less variability was observed in α_S-casein concentrations than in the other casein components.     

Preparation of whole gamma-casein by treatment with calcium phosphate gel

Preparations of crude whole γ-casein, containing approximately 32% γ-casein, were treated with calcium phosphate gel to develop a batch-type procedure for isolation of γ-casein. The crude whole γ-casein was dissolved to a final concentration of 2% in a .02 M potassium phosphate buffer, pH 6.8, containing 4.5 M urea and .005 M potassium chloride. β-casein contamination was progressively removed by five successive calcium phosphate gel treatments (1.5 g gel/g protein) . However, several variations of this procedure failed to remove completely the small amounts of temperature-sensitive caseins. The final product contained 94.8% y-casein but must be labeled whole y-casein due to the small amounts of the temperature-sensitive caseins. A yield of approximately 15% of the whole γ-casein originally present in whole casein could be recovered with this procedure.     

Rennet-induced coagulation of enzymatically cross-linked casein micelles

The enzymatic cross-linking of casein micelles with transglutaminase had an adverse influence on rennet-induced coagulation. Incubation with transglutaminase at 30 °C progressively reduced the levels of monomeric caseins and increased rennet flocculation time (RFT) in a Berridge test. For incubation up to 3 h at 30 °C, the reciprocal of the RFT was linearly correlated with the level of residual monomeric κ-casein, indicating that at complete cross-linking flocculation is absent. After treatment for 4–24 h at 30 °C, no residual monomeric κ-casein was detected and no rennet-induced flocculation of the casein micelle suspension was observed. Monitoring rennet-induced coagulation by diffusing wave spectroscopy revealed that transglutaminase-induced inhibition of rennet-induced coagulation of casein micelles is primarily due to an inhibition of the secondary phase of rennet coagulation, i.e., the gelation and gel-firming phase of the casein micelle coagulation. The gelation and fusion of κ-casein-depleted para-casein micelles as in normal milk appears to be absent if the casein macropeptide remains attached to the casein micelle.     

Formation of whey protein/κ-casein complexes in heated milk: Preferential reaction of whey protein with κ-casein in the casein micelles

Studies of the formation of soluble κ-casein/whey protein (WP) complexes in heated (90 °C 10 min⁻¹) milk and related mixtures of proteins have been made. The use of milk samples containing different genetic variants, and having different compositions, allowed the effects of changing the natural protein balance on the formation of particles to be investigated. In addition, studies were made of the effects of addition of WP or of purified κ-casein to the milk samples. The addition of WP caused an increase in the amount and the size of the complexes, but addition of κ-casein to the milk had little or no effect on the complex formation, nor did it seem that the added κ-casein could react with the WP in the milk. Conversely, in systems where the casein micelles were absent, the purified κ-casein reacted well with WP, suggesting that in milk heated at its normal pH the WP react preferentially with the κ-casein on the casein micelles.     

Production,analysis and in vivo evaluation of novel angiotensin-I-converting enzyme inhibitory peptides from bovine casein

Novel angiotensin-I-converting enzyme inhibitory peptides were isolated from bovine casein hydrolysate prepared by AS1.398 neutral protease. The active hydrolysate obtained at 12 h hydrolysis showed the highest ACE-inhibitory activity and was further consecutively separated by ultrafiltration, and the 3 kDa permeate showed the highest ACE-inhibiting activity. This active fraction was further purified to yield two novel ACE-inhibiting peptides, whose amino acid sequences were Arg-Tyr-Pro-Ser-Tyr-Gly (κ-casein; f25–30) and Asp-Glu-Arg-Phe (κ-casein; f15–18), respectively. The IC₅₀ value of the peptides were 54 ± 1.2 μg/mL and 21 ± 0.8 μg/mL, respectively. The Lineweaver–Burk plots revealed that the peptides acts as a non-competitive inhibitor. Antihypertensive effect in spontaneously hypertensive rats also revealed that single and repeated oral administrations of hydrolysates of bovine casein decreased systolic blood pressure significantly in spontaneously hypertensive rats (P < 0.01, P < 0.05). These results suggested that the peptide derived from peptides from bovine casein would be a beneficial ingredient for functional food or pharmaceuticals against hypertension.     

Effect of NaCl on some physico-chemical properties of concentrated bovine milk

The influence of added NaCl (75–850 mmol L⁻¹) on some physicochemical properties of 2× or 3× concentrated milk (18 or 27%, w/v, total solids, respectively) was investigated. Adding NaCl did not influence average casein micelle size, but reduced the net-negative charge on the casein micelles and milk pH. The level of soluble and ionic calcium was increased by addition of NaCl, but the level of soluble inorganic phosphorus was not influenced. Addition of NaCl shifted the maximum in the pH–heat coagulation time (HCT) profile of 2× or 3× concentrated milk to a higher pH value and certain concentrations increased the maximum HCT, probably due to the fact that NaCl reduced the extent of heat-induced dissociation of κ-casein. Added NaCl reduced the ethanol stability, with the extent of this effect increasing with the concentration of NaCl. The key-mechanism though which added NaCl induces changes in the physico-chemical stability of casein micelles appears to be through changes in the charge on the casein micelles.     

Calcium sensitivity and molecular weight of alpha-s5-casein

Alpha_s5-casein was more sensitive to calcium than α_s1-casein, requiring 2 mM calcium chloride for 80% protein precipitation as compared to 8 mM for α_s1-casein. Ability of α_s5-casein to interact with κ-casein forming micelles resistant to precipitation with calcium was less than that of α_s1-casein.Molecular weights determined by the differential boundary method in an ultracentrifuge were 65,750 and 31,800 for α_s5-casein and for reduced α_s5-casein (the equimolar mixture of α_s3- and α_s4-caseins). Elution of α_s5-casein at the second half of the κ-casein rich peak on Sephadex G-100 at pH 10.8 was ascribed to the molecular size of α_s5-casein.     

The effect of ultra high-pressure homogenization (UHPH) on rennet coagulation properties of unheated and heated fresh skimmed milk

The effect of ultra-high pressure homogenization at a pressure of 179 MPa on the renneting of milk has been studied. The homogenization has a small effect on the diameters of casein micelles, because of the loss of some of their surface κ-casein. This modification of the structure leads to a slightly decreased rennet coagulation time. Interactions between the casein micelles in homogenized and unhomogenized milk samples started at a degree of proteolysis of the κ-casein of about 65–70%, although aggregation of the micelles did not start until over 90% proteolysis. Homogenization improved the coagulation properties of heated milk only slightly; however, it was shown that the removal of stabilizing repulsions between the casein micelles in the heated milk seemed to proceed in the same way as in unheated milk. The removal of the κ-casein has the same effect in heated and unheated milk samples, and the casein micelles are destabilized; it is only in the final aggregation step that the two milks differ.     

Formation of soluble protein complexes and yoghurt properties influenced by the addition of whey protein concentrate

The effects of whey protein concentrate (WPC) on the formation of soluble protein complexes and yoghurt texture were evaluated. Skim milk (SM) and skim milk enriched with 1% WPC (SM + 1%WPC) or 2% WPC (SM + 2%WPC) were left unheated or heated and then made into yoghurt gels. Yoghurt prepared from heated SM + 2%WPC had significantly higher storage modulus, water holding capacity and firmness values and a denser microstructure than those prepared only from skim milk. Electrophoretic analysis of the milk showed that the level of β-lactoglobulin and κ-casein in the serum phase increased with increasing WPC concentration, indicating that the content of disulfide-linked β-lactoglobulin and κ-casein was higher in SM + 2%WPC than in SM, suggesting that more soluble protein complexes had been formed. Consequently, yoghurt prepared from heated SM enriched with WPC may have more bonds and more protein complexes in the protein network than yoghurt prepared only from SM, thus resulting in firmer gels.Practical applicationsYoghurt, one of the most popular fermented milk products, is of high economic importance to the dairy industry worldwide. In particular, high-protein yoghurt, such as Greek-style or set-type yoghurt, has been driving its ongoing popularity over recent years. In current industrial production of high-protein yoghurt, protein fortification and heat treatment of milk are two of the most important processing parameters affecting yoghurt texture. Whey protein concentrate has been added to milk to reduce whey separation and to increase the firmness of the yoghurt. From a technological point of view, the interaction of the denatured whey proteins with casein micelles or with κ-casein in the serum phases is regarded as responsible for obtaining a good yoghurt structure. The present research has shown that it is possible to produce yoghurt with a range of textural properties by precisely controlling the rate of whey protein fortification during its manufacture. Therefore, this study provides a better understanding of the effect of WPC fortification and aims to extend this insight for the production of good-quality yoghurt.     

热处理对牛乳酪蛋白胶束结构影响的研究进展

概述了热处理过程中对牛乳中酪蛋白内部作用力、酪蛋白各个单体以及胶束结构的影响。指出热处理的温度和处理时间的不同,导致酪蛋白的疏水作用及二硫键的变化、组成酪蛋白胶束的各个单体(αs-酪蛋白、β-酪蛋白、κ-酪蛋白)离解程度及结构的差异、酪蛋白胶束尺寸及胶束间作用力的改变。这对于牛乳酪蛋白的应用和整个胶束结构在热处理过程中的研究具有指导意义。     

Ethanol stability of casein micelles cross-linked with transglutaminase

The influence of transglutaminase (TGase)-induced cross-linking on the ethanol stability of skimmed milk was investigated. The stability of milk against ethanol-induced coagulation increased in sigmoidal fashion with milk pH (5.0–7.5) for all samples; ethanol stability also increased upon incubation (0–24 h) with 0.05 g L⁻¹ TGase at 30 °C. In untreated milk, addition of ethanol induced a collapse of the polyelectrolyte brush of κ-casein on the micelle surface, thereby facilitating micellar aggregation. Dynamic and static light scattering measurements indicated that in TGase-treated milk, the ethanol-induced collapse of the polyelectrolyte brush was far less than in untreated milk, suggesting that the increased ethanol stability of TGase-treated casein micelles is caused by the cross-linking of the polyelectrolyte brush on the micellar surface.     

Heat-Induced Protein Changes in Milk Processed by Vat and Continuous Heating Systems

This study investigated and compared the effects of heating system and residence times on the physicochemical properties and interactions of casein and whey proteins in heated milk. Milk was processed by vat heating (85°C for 10 to 40 min), HTST heating (98°C for .5 to 1.87 min), UHT heating (140°C for 2 to 8 s), cooled, and fractionated into casein and whey by isoelectric precipitation.β-Lactoglobulin A and B variants were partially denatured by HTST and UHT heating and totally denatured by vat heating. Increasing residence time caused significant (P<.01) increases in denaturation of both β-lactoglobulin variants in UHT and HTST heating systems and of α-lactalbumin in the vat heating system. Surface hydrophobicity and sulfhydryl content were negatively correlated with whey protein denaturation. Sodium dodecyl sulfate electrophoresis of the casein fraction of heated milk indicated the presence of a high molecular weight component that would not enter the gel. Addition of 2-mercaptoethanol to heated casein samples dissociated this component, with the concurrent appearance of β-lactoglobulin and α-lactalbumin bands. In HTST and UHT heating systems, ratio of β-lactoglobulin to κ-casein increased linearly from the complex with increasing residence time.     

Enhancement of transglutaminase-induced protein cross-linking by preheat treatment of cows’ milk: A statistical approach

Optimization of heat treated milk towards protein cross-linking induced by transglutaminase was carried out. Capillary electrophoresis was employed to study the extent of cross-linking under different preheating temperatures (70–90 °C) and times (15–60 min). The experiments were arranged according to a central composite statistical design (3²+centre points). Response surface methodology was used to assess factor interactions and empirical models regarding relative peak area (%) of individual protein (α_s2-casein, α_s1-casein, α_s0-casein, κ-casein, β-casein A¹, β-casein A², α-lactalbumin and β-lactoglobulin) and total α_s-caseins, total β-caseins and whey proteins (sum of α-lactalbumin and β-lactoglobulin). Multi-response optimization was also performed on the total α_s-caseins, total β-caseins, κ-casein and whey proteins data set of the factorial design. The desirability function was the statistical tool employed in this multi-optimization step. The optimum preheating conditions that maximized the cross-linking reactions catalyzed by transglutaminase were achieved within 60 min at 84.5 °C.     

Adsorption of Positively-Charged β-Lactoglobulin Derivatives to Casein Micelles

Positively-charged β-lactoglobulin derivatives prepared by amidation or esterification of available carboxyl groups of the native protein were bound strongly by casein micelles. Increasing amounts of these additives progressively decreased rennet coagulation time of casein micelles. Higher concentrations of positively-charged proteins coagulated casein micelles without addition of rennet extract. Of the modified proteins tested, approximately 1.0 g amidated β-lactoglobulin, 2.0 g ethyl-esterified β-lactoglobulin, or 1.0 g methyl-esterified β-lactoglobulin would be required to coagulate 100 ml of casein micelles at concentrations in milk.     

pH stability and influence of salts on activity of a milk-clotting enzyme from Solanum dubium seeds and its enzymatic action on bovine caseins

In this study we investigated the pH stability and effect of salts on the activity of a partially purified enzyme from Solanum dubium seeds as well as its hydrolytic power on caseins and caseins components. The seeds of S. dubium were blended and extracted using 50 g/L NaCl in 50 mmol/L acetate buffer, pH 5.0. The enzyme was then partially purified using ammonium sulfate. The results obtained showed that both NaCl and CaCl₂ enhanced the proteolytic activity of the enzyme and the enhancement was found to be significant when NaCl was used. Moreover, the stimulatory effect was found to be concentration dependent. The proteolysis of bovine whole casein and casein subunits by the enzyme during incubation was studied by SDS-PAGE. The results obtained revealed that both κ-casein and β-casein are the most susceptible to hydrolysis than α-casein. The three main casein components α-, β-, and κ-caseins were sensitive to the action of the enzyme and the order of hydrolysis obtained was κ- casein, β- casein, and α- caseins.     

Molecular Weight of αs3- and αs4-Caseins by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Molecular weights of both bovine α_S3- and α_s4-casein were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Analysis by least squares of natural logarithm of molecular weight versus distance of migration gave a molecular weight of 27,800 ± 900 daltons for each protein. The value agrees closely with the molecular weight (26,000 daltons) for bovine α_S3-casein reported by Richardson and Creamer and is less than 31,800 daltons for reduced α_S5-casein reported by Toma and Nakai.     

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.     

An FTIR spectroscopy study of the interaction between αs-casein-bound phosphoryl groups and chitosan

Fourier-transform infrared spectroscopy was used to study the nature of the linkage and interactions of phosphate ester bonds in α_s-casein under precipitation by chitosan. We have found that the dianionic stretching band of the covalently bound phosphate in α_s-casein at 976 cm⁻¹ is sensitive to the ionization state and the binding of Ca²⁺ or chitosan. Thus, the neutralization of the negative charges of carboxylates and phosphates by lowering the pH of α_s-casein solution from 6.8 to 2.0 led to a dramatic reduction of this signal. Precipitating amounts of Ca²⁺ caused a shift in the phosphate signal from 976 to 986 cm⁻¹ indicating a direct electrostatic interaction between Ca²⁺ and phosphate. The interaction of α_s-casein with low molecular weight chitosan showed a small shift (ca. 2 cm⁻¹) in the phosphate peak position as compared with pure α_s-casein with a pronounced reduction in the phosphate peak amplitude that was about a half of that of casein alone. When α_s-casein was precipitated with high molecular weight chitosan, a more noticeable effect occurred as this complex showed only around 25% of the phosphate peak amplitude. The interactions between the phosphate groups covalently bound to α_s-casein and the amino groups in chitosan seem to induce changes similar to those observed upon protonation of the negative charges of phosphate.     

Primary proteolysis and textural changes during ripening in Cheddar cheeses manufactured to different fat contents

The effects of varying fat content in Cheddar cheese, from 6.3 to 32.5 g 100 g⁻¹, on changes in pH, primary proteolysis and texture were monitored over a 225 d ripening period. Reduction in the fat content resulted in significant (P<0.05) increases in pH, moisture and protein contents and decreases in the concentration of moisture in the non-fat substance. The increase in pH as the fat content increased was attributed to the concomitant decrease in the lactate-to-protein ratio. Polyacrylamide gel electrophoresis showed that the concentration of intact casein decreased in all cheeses during ripening and that the rate of decrease was not affected by the fat content. However, for a given concentration of casein, α_s1-casein was degraded more slowly, and β-casein more rapidly, as the fat content was reduced. The slower degradation of α_s1-casein with decreased fat content coincided with a decrease in the ratio of residual chymosin activity to protein in the cheese. At most ripening times, reduction in the fat content resulted in significant increases in the concentration of intact casein, fracture stress, fracture strain, and cheese firmness. The effects of fat reduction on proteolysis and rheology are probably due to the interactive effects of the concomitant changes in composition.