Bovine milk procathepsin D: Presence and activity in heated milk and in extracts of rennet-free UF-Feta cheese

Milk samples were heat-treated at 72, 85 and 99°C for 15 or 60 s, and the effect on the stability of the milk acid proteinase zymogen procathepsin D was studied by combining immunoblotting using antibodies directed against bovine cathepsin D and its propeptide and by measuring residual procathepsin D-derived activity. Approximately half of the procathepsin D-derived activity detected in milk serum remained after heat treatment at 72°C/15 s or 72°C/60 s, while heat treatment at increased temperature further reduced the detectable activity. In accordance, immunoreactive procathepsin D was detected in serum from milk heated at 72°C/15 s and 72°C/60 s, while very low amounts of immunoreactivity were observed after treatment at higher temperatures. Contrary to the decrease in milk serum, the amount of procathepsin D antigen associated with casein micelles slightly increased with the temperature of the heat treatment, but still the measurable proteolytic activity derived from procathepsin D in the casein micelle samples decreased with temperature treatment. Moreover, the presence of procathepsin D and derived proteolytic activity was demonstrated in rennet free UF-Feta cheese. These results correlated with the finding of α_s1-I and para-κ-caseins in rennet free cheese. This is the first demonstration of procathepsin D in cheese, and of activity derived from indigenous procathepsin D in milk contributing to the proteolysis process in UF-products.     

Cathepsin D activity in quarg

Quarg cheese was produced from raw skim milk, pasteurised skim milk, raw skim milk with rennet added and ultrafiltrated raw skim milk. Quarg was also produced from raw skim milk with pepstatin added at curd cutting and from ultrafiltration retentate of raw milk with added pepstatin. No starter bacteria were used in this model system, with the reduction of pH being achieved by addition of glucono- δ-lactone. Yields ranged between 20.25 and 23.5%, with protein levels of 13.6–15.7%. Proteolysis occurred during storage of all experimental cheese samples for 3 m at 8°C. By immunoblotting using antibodies against bovine cathepsin D, immunoreactive procathepsin D was identified in all cheese samples. Presence of cathepsin D or procathepsin D-derived activity was confirmed by a specific enzyme assay in all samples, except those which contained pepstatin. Inhibition of cathepsin D-catalysed proteolysis by pepstatin was observed in chromatograms of water-soluble extracts analysed by reverse-phase HPLC. Peptides thought to be produced as a result of cathepsin D activity were observed in cheese made from both raw and pasteurised milk, suggesting that the activity at least partially survived pasteurisation.     

Ultrafiltration of cheese milk: Effect on plasmin activity and proteolysis during cheese ripening

Havarti 45+ cheese was manufactured from milk concentrated 1.8–4.6-fold by ultrafiltration (UF) and from normal milk, and the effect of concentration factor on plasmin activity and subsequent proteolysis in cheese during ripening was examined. There was decreased plasmin activity and a reduced rate of proteolysis of α_S2-casein and β-casein in the UF-cheeses, compared with the corresponding controls, independent of concentration factor. The decreased plasmin activity and slower breakdown of α_S2-casein and β-casein in UF-cheeses compared with traditional cheeses can be partly explained by the inclusion of an increased amount of plasmin inhibitors into the UF-cheeses. However, it is suggested that the differences in plasmin activity and proteolysis arise mainly as a result of inactivation of the plasminogen activation system during UF-concentration, due to a combination of time, temperature and the presence of air in the UF-equipment. The effect of milk treatment on the plasminogen activation system should be further investigated.     

Effect of protein composition on the cheese-making properties of milk from individual dairy cows

The objective of this study was to evaluate the effect of variations in milk protein composition on milk clotting properties and cheese yield. Milk was collected from 134 dairy cows of Swedish Red and White, Swedish Holstein, and Danish Holstein-Friesian breed at 3 sampling occasions. Concentrations of α_S1-, β-, and κ-casein (CN), α-lactalbumin, and β-lactoglobulin (LG) A and B were determined by reversed phase liquid chromatography. Cows of Swedish breeds were genotyped for genetic variants of β- and κ-CN. Model cheeses were produced from individual skimmed milk samples and the milk clotting properties were evaluated. More than 30% of the samples were poorly coagulating or noncoagulating, resulting in weak or no coagulum, respectively. Poorly and noncoagulating samples were associated with a low concentration of κ-CN and a low proportion of κ-CN in relation to total CN analyzed. Furthermore, the κ-CN concentration was higher in milk from cows with the AB genotype than the AA genotype of κ-CN. The concentrations of α_S1-, β-, and κ-CN and of β-LG B were found to be significant for the cheese yield, expressed as grams of cheese per one hundred grams of milk. The ratio of CN to total protein analyzed and the β-LG B concentration positively affected cheese yield, expressed as grams of dry cheese solids per one hundred grams of milk protein, whereas β-LG A had a negative effect. Cheese-making properties could be improved by selecting milk with high concentrations of α_S1-, β-, and κ-CN, with high κ-CN in relation to total CN and milk that contains β-LG B.     

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.     

Relationships Between Milk Protein Polymorphisms and Cheese Yielding Capacity

A total of 31 Holstein cows were selected for their phenotypes for β-casein, κ-casein, and κ-lactoglobulin. Over 14 mo, milk samples were collected individually on a monthly basis from each cow during lactation. Effects of genetic variants of milk proteins on cheese yielding capacity were investigated. To minimize variations due to cheese making practices, cheese was made under laboratory conditions. Least-squares analysis of the data indicated that higher cheese yields were obtained with β-casein A1A1, κ-casein BB, and β-lactoglobulin BB when compared with other phenotypes of the respective proteins under study. Regression analyses indicated that each percentage increase in fat content in milk resulted in an increase of 1.07, 1.34, and 1.33% in actual, adjusted, and theoretical yield. For a similar increase of casein in milk, there was 1.59, 1.26, and 1.81% increase in actual, adjusted, and theoretical yield, respectively. Relative proportions of different caseins and whey proteins also had some influence on the yields. Relationships were negative between initial milk pH and yields.     

Relationships Between Milk Protein Polymorphisms and Major Milk Constituents in Holstein-Friesian Cows

Milk samples from 1,908 Holstein Friesian cows were phenotyped for genetic variants of α_s1 -casein, β-casein, κ-casein, and β-lactoglobulin. The relationships between milk protein polymorphism and test day milk yield and composition were investigated. After adjustments were made for environmental effects of herd, parity number, month of test, stage of lactation, age of sample at testing, and somatic cell count, milk protein phenotypes were found to be associated with milk yield; concentrations in fat, protein, casein, and whey protein; and proportion of casein in protein. Higher test-day milk production was associated with α_s1-casein BB, β-casein A1A3, κ-casein AA, and β-lactoglobulin AA phenotypes. Fat and protein concentrations were highest in milk from cows of α_s1-casein BC, β-casein A1B, and κ-casein BB phenotypes. β-Lactoglobulin BB milk was associated with higher percentages of fat and casein and with lower percentages of total protein and whey protein. Based on desirable fat and casein contents of milk for cheese production, it would be advantageous to select for cows bearing κ-casein BB and β-lactoglobulin BB phenotypes.     

Study on milk-clotting mechanism of rennet-like enzyme from glutinous rice wine: proteolytic property and the cleavage site on kappa-casein

Chinese Royal cheese, an ancient and attractive dairy product now in China, is made from milk coagulated with glutinous rice wine. In this paper, it was mainly studied on the proteolytic property toward proteins of bovine milk including caseins (CN) and whey proteins and the cleavage bond on the κ-CN of rennet-like enzyme purified from glutinous rice wine by ion-exchange chromatography. Compared with whey protein, the rennet-like protease has substrate specificity toward CN but with different hydrolysis degrees among κ-, α-, and β-CN, and the α-CN was almost completely degraded, whereas κ- and β-CN partly showed hydrolysis in 12 h. The analysis for enzyme digestion by electrospray tandem mass spectrometry, Q-TOF2, and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry revealed that the cleavage of protease from glutinous rice wine on κ-CN mainly happens at the Thr₉₄-Met₉₅ bond, which is different from the most chymosin-sensitive bond, Phe₁₀₅-Met₁₀₆.     

Variation in activity and heterogeneity of bovine milk proteases with stage of lactation and somatic cell count

Samples of prepartum secretion, colostrum and mature milk were analysed for variation in activity of cathepsin D and cysteine protease due to lactation stage and somatic cell count (SCC). In spite of considerable variation between animals, cathepsin D activity was found to be highest in prepartum secretion, lowest in colostrum and at intermediate levels in milk throughout lactation. Levels of cysteine protease activities were highest 1 week postpartum and lowest in early/mid-lactation milk. The activity of an unidentified milk protease with specificity different from cathepsin D and cysteine proteases was also detected in milk, but had very low activity in prepartum secretions and colostrum. By immunoblotting, prepartum secretion was found to contain both cathepsin D and procathepsin D, while only procathepsin D could be detected in colostrum and milk. Cathepsin D activity was positively correlated with SCC, and immunoblotting indicated that the elevated activity derived from cathepsin D was due to increased level of immunoreactive procathepsin D in high SCC milk, and not to increased levels of mature cathepsin D. This indicates that activation of procathepsin D does not occur at elevated SCC.     

Effects of genetic type,stage of lactation,and ripening time on Caciocavallo cheese proteolysis

The objective of this experiment was to evaluate the effects of genetic type, stage of lactation, and ripening time on proteolysis in Caciocavallo cheese. One hundred twenty Caciocavallo cheeses made from the milk of 2 breeds, Italian Brown and Italian Holstein and characterized by different stages of lactation were obtained and ripened for 1, 30, 60, 90, and 150 d. Cheese proteolysis was investigated by ripening index (ratio of water-soluble N at pH 4.6 to total protein, %) and by the study of degradation of the protein fractions (α_S1-, β-, and para-κ-casein), which was determined by densitometric analysis of isoelectric focusing results. The statistical analysis showed a significant effect of the studied factors. Ripening index was higher in Italian Brown Caciocavallo cheese and in cheeses made with early lactation milk, whereas casein solubilization was greater in the first 2 mo of ripening. Isoelectric focusing analysis of cheese samples during ripening showed extensive hydrolysis of caseins. In particular, the protein fraction that underwent major degradation by proteolytic enzymes was α_S1-casein, followed by β-casein, whereas para-κ-casein was less degraded. Italian Brown cheese showed a lower residual quantity of β- and para-κ-casein, whereas Italian Holstein cheese showed a lower residual quantity of α_S1-casein. In addition, significant interactions of both first and second order were found on both ripening index and degradation of protein fractions. This study demonstrated that the analyzed factors influenced proteolysis of Caciocavallo cheese, which forms the basis of new knowledge that could lead to the production of a pasta filata cheese with specific characteristics.     

Thermal inactivation kinetics of bovine cathepsin D

Cathepsin D, the principal indigenous acid proteinase in bovine milk, is a lysosomal proteinase, which exists in milk in four forms, including the inactive zymogen procathepsin D. The thermal inactivation kinetics of bovine cathepsin D, isolated from spleen and milk, were studied under isothermal conditions, using a specific HPLC assay to determine residual activity. Inactivation of the blood enzyme preparation followed first order kinetics, with z-values in phosphate buffer (pH 6.7) and skimmed milk of 6.5 and 7.6 degrees C, respectively, the enzyme being far more stable in the latter environment. Inactivation kinetics of the enzyme purified from milk were more complex, and could be best approximated by a double exponential model. Again, stability was higher in milk than in buffer. The double exponential model may indicate differing heat stabilities of isoforms of the enzyme, or stabilization of the enzyme by some milk constituent. It is clear that the enzyme can survive, at least partially, processes such as heating at 55 degrees C for 30 min during manufacture of high-cook cheese varieties (45% survival), and HTST pasteurization (8% survival), and thus may contribute to proteolysis in a range of dairy products.     

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.     

Primary proteolysis studied in a cast cheese made from microfiltered milk

Primary proteolysis was studied in a starter-free cheese model made from microfiltered (MF) milk (19.0% casein,<0.2% whey proteins). Specificity of plasmin and chymosin activity was investigated in the pH range 5.0–6.0, by analysis of peptide composition using high-performance liquid chromatography and liquid chromatography–mass spectrometry. Hydrolysis experiments with purified caseins were performed to aid identification of peptides released by specific activities. Plasmin had no activity in cheese below pH 5.4, while its activity increased from pH 5.4 to 6.0. Chymosin activity on the Phe₂₃–Phe₂₄ bond of α_S1-casein had an optimum pH around 5.3, while release of the bitter peptide β-casein (f193–209), effected by chymosin, was highest at pH 6.0. At pH <5.3, the specificity of chymosin on α_S1-casein changed, and the peptide bond Leu₂₀–Leu₂₁ was cleaved at an increasing rate with decreasing pH. Demineralisation of the MF retentate generally increased proteolytic activity.     

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6 h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12 h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α_S1-casein during incubation of milk samples at peak somatic cell counts, with that of β-casein being faster than that of α_S1-casein. Two-dimensional gel electrophoretograms of milk 6 h after infusion with the toxin confirmed hydrolysis of β- and α_S1-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C₁₈ columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α_s1- and β-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.     

A study of polymorphism in milk proteins from local and imported dairy sheep in Australia by capillary electrophoresis

Casein and whey protein fractions of milk obtained from 47 ewes of five breeds or crossbreeds (Awassi, Merino, East Friesian×Merino, Awassi×Merino and Awassi×East Friesian) were analysed by capillary electrophoresis (CE). The experiments were performed on a Beckman P/ACE™ system 5510 with an uncoated fused-silica capillary and a low pH buffer containing urea and a polymeric additive. The four major caseins (α_s1-, α_s2-, β- and κ-casein) in an acid precipitate were well separated, as were the two whey proteins, α-lactalbumin (α-La) and β-lactoglobulin (β-Lg). The electromigration of the proteins was in the order of α-La, β-Lg, α_s2-CN, α_s1-CN, κ-CN and β-CN. The milk samples were composed of the same variant of α-La and two different genotypes (A and B) of β-Lg while the β-Lg AB genotype was evident in the milk of some animals. The α_s1-CN fractions displayed considerable heterogeneity with at least 4 different peaks, representing 4 different variants. A fifth peak, corresponding to the Welsh variant (or α_s1-CN D), was present in 90% of the ewes’ milk samples. The κ-CN fraction was resolved as a single peak, while the β-CN revealed significant heterogeneity with 3 variants. It appears that the presence of the α_s1-CN Welsh variant in Merino ewe and its crosses with Awassi and East Friesian ewes adversely affected milk composition and yield.     

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.     

Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties

Water-soluble extracts of 9 Italian cheese varieties that differed mainly for type of cheese milk, starter, technology, and time of ripening were fractionated by reversed-phase fast protein liquid chromatography, and the antimicrobial activity of each fraction was first assayed toward Lactobacillus sakei A15 by well-diffusion assay. Active fractions were further analyzed by HPLC coupled to electrospray ionization-ion trap mass spectrometry, and peptide sequences were identified by comparison with a proteomic database. Parmigiano Reggiano, Fossa, and Gorgonzola water-soluble extracts did not show antibacterial peptides. Fractions of Pecorino Romano, Canestrato Pugliese, Crescenza, and Caprino del Piemonte contained a mixture of peptides with a high degree of homology. Pasta filata cheeses (Caciocavallo and Mozzarella) also had antibacterial peptides. Peptides showed high levels of homology with N-terminal, C-terminal, or whole fragments of well known antimicrobial or multifunctional peptides reported in the literature: α_S1-casokinin (e.g., sheep α_S1-casein (CN) f22-30 of Pecorino Romano and cow α_S1-CN f24-33 of Canestrato Pugliese); isracidin (e.g., sheep α_S1-CN f10-21 of Pecorino Romano); kappacin and casoplatelin (e.g., cow κ-CN f106-115 of Canestrato Pugliese and Crescenza); and β-casomorphin-11 (e.g., goat β-CN f60-68 of Caprino del Piemonte). As shown by the broth microdilution technique, most of the water-soluble fractions had a large spectrum of inhibition (minimal inhibitory concentration of 20 to 200 μg/mL) toward gram-positive and gram-negative bacterial species, including potentially pathogenic bacteria of clinical interest. Cheeses manufactured from different types of cheese milk (cow, sheep, and goat) have the potential to generate similar peptides with antimicrobial activity.