High pressure treatment of bovine milk: effects on casein micelles and whey proteins

Effects of high pressure (HP) on average casein micelle size and denaturation of alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) in raw skim bovine milk were studied over a range of conditions. Micelle size was not influenced by treatment at pressures <200 MPa, but treatment at 250 MPa increased micelle size by approximately 25%, while treatment at > or = 300 MPa irreversibly reduced it to approximately 50% of that in untreated milk. The increase in micelle size after treatment at 250 MPa was greater with increasing treatment time and temperature and milk pH. Treatment times > or = 2 min at 400 MPa resulted in similar levels of micelle disruption, but increasing milk pH to 7.0 partially stabilised micelles against HP-induced disruption. Denaturation of alpha-la did not occur < or = 400 MPa, whereas beta-lg was denatured at pressures >100 MPa. Denaturation of alpha-la and beta-lg increased with increasing pressure, treatment time and temperature and milk pH. The majority of denatured beta-lg was apparently associated with casein micelles. These effects of HP on casein micelles and whey proteins in milk may have significant implications for properties of products made from HP-treated milk.     

Kinetics of heat-induced whey protein denaturation and aggregation in skim milks with adjusted whey protein concentration

Microfiltration and ultrafiltration were used to manufacture skim milks with an increased or reduced concentration of whey proteins, while keeping the casein and milk salts concentrations constant. The skim milks were heated on a pilot-scale UHT plant at 80, 90 and 120 degrees C. The heat-induced denaturation and aggregation of beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and bovine serum albumin (BSA) were quantified by polyacrylamide gel electrophoresis. Apparent rate constants and reaction orders were calculated for beta-lg, alpha-la and BSA denaturation. Rates of beta-lg, alpha-la and BSA denaturation increased with increasing whey protein concentration. The rate of alpha-la and BSA denaturation was affected to a greater extent than beta-lg by the change in whey protein concentration. After heating at 120 degrees C for 160 s, the concentration of beta-lg and alpha-la associated with the casein micelles increased as the initial concentration of whey proteins increased.     

Peptides released from acid goat whey by a yeast-lactobacillus association isolated from cheese microflora

Seven lactobacilli and a variety of microflora extracted from twenty five commercial cheeses were grown on unsupplemented acid goat whey and screened for their capacity to hydrolyse whey proteins [alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg)] and to generate peptides. Fermentations were performed aerobically or anaerobically at 37 degrees C using crude or pre-heated whey (10 min at 65, 75 or 85 degrees C). Under aerobic conditions, growth of lactobacilli was poor and protein hydrolysis did not occur. Anaerobic conditions slightly increased lactobacilli growth but neither beta-lg hydrolysis nor peptide generation were observed. More than 50% of alpha-la was digested into a truncated form of alpha-la (+/- 12 kDa) in crude whey and whey pre-heated at 65 degrees C. Twenty-five microflora extracted from raw milk cheeses were screened for their proteolytic activities on acid goat whey under the conditions previously described. Eight of them were able to hydrolyse up to 50% of alpha-la mainly during aerobic growth on crude or pre-heated whey. The corresponding hydrolysates were enriched in peptides. The hydrolysate involving microflora extracted from Comté cheese after or at 18 months ripening was the only one to exhibit hydrolysis of both alpha-la and beta-lg. Microbiological analysis showed that microorganisms originating from Comté cheese and capable of growth on unsupplemented whey consisted of Candida parapsilosis and Lactobacillus paracasei. Fermentation kinetic profiles suggested that peptides were released from alpha-la hydrolysis. The co-culture of both microorganisms was required for alpha-la hydrolysis that occurred concomitantly with the pH decrease. During whey fermentation, Cand. parapsilosis excrete at least one protease responsible for alpha-la hydrolysis, and Lb. paracasei is responsible for medium acidification that is required for protease activation.     

Disruption and reassociation of casein micelles during high pressure treatment: influence of whey proteins

In the study presented in this article, the influence of added alpha-lactalbumin and beta-lactoglobulin on the changes that occur in casein micelles at 250 and 300 MPa were investigated by in-situ measurement of light transmission. Light transmission of a serum protein-free casein micelle suspension initially increased with increasing treatment time, indicating disruption of micelles, but prolonged holding of micelles at high pressure partially reversed HP-induced increases in light transmission, suggesting reformation of micellar particles of colloidal dimensions. The presence of alpha-la and/or beta-lg did not influence the rate and extent of micellar disruption and the rate and extent of reformation of casein particles. These data indicate that reformation of casein particles during prolonged HP treatment occurs as a result of a solvent-mediated association of the micellar fragments. During the final stages of reformation, kappa-casein, with or without denatured whey proteins attached, associates on the surface of the reformed particle to provide steric stabilisation.     

Process steps for the preparation of purified fractions of alpha-lactalbumin and beta-lactoglobulin from whey protein concentrates.

Fractions enriched with alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were produced by a process comprising the following successive steps: clarification-defatting of whey protein concentrate, precipitation of alpha-lactalbumin, separation of soluble beta-lactoglobulin, washing the precipitate, solubilization of the precipitate, concentration and purification of alpha-la. The present study evaluated the performance of the process, firstly on a laboratory scale with acid whey and then on a pilot scale with Gouda cheese whey. In both cases soluble beta-lg was separated from the precipitate using diafiltration or microfiltration and the purities of alpha-la and beta-lg were in the range 52-83 and 85-94% respectively. The purity of the beta-lg fraction was higher using acid whey, which does not contain caseinomacropeptide, than using sweet whey. With the pilot scale plant, the recoveries (6% for alpha-la; 51% for beta-lg) were disappointing, but ways of improving each step in the process are discussed.     

Caprine immunoglobulin G,beta-lactoglobulin,alpha-lactalbumin and serum albumin in colostrum and milk during the early post partum period

Colostrum and milk samples from 20 goats were analysed for concentrations of immunoglobulin G (IgG), beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and serum albumin (CSA) throughout the first 14 milkings post partum (7 d of lactation) using single radial immunodiffusion assay. Concentrations (mg/ml, means +/- SD) at first milking were IgG 47.9 +/- 25.5, beta-lg 30.7 +/- 10.4, alpha-la 2.77 +/- 0.82 and CSA 2.97 +/- 2.46 mg/ml. Large variations were recorded for IgG concentrations (19.9-94.5 mg/ml) and beta-lg (9.3-49.8 mg/ml). Concentrations of IgG, beta-lg and CSA dropped abruptly in the subsequent milkings and alpha-la concentration decreased slowly. Mean IgG concentration was < 2 mg/ml after 7 milkings and < 1 mg/ml after 11 milkings. However, IgG concentration does not differ significantly, at the 1% level, from milkings 7-14. The contribution of beta-lg to the increase in whey proteins in early milks was greater than that of IgG from milkings 5 to 14. The results were tabulated to make it possible to calculate the excess of whey proteins that would be obtained if early milks were illegally added to milk supply.     

Bovine immunoglobulin G, beta-lactoglobulin, alpha-lactalbumin and serum albumin in colostrum and milk during the early post partum period.

Colostrum and milk samples from 60 Holstein-Friesian cows were analysed for concentrations and yields of immunoglobulin G (IgG), beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and serum albumin (BSA) throughout the first 16 milkings post partum (8 d of lactation) using a single radial immunodiffusion assay. Concentrations (mg/ml, means +/- SD) at first milking were IgG 59.8 +/- 28.5, beta-lg 14.3 +/- 4.6, alpha-la 2.04 +/- 0.6, BSA 1.21 +/- 0.44. Large variations were recorded for IgG concentrations (15.3-176.2 mg/ml) and yields (0.2-925 g). Cows in their first lactation produced significantly lower concentrations and yields of colostral IgG than cows in later lactations. A colostral yield of IgG below the 100 g required to prevent calf hypo-gamma-globulinaemia was found in 18.3% of the cows. The concentrations of IgG, beta-lg and BSA dropped abruptly in subsequent milkings and alpha-la concentration decreased slowly. The mean IgG concentration was < 2 mg/ml after eight milkings and < 1 mg/ml after fifteen milkings. However, IgG concentration did not differ significantly, at the 1% level, during milkings 11-15. The results were tabulated to make it possible to calculate the excess of whey proteins that would be obtained if early milks were illegally added to the milk supply.     

High-pressure treatment of milk in industrial and pilot-scale equipments: effect of the treatment conditions on the protein distribution in different milk fractions

The effects of two different high-pressure (HP) equipments, operating at industrial- and pilot scales, and of the HP-release rate on the contents of non-sedimentable proteins and denatured whey proteins were investigated after treatments of skim milk—from 250 to 650 MPa. Non-sedimentable caseins and denatured whey proteins significantly increased with the pressure level. The industrial-scale equipment produced lower micellar disintegration than the pilot-scale equipment with similar degrees of whey protein denaturation. Ultracentrifugation supernatants obtained from skim milk at 100,000×g and 20 °C for 1 h were also HP-treated for comparative purposes, showing that, in skim milk, the presence of casein promoted the denaturation of whey proteins, although the extent of whey protein denaturation did not influence the release of casein to the soluble phase. Furthermore, most denatured whey proteins remained soluble after treatment in both equipments. In the pilot-scale equipment, the pressure-release rate influenced casein solubilization and whey protein denaturation.     

High-pressure-induced changes in the rennet coagulation properties of bovine milk

The mechanism of high-pressure (HP)-induced changes in rennet coagulation properties of milk, particularly the role of whey protein-casein micelle associations, was studied. Treatment at 100 or 250 MPa reduced the rennet coagulation time (RCT) of raw skimmed bovine milk, compared with untreated milk. Treatment at 400 MPa had little effect, but at 600 MPa, RCT increased considerably. HP-induced increases in RCT did not occur in serum protein-free milk or milk treated with the sulphydryl-oxidising agent KIO₃, which prevents association of denatured β-lactoglobulin with casein micelles. Treatment at 5 or 10 °C at 250–600 MPa resulted in shorter RCT than treatment at 20 °C. In milk without KIO₃, coagulum strength was highest after treatment at 250 or 400 MPa, whereas in milk with KIO₃ it was highest after treatment at 400 MPa. These results indicate the significance of HP-induced association of whey proteins with casein micelles for rennet coagulation properties of milk.     

High-pressure-induced changes in bovine milk: a review

High-pressure (HP) treatment of food products is a novel processing technique during which the product is treated in a vessel of suitable strength at a high pressure, generally in the range 100–1000 MPa. As a result, several constituents and properties of the treated product are altered. HP-induced changes in the constituents and properties of milk are arguably among the most extensive of the range of food products studied to date. HP treatment of milk induces solubilization of minerals associated with the casein micelles, denatures whey proteins and, depending on pressure, can either induce aggregation or disruption of the casein micelles. These HP-induced changes in milk constituents affect the properties of the milk; cheesemaking properties of milk can be enhanced considerably, indicating potential application of HP treatment in this area; furthermore, encouraging results have also been reported for HP treatment of milk prior to yogurt manufacture. HP treatment of milk also affects its microflora; however, considerable variation in baroresistance between bacterial species and strains exists. Further applied research appears warranted to establish the full commercial potential of HP treatment of milk.     

Kinetics of changes in plasmin activity and proteolysis on heating milk

Heat-induced inactivation of bovine plasmin, denaturation of beta-lactoglobulin (beta-lg), the interactions between both species and casein micelles and the subsequent net effect on proteolysis of beta-casein was studied in a model system consisting of phosphocasein and beta-lg in synthetic milk ultrafiltrate. The inactivation of plasmin and denaturation of beta-lg were first order reactions, with the rate of inactivation of plasmin being greater than the rate of denaturation of beta-lg. The predominant mechanism involved in the denaturation of plasmin in the temperature range 65-80 degrees C was its interaction with beta-lg (kr at 60 degrees C, 0.0526; Ea, 176 KJ/mol). At the point of complete inactivation of plasmin approximately 45% of the beta-lg remained undenatured. Thermal inactivation of plasmin through other mechanisms was negligible. The association of beta-lg with the casein micelles at 60 degrees C had a rate constant of 3.71 x 10-5 min-1 and an Ea of 259 KJ/mol; thermal denaturation of beta-lg was of much less importance, with a rate constant at 60 degrees C of the order of 1 x 10-10 min-1 and an Ea of 250 KJ/mol. On denaturation of all beta-lg in the system, a maximum of approximately 55% was associated with the casein micelles. The effect of heating on the subsequent hydrolysis of beta-casein indicated that the level of plasmin activity was the most important factor affecting proteolysis, while the interaction of beta-lg with the casein micelles had limited effect. Overall, thermal stability of plasmin in milk is very much dependent upon its interaction with beta-lg.     

High pressure-induced changes in milk proteins and possible applications in dairy technology

This paper reviews the newest information on the effects of high pressure (HP) on whey proteins, caseins and milk enzymes, and discusses their influence on milk properties. HP treatments cause substantial modification to milk proteins and to the mineral balance of milk. Casein micelles disaggregate into smaller particles or aggregate, depending the intensity and the temperature of the HP treatment. Whey proteins are denatured, possibly interacting with the remnants of the casein micelles, and give aggregated forms different from those produced by heat treatment. These events influence rennet coagulation properties of milk, with micellar disintegration favoring coagulation and whey protein denaturation hindering the aggregation of renneted micelles and enhancing cheese yield. HP treatment of milk favors acid coagulation and produces acid gels whose structure is greatly determined by the different micellar sizes attainable and the degree of whey protein denaturation. Milk gels can also be formed from concentrated milk under HP, providing new structures inaccessible via conventional methods.     

Impaired rennetability of heated milk; study of enzymatic hydrolysis and gelation kinetics

Casein micelles in milk are stable colloidal particles with a stabilizing hairy brush of kappa-casein. During cheese production rennet cleaves kappa-casein into casein macropeptide and para-kappa-casein, thereby destabilizing the casein micelle and resulting in aggregation and gel formation of the micelles. Heat treatment of milk causes impaired clotting properties, which makes heated milk unsuitable for cheese production. In this paper we compared five different techniques, often described in the literature, for their suitability to quantify the enzymatic hydrolysis of kappa-casein. It was found that the technique is crucial for the yield of casein macropeptide and this yield then affects the calculated enzymatic inhibition caused by heat treatment, ranging from 5 to 30%. The technique, which we found to be the most reliable, demonstrates that heat-induced calcium phosphate precipitation does not affect the enzymatic cleavage, while whey protein denaturation causes a very slight reduction of enzyme activity. By using diffusing wave spectroscopy, a very sensitive technique to monitor gelation processes, we demonstrated that heat-induced calcium phosphate precipitation does not affect the clotting. Whey protein denaturation does not affect the start of flocculation but has a clear effect on the clotting process. This work adds to a better understanding of the processes causing the impaired clotting properties of heated milk.     

The dominating effect of ionic strength on the heat-induced denaturation and aggregation of β-lactoglobulin in simulated milk ultrafiltrate

The denaturation/aggregation behaviour of heated (78 °C, 10 min) β-lactoglobulin (1%, w/w) was examined as a function of heating pH (5.0–7.0), in the presence of different salts. Heating β-lactoglobulin in the presence of calcium (5 mm) significantly increased the level of aggregated protein at most heating pH values, compared to heating in water or sodium chloride (100 mm). Heating β-lactoglobulin in the presence of calcium (5 mm) and phosphate (5 mm), resulted in similar denaturation levels in the pH range 5.0–5.8 as in the presence of calcium (5 mm) alone but reduced denaturation in the pH range 6.0–7.0, probably due to the formation of insoluble calcium phosphate. The addition of NaCl (100 mm) counteracted the aggregation promoting properties of the calcium and calcium/phosphate systems. Heating β-lg in a simulated milk ultrafiltrate solution was similar to heating in NaCl alone. This suggested that Ca²⁺ effects alone may not explain the heat-induced denaturation/aggregation behaviour of β-lactoglobulin in milk whey systems.     

Plasmin activity, β-lactoglobulin denaturation and proteolysis in high pressure treated milk

The effects of high pressure (HP) on plasmin activity, β-lactoglobulin denaturation and proteolysis during subsequent storage of HP treated milk, were studied. Fresh raw milk samples were exposed to a range of pressures from 50 to 800 MPa, for times of 1, 10 or 30 min, at 20°C. Residual plasmin activity and whey protein denaturation were measured immediately post HP-treatment. Indices of proteolysis were measured during post-HP storage. Treatment at pressures >300 MPa resulted in extensive β-lactoglobulin denaturation. Plasmin activity decreased in milk treated at pressures 400 MPa; the loss of activity was not well correlated with β-lactoglobulin denaturation. Compared to raw milk, treatment at 50 MPa had little effect on proteolysis during storage of treated milk measured as increases in pH 4.6-soluble N and liberation of proteose peptones, but at pressures of 300–400 MPa, proteolysis was increased relative to raw milk. After pressurisation >500 MPa, proteolysis during storage of milk was less than that observed in raw milk. Overall, HP influenced proteolysis in milk in a way which is different from that produced by heat, in terms of subsequent susceptibility of casein to proteolysis during storage or incubation. In particular, HP treatment at pressures of 300–500 MPa can increase proteolysis in milk, possibly through changes in micelle structure facilitating increased availability of substrate bonds to plasmin, which has implications for products prepared from milk thus treated.     

Characterization of a goat whey peptic hydrolysate produced by an ultrafiltration membrane enzymic reactor

Goat whey was hydrolysed by pepsin in an ultrafiltration membrane enzymic reactor coupled with a 30 kDa mineral membrane. Peptides collected in the permeate were resolved using reversed-phase HPLC. Their sequences were determined by amino acid analysis, second order derivative spectra analysis and mass spectrometry. Owing to the resistance of beta-lactoglobulin (beta-lg) towards pepsin, the majority of peptides identified were derived from alpha-lactalbumin (alpha-la). Pepsin showed a broad specificity of hydrolysis sites and generated a wide range of products from dipeptides to very large peptides containing disulphide bridges. The molecular masses of peptides resulting from alpha-la degradation were between 150 and 6900 Da: 36% were < 600 Da. 24% were 600-2000 Da and 40% were > 2000 Da.     

Effects of calcium and high pressure on soybean proteins: A calorimetric study

The effects of calcium and high pressure (HP) treatment on the thermal properties of soybean proteins were analyzed in soybean protein isolate (SPI), a β-conglycinin-enriched fraction (7SEF), a glycinin-enriched fraction (11SEF), and whey protein concentrate (WPC). For β-conglycinin, the temperature of denaturation (Td) decreased with up to 12.5 mM or 6.2 mM calcium in SPI and 7SEF, respectively. This parameter increased when calcium was more concentrated. The Td of glycinin increased for every assayed calcium concentration. The values of changes in Td (ΔTd) depended on calcium concentration and the proportion of β-conglycinin and glycinin in the samples. Activation energy decreased for glycinin in the presence of calcium. HP treatment promoted denaturation of β-conglycinin and glycinin. Calcium protected both proteins in SPI, 7SEF and 11SEF at 200 MPa, and protected glycinin in SPI and 7SEF at 400 and 600 MPa. Nevertheless, calcium increased the degree of denaturation of β-conglycinin in 7SEF at 600 MPa. In the absence of calcium, partially-HP-denatured polypeptides exhibited the same or lower Td than controls, whereas in its presence, they exhibited higher Tds than their respective controls.     

Effect of beta-lactoglobulin on plasma retinol and triglyceride concentrations, and fatty acid composition in calves

Beta-lactoglobulin (beta-lg) is the main protein of ruminant milk whey. Although beta-lg can bind in vitro to a variety of hydrophobic substrates, mainly retinol and long-chain fatty acids, its physiological function is still unknown. In Exp. 1, we investigated the effect of beta-lg on the plasma retinol concentration in preruminant calves. Holstein male calves (n = 20) were fed Holstein whole milk at 40 g/kg body weight (BW) plus vitamin A acetate (500,000 i.u.) with or without beta-lg (0.4 g/kg BW). The plasma retinol concentration of 10-d-old calves was greater (P < 0.05) in the beta-lg-fed group) than in the control group during the period from 8 to 12 h and at 24 h after the feeding. The postprandial change of plasma retinol in 40-d-old calves fed milk with beta-lg was higher (P < 0.05) than that in the control calves only at 12 h after the feeding. In Exp. 2, Holstein male calves (n = 18) were used to investigate the effect of beta-lg on plasma triglyceride concentration and fatty acid composition. Calves were fed Holstein whole milk at 40 g/kg BW plus milk fat prepared from whole milk at 2 g/kg BW with or without beta-lg (0.4 g/kg BW). Plasma triglyceride concentration at age 10 d was higher (P < 0.05) in the beta-lg-fed group than in the controls during the periods from 1 to 2 h and from 7 to 11 h after the feeding. At age 40 d, plasma triglyceride in the beta-lg-fed group was higher (P < 0.05) than in the control group only at 9 h. Ratios of palmitic, stearic, and oleic acids to total plasma lipids were higher (P < 0.05) in the calves fed beta-lg milk than in the control calves at age 10 d. These results suggest that beta-lg enhances the intestinal uptake of retinol, triglyceride, and long-chain fatty acids in preruminant calves.     

Effects of milk protein genetic variants on milk yield and composition

Effects of genetic variants of the milk proteins, alpha S1-casein, beta-casein, kappa-casein and beta-lactoglobulin (beta-lg), on milk yield and composition, particularly the protein composition, were investigated in milk samples from 289 Jersey and 249 Friesian cows in eight commercial herds. Milk protein genotypes had no significant effect on yields over a complete lactation of milk and fat, but significant differences in fat content were detected for beta-casein (B, A1B, A2 greater than A1A2) and beta-lg (B, AB greater than A) variants. Significant differences between beta-lg variants were also found with total solids (B, AB greater than A), casein (B, AB greater than A), whey protein (A greater than AB greater than B) and beta-lg (A greater than AB, AC greater than B greater than BC) concentrations. Casein genotypes were not significantly different in total protein and casein concentrations but many differences were found in casein composition. alpha S1-Casein variants significantly affected alpha S1-casein (BC greater than B) and kappa-casein (B greater than BC) concentrations. beta-Casein variants affected concentration and proportion of beta-casein (A1B, A2B greater than A1, A1A2, A2, B), alpha S1-casein (A1, A2 greater than B) and kappa-casein (B greater than A2) and concentration of whey protein (A1 greater than most other beta-casein variants). kappa-Casein variants affected concentration and proportion of kappa-casein (B greater than AB greater than A), proportion of alpha S1-casein (A greater than AB greater than B) and concentrations of beta-lg (A greater than AB, B) and alpha-lactalbumin (A, AB greater than B). Differences in milk composition were found between breeds, herds and ages, and with stage of lactation. The potential use of milk protein genotypes as an aid in dairy cattle breeding is discussed.     

Effects of mineral salts and calcium chelating agents on the gelation of renneted skim milk

The effects of adding CaCl2, orthophosphate, citrate, EDTA, or a mixture of these, to reconstituted skim milk (90 g of solids/kg solution) on the gelation of renneted milk were mediated by changes in Ca2+ activity and the casein micelle. At pH 6.65, the addition of citrate or EDTA, which removed more than 33% of the original colloidal calcium phosphate with the accompanying release of 20% casein from the micelle, completely inhibited gelation. Reformation of the depleted colloidal calcium phosphate and casein in the micelle, by the addition of CaCl2, removed this inhibition. When the minimum requirements for colloidal calcium phosphate and casein in the micelle were met, the coagulation time decreased with increasing Ca2+ activity, leveling off at high Ca2+ activity. The storage modulus of renneted gels, measured at 3 h, increased with increasing colloidal calcium phosphate content of micelles up to a level at which it was approximately 130% of the original colloidal calcium phosphate in the micelles. Further increases in colloidal calcium phosphate by the addition of CaCl2, orthophosphate, or mixtures of these, which did not change the proportion of casein in the micelle, decreased the storage modulus. The gelation of the renneted milk was influenced by Ca2+ activity, the amounts of colloidal calcium phosphate, and casein within the micelle, with the effects of colloidal calcium phosphate and casein within the micelle clearly dominating the storage modulus. These results are consistent with the model of Horne (Int. Dairy J. 8:171-177, 1998) which postulates that, following cleavage of the stabilizing K-casein hairs by rennet, the properties of the rennet gel are determined by the balance between the electrostatic and hydrophobic forces between casein micelles.