Proteolytic activity of two commercial proteinases from Aspergillus oryzae and Bacillus subtilis on ovine and bovine caseins.

Electrophoretic analysis of the action of two commercial enzymes, Neutrase 0.5 and MKC Fungal Protease, on whole casein and alpha s-, beta- and kappa-caseins from cows' and ewes' milk showed that Neutrase 0.5 chiefly degraded beta-casein, giving rise to peptides soluble at pH 4.6 detectable by PAGE. In contrast, although MKC Fungal Protease caused intense hydrolysis of bovine beta-casein, in ovine casein it resulted in more active degradation of alpha s- than beta-casein. The latter enzyme did not produce peptides soluble at pH 4.6 detectable by PAGE. Both enzymes degraded kappa-casein, yielding a breakdown product that exhibited an electrophoretic mobility similar to that of the breakdown product produced by the action of commercial rennet.     

Bioactive peptides derived from milk proteins and their health beneficial potentials: an update

It has been well recognized that dietary proteins provide a rich source of biologically active peptides. Today, milk proteins are considered the most important source of bioactive peptides and an increasing number of bioactive peptides have been identified in milk protein hydrolysates and fermented dairy products. Bioactive peptides derived from milk proteins offer a promising approach for the promotion of health by means of a tailored diet and provide interesting opportunities to the dairy industry for expansion of its field of operation. The potential health benefits of milk protein-derived peptides have been a subject of growing commercial interest in the context of health-promoting functional foods. Hence, these peptides are being incorporated in the form of ingredients in functional and novel foods, dietary supplements and even pharmaceuticals with the purpose of delivering specific health benefits.     

Experimental evaluation of the allergen activity of a peptide preparation produced from an enzymatic hydrolysate of milk proteins using ultrafiltration]

Noninbred guinea-pig experiments were performed to investigate specific potential allergenicity of cow milk proteins (CMP) consisting of casein (80%) and serum proteins (20%), of low-molecular peptides (LMP 20/80) produced from CMP 20/80 by enzymatic hydrolysis and ultrafiltration; to evaluate nonspecific effect of test diet CMP 20/80 and LMP 20/80 on anaphylactic response of the animals to ovalbumin and histamine LD50. LMP 20/80 versus CMP 20/80 had a hypoallergenic effect. Potential mechanisms of low anaphylactic sensitivity induced by LMP 20/80 are discussed.     

Mucosal immunomodulation by the non-bacterial fraction of milk fermented by Lactobacillus helveticus R389

The health promoting effects ascribed to probiotic bacteria and fermented dairy products arise not only from bacteria themselves but also from metabolites derived from milk fermentation. Exopolysaccharides produced during milk fermentation and peptides derived from major milk proteins, when released during fermentation, are potential modulators of various regulatory processes in the body. The aim of this work was to increase the knowledge of the previously observed immunomodulating capacity of milk fermented by Lactobacillus helveticus R389 by the study of the mucosal immunomodulation exerted by the non-bacterial fraction of the milk fermented at a constant pH6 (NBFpH6). The effects on IL-6 production by small intestine epithelial cells, the profile of IgA+ and cytokine+ cells (IL-2, IL-6, IL-10, TNF-alpha and IFN-gamma) induced in the gut lamina propria, and the levels of total and specific secretory IgA in the lumen of BALB/c mice that received NBFpH6 for 2, 5 or 7 days were examined. There was an increase in the number of IgA+, IL-10+, IL-2+ and IL-6+ cells after all feeding periods. Total S-IgA in the small intestine lumen increased in mice that received NBFpH6 for 2 days. However, no specific antibodies against NBFpH6 were detected. Feeding of NBFpH6 for 7 days significantly (P<0.05) enhanced IL-6 secretion by small intestine epithelial cells. NBFpH6 induced a non-specific mucosal response that was down-regulated for protective immunity, enhancing IL-6 production by epithelial cells and IgA production in the small intestine. These events improve the immunological defenses at the intestinal level, increasing host protection against pathologies. Because mucosal immune responses induced by certain dietary antigens play a large part in the prevention of gastrointestinal diseases, the oral administration of a mucosal adjuvant such as NBFpH6 may positively affect the milieu of the intestinal lumen. The opportunity exists then to manipulate the constituents of the lumen of the intestine through dietary means, thereby enhancing the health condition of the host.     

Managing diet quality for Cheddar cheese manufacturing milk. 1. The influence of protein and energy supplements.

The effects of supplementing cows' diets with protein and energy on milk composition and the composition and yield of Cheddar cheese were investigated. This research addresses the problems of seasonal reduction in the capacity of cheese curds to expel moisture as observed in parts of south-eastern Australia. Milk was collected from cows offered a basal diet of silage and hay supplemented with different sources and levels of dietary protein and energy. The protein supplements were sunflower, canola, cottonseed meal and lupin, and the energy supplements were maize grain, oats, wheat and barely. This milk was used to manufacture Cheddar cheese on a pilot scale. Cheese moisture content was dependent on the source and level of dietary protein and energy. Milk from cows offered the lupin protein supplements and wheat energy supplements consistently produced cheese with a lower moisture content and moisture in fat-free matter. Milk from these supplemented diets had increased casein concentrations and higher proportions of alpha S2-casein than milk from the poor quality control diet. Cheese yield was directly related to the total casein concentration of milk, but was not influenced by differences in casein composition. Supplementing the cows' diets increased the inorganic P, Mg and Ca concentrations in milk. A low inorganic P concentration in milk from cows offered the control diet was caused by a low intake of dietary P. These findings showed that changes in the mineral and casein composition of milk, associated with diet, could influence the composition of Cheddar cheese.     

Bioactive peptides derived from milk proteins. Structural,physiological and analytical aspects

The primary function of dietary proteins is to supply the body adequately with indispensable amino acids and organic nitrogen. Little attention has been paid up to date to milk proteins, in particular caseins, that are currently the main source of biologically active peptides, although other animal as well as vegetable proteins are known to contain potentially bioactive sequences. Such regulatory peptides can be released by enzymatic proteolysis of caseins in vitro and in vivo and may act as potential physiological modulators of metabolism during the intestinal digestion of the diet. It has been proved that bioactive peptides derived from caseins, such as β-casomorphins and phosphopeptides, can be released during gastrointestinal passage. It is also evident that peptides originating from food proteins should be taken into account as potential modulators of various regulatory processes in the body. The possible regulatory effects concern nutrient uptake (phosphopeptides, casomorphins), postprandial hormone secretion (casomorphins), immune defense (immunopeptides, casokinins, casomorphins) and neuroendocrine information transfer (casokinins). The advances in the research field of bioactive peptides are driven by a molecular understanding of biological processes, and analytical techniques are a critical component of this understanding. Different up-to-date methods, including peptide synthesis and immunochemistry, have been applied to the chemical characterization of bioactive peptides. Especially casein derived peptides have already found interesting applications, both as dietary supplements (phosphopeptides) and as pharmaceutical preparations (phosphopeptides, β-casomorphins). The question of'what kinds of bioactive peptides are beneficial and desirable as food constituents or as drugs' should be always carefully examined. However, the possibilities for the design of dietary products and ‘natural’ drugs look promising.     

Proteolytic activity of lactic acid bacteria in skim milk with special reference to the biodegradation of casein fractions

Streptococci and lactobacilli were assayed for their proteolytic activity in pasteurised (95 degrees C for 30 min) fresh Friesian cows' skim milk incubated at 30 degrees C for 48 h. Lactobacilli were more proteolytic than the streptococci except S. faecalis subsp. liquefaciens. S. faecalis and S. thermophilus followed S. lactis subsp. diacetylactis in the proteolytic activity. Electrophoretic analysis of the precipitated casein revealed K-, pre-beta- and the small slow band of alpha s1-casein to be the most fractions and beta-casein the least fraction susceptible to the biodegradation. S. faecalis subsp. liquefaciens was the only organism able to degrade beta-casein to 3 fractions within 48 h. S. lactis with its subsp. diacetylactis was characterized by its inability to degrade gamma-casein. Addition of 2.0 g glucose, 0.5 g yeast extract, 2.0 mmol Mg2+, 0.5 mmol Mn2+ and 0.1 mmol Fe2+/l of skim milk culture of each L. casei and L. plantarum increased acid formation but decreased proteolysis. 6% NaCl was inhibitory to both. After 60 days at 18 degrees C, a fraction, probably derived from beta-casein, was noticeable. The large intense band of alpha s1-casein appears to be degraded gradually to only small peptides which in turn are transferred immediately into the cells. The hydrolysis of gamma-casein and beta-casein also appears to be inhibited by exogenously supplied NaCl and nutrients, respectively.     

Development of a cows' milk identification test (COMIT) for field use

A cows' milk identification test (COMIT) has been successfully developed for the detection of defined amounts of cows' milk (3-100%) in ewes' milk. The assay uses polyclonal antibodies raised in goats against bovine whey proteins. The test, an agar-gel immunodiffusion technique, uses agar plates with a printed template for correct placement of the test components (antisera discs, positive and negative milk reference discs and sample discs). The presence of cows' milk in the sample was recorded as positive when the precipitin lines appearing between the sample disc and the antisera disc fuse completely with those from the positive cows' milk reference disc. This test is reliable, practical and easy to perform in the field and in dairy factories. Furthermore, the nature and stability of the components of the test make it suitable for commercial development into kits which should be highly practical in any kind of inspection programme concerned with milk species identification.     

Managing diet quality for cheddar cheese manufacturing milk. 2. Pasture v. grain supplements.

The effects of supplementing a basal diet of silage and hay with increasing amounts of harvested spring pasture, or with lupin and wheat, on the composition of milk and the consequent effects on cheese composition and yield were investigated in an indoor feeding study. Milk was collected from five groups of eight cows in mid lactation offered different diets and manufactured into Cheddar cheese on a pilot scale. Milk from cows given the lupin-wheat (LW) and the high pasture level (HP) diets produced low moisture cheese. Cheese produced with milk from cows given the control diet was high in moisture content compared with that made with milk from cows offered the LW diet. Cheese yields from the milk of cows offered the HP and LW diets were greater than from the milk of cows on the control diet, and were associated with the higher casein concentrations of these milks. Casein number was higher in milk from diets supplemented with pasture but was not an indicator of the functional properties of milk that affected cheese moisture. The proportion of beta-casein in milk from cows offered the HP diet was higher and that of gamma-casein lower than in milk from cows given the LW supplement, although cheese moisture content was similar with both diets. Milk from cows offered the HP diet had a greater inorganic P concentration than that from cows given the LW diet, although the dietary intake of P was higher for the LW diet. The significance of the effect of dietary P intake on the concentration of inorganic P in milk and hence its suitability for cheesemaking was apparent when dietary P intake was low, as shown in milk produced by cows offered the control diet.     

Functional role of bioactive peptides with special reference to cheeses

Growing perception about diet in relation to health has extended the necessity to explore the biologically active components present in native foods. In this review, bioactive peptides released from cheeses that may have important physiological functions are discussed. Bioactive encrypted peptides can be generated from precursor milk proteins during food processing via enzymatic hydrolysis and fermentation. Generally, biofunctionalities of peptides are latent within precursor proteins. Bioactive peptides liberated from cheeses exhibit numerous potential therapeutic roles: for example, angiotensinogen‐converting enzyme (ACE) inhibition, antioxidant, anti‐thrombotic, anti‐microbial, anti‐cancer and anti‐inflammatory. This article critically focuses on the functional roles of bioactive peptides derived from different cheeses.     

Mare's and cow's milk: promote similar metabolic effects and expression of innate markers in Caco-2 cells?

Mare's milk (MM) is very similar to human breast milk, as it consists of low concentrations of proteins and fat and high concentrations of lactose. Thus, MM can be considered as an alternative to cow's milk (CM) for human nutrition. In this study, proteolytic resistant peptides to gastrointestinal enzymes were measured by reverse phase-high-performance liquid chromatography (RP-HPLC-PDA-UV) to determine their relative uptake by intestinal epithelial (Caco-2) cells. Alterations in mitochondrial enzyme activities (MTT test), inner mitochondrial membrane potential (ΔΨm), cell cycle progression and the expression (mRNA) of inflammatory (IL-1β, TNF-α and NF-κB) markers were studied. This is the first study comparing the effects of proteolytic resistant peptides from the whey proteins of mare's milk to those originating from cow's milk on intestinal cells. Similar proteolytic resistant peptide patterns, but lower uptake rates, were observed for MM peptides (7–40%) compared to peptides from CM (40–60%), although both types of peptides, probably derived from α-lactalbumin, were still capable of impairing ΔΨm in intestinal cells (CM > MM).Both MM and CM increased the expression of TNF-α, whereas IL-1β levels were not affected. CM increased NF-κB mRNA levels. Overall, our data indicate that proteolytic resistant peptides from both MM and CM exert similar metabolic effects on mitochondrial metabolism (up to 30%) in intestinal Caco-2 cells. However, the low uptake rates of peptides from MM may be beneficial to the human diet, an alternative to CM. Further in vivo studies are needed to better understand the potential clinical relevance of the control and/or regulation of metabolic processes related to perturbations in the gut immune system associated with lymphoid tissue.     

Phosphorylation of beta-casein and alpha-lactalbumin by casein kinase from lactating bovine mammary gland

Two milk proteins, beta-casein and alpha-lactalbumin, were compared as substrates for casein kinase from bovine mammary gland. beta-Casein could be rephosphorylated after removal of its phosphate groups, whereas alpha-lactalbumin was an effective substrate after the protein had been reduced and carboxymethylated. The native proteins could not be phosphorylated. Magnesium2+, Ca2+, and Mn2+ stimulated phosphorylation of the modified proteins. Calcium2+ was the most effective cation for alpha-lactalbumin and Mn2+ for beta-casein. Michaelis constants were 144 microM for alpha-lactalbumin in the presence of Ca2+ and 142 microM for beta-casein in the presence of Mn2+; however, the maximum velocity for alpha-lactalbumin was three times that of beta-casein. After phosphorylation with [gamma-32P] ATP, partial hydrolysis showed that only serine residues were phosphorylated in both proteins. Chymotryptic peptides of phosphorylated alpha-lactalbumin and tryptic peptides of phosphorylated beta-casein were examined by HPLC and selected peptides were analyzed for amino acid content. Comparison of the analyses with sequence data showed that serine at position 47 in alpha-lactalbumin is the major site of phosphate incorporation. Dephosphorylated beta-casein was only partially rephosphorylated. However, the sites identified correspond to the phosphorylated residues in native beta-casein, namely, serine at position 35 and the cluster of four serines between residues 15 and 20.     

Identification of Anticancer Peptides from Bovine Milk Proteins and Their Potential Roles in Management of Cancer: A Critical Review

Cancer is the most widely recognized reason for human deaths globally. Conventional anticancer therapies, including chemotherapy and radiation, are very costly and induce severe side effects on the individual. The discovery of natural anticancer compounds like peptides may thus be a better alternative for cancer prevention and management. The anticancer peptides also exist in the amino acid chain of milk proteins and can be generated during proteolytic activities such as gastrointestinal digestion or food processing including fermentation. This paper presents an exhaustive overview of the contemporary literature on antitumor activities of peptides released from milk proteins. In addition, caseins and whey proteins have been evaluated for anticancer potential using the AntiCP database, a web‐based prediction server. Proline and lysine, respectively, dominate at various positions in anticancer peptides obtained from caseins and whey proteins. The remarkable number of potential anticancer peptides revealed milk proteins as favorable candidates for the development of anticancer agents or milk and milk products for reduction of cancer risks. Moreover, anticancer peptides liberated from milk proteins can be identified from fermented dairy products. Although current findings of correlation between dairy food intakes and cancer risks lack consistency, dairy‐derived peptides show promise as candidates for cancer therapy. This critical review supports the notion that milk proteins are not only a nutritious part of a normal daily diet but also have potential for prevention and/or management of cancer.     

Bioactive peptides: Production and functionality

Milk proteins exert a wide range of nutritional, functional and biological activities. Many milk proteins possess specific biological properties that make these components potential ingredients of health-promoting foods. Increasing attention is being focused on physiologically active peptides derived from milk proteins. These peptides are inactive within the sequence of the parent protein molecule and can be liberated by (1) gastrointestinal digestion of milk, (2) fermentation of milk with proteolytic starter cultures or (3) hydrolysis by proteolytic enzymes. Milk protein derived peptides have been shown in vivo to exert various activities affecting, e.g., the digestive, cardiovascular, immune and nervous systems. Studies have identified a great number of peptide sequences with specific bioactivities in the major milk proteins and also the conditions for their release have been determined. Industrial-scale technologies suitable for the commercial production of bioactive milk peptides have been developed and launched recently. These technologies are based on novel membrane separation and ion exchange chromatographic methods being employed by the emerging dairy ingredient industry. A variety of naturally formed bioactive peptides have been found in fermented dairy products, such as yoghurt, sour milk and cheese. The health benefits attributed to peptides in these traditional products have, so far, not been established, however. On the other hand, there are already a few commercial dairy products supplemented with milk protein-derived bioactive peptides whose health benefits have been documented in clinical human studies. It is envisaged that this trend will expand as more knowledge is gained about the multifunctional properties and physiological functions of milk peptides.     

Effects of 5-aminolevulinic acid supplementation on milk production,iron status,and immune response of dairy cows

The objective of this study was to investigate the effect of 5-aminolevulinic acid (5-ALA) as a dietary supplement on milk yield and composition as well as iron status and immune response in lactating dairy cows. In this study 13 lactating Holstein cows were randomly assigned to either a control group or a treatment group supplemented with 10 mg of 5-ALA per kilogram of dry matter. During feeding, 5-ALA was mixed with a small amount of the total mixed ration and top-dressed. The experiments followed a crossover design with 2 periods. Each period consisted of an adaptation period of 12 d and a test period of 2 d. Dairy cows fed the diet supplemented with 5-ALA exhibited increased counts of white blood cells and granulocytes compared with the control group. The rate of phagocytosis and mitogen-induced proliferation of peripheral blood mononuclear cells in cows fed 5-ALA were higher than in cows fed a basal diet. However, 5-ALA did not affect iron status or plasma biochemical composition. Supplementation with 5-ALA improved milk protein and milk casein contents; however, it had no effect on milk production, milk fat, lactose, total solids, or solids-not-fat, compared with the control. We conclude that dietary supplementation of 5-ALA to lactating dairy cows may have a positive effect on milk protein synthesis and the immune response.     

Rabbit whey acidic protein concentration in milk, serum, mammary gland extract, and culture medium.

Rabbit whey acidic protein has been purified from whey using an AcA54 column. The purified whey acidic protein had an amino acid composition in agreement with the previously defined cDNA sequence. An antibody against whey acidic protein was raised in guinea pig. This antibody did not crossreact with mouse or cow milk or with rabbit alpha s1-casein and beta-casein. Whey acidic protein concentration was measured in rabbit milk using the antibody with a radioimmunoassay. The concentration of whey acidic protein in rabbit milk was 15 mg/ml, whereas the concentrations of alpha s1-casein and beta-casein were 16 and 45 mg/ml, respectively. The concentration of the three proteins was also evaluated in culture medium of rabbit primary mammary cells. The three proteins were induced by prolactin alone. Glucocorticoids amplified the prolactin effect on whey acidic protein more intensively than on caseins. The three proteins were present in mammary extract from virgin rabbit. The concentration of these proteins was lower at d 8 and 14 of pregnancy, and it was very high at d 25 of pregnancy. Whey acidic protein was undetectable in blood of virgin, weaned, and midpregnant females and of males. Whey acidic protein was present in blood of lactating rabbits, but alpha s1-casein and beta-casein were not detectably present in rabbit blood at the examined physiological states.     

Structures of the triglycerides of cows' milk, fortified milks (including. infant formulae), and human milk

The positional distributions of fatty acids in triglycerides from cows' milk, cows' milk enriched in linoleic acid by dietary means, and two commercial infant formulae fortified with vegetable fats have been determined for comparison with human milk. The results are discussed in terms of the digestibility of the fats and of the requirements of the human infant for linoleic acid .     

Functional Significance of Bioactive Peptides Derived from Milk Proteins

Bioactive peptides can be defined as protein fragments with potential biological activities. Milk proteins are precursors of many different biologically active peptides. Bioactive peptides from milk proteins are considered potential modulators of various regulatory processes in the body. They mediate physiological functions in cardiovascular, nervous, gastro intestinal and immune systems. The functional significance of bioactivities depends on peptide fragment. Bioactive peptides encrypted in major milk proteins are latent within the sequence of the parent protein molecule. They can be liberated by (i) gastro intestinal digestion of milk, (ii) fermentation of milk with proteolytic starter cultures, and (iii) hydrolysis by proteolytic enzymes. In relation to their mode of action, bioactive peptides may reach target sites at the luminal side of intestinal tract, or after absorption, in peripheral organs. The production, functionalities, and mode of action of bioactive milk peptides as well as latest peptide products and ingredients are reviewed.     

Variation in milk protein concentrations associated with genetic polymorphism and environmental factors

Concentrations of alpha s-casein, beta-casein, kappa-casein, beta-lactoglobulin, alpha-lactalbumin, serum albumin, and immunoglobulin in milk from 1888 Holstein cows were determined monthly over the lactation period. Cows were phenotyped for genetic variants of alpha s1-casein, beta-casein, kappa-casein, and beta-lactoglobulin. Least squares analyses showed variations in individual proteins due to parity number, month of test, stage of lactation, somatic cell count, fat content, milk yield, and phenotypes of cows for milk proteins. beta-Casein declined and serum proteins increased with advancing age of cows. Concentration of individual proteins decreased during the first 2 to 3 mo in lactation and then increased as lactation progressed. alpha s1-Casein variants significantly affected concentrations of alpha s-casein (BC greater than BB greater than AB) and beta-lactoglobulin (AB greater than BB greater than BC). Variant B for beta-casein is associated with lower alpha s-casein, beta-lactoglobulin, immunoglobulins, and higher beta-casein and alpha-lactalbumin concentrations than variant A1, A2, or A3. Milk from BB kappa-casein, and BB beta-lactoglobulin cows contained more alpha s-casein, kappa-casein, and less beta-lactoglobulin than milk from AA cows for the two proteins. Concentrations of all proteins were negatively correlated with milk production. Increased somatic cell counts were associated with lower beta-casein and higher concentrations of other proteins. Fat content of milk was positively correlated with the three casein fractions and beta-lactoglobulin.     

Goats' milk of defective alpha(s1)-casein genotype decreases intestinal and systemic sensitization to beta-lactoglobulin in guinea pigs

Contradictory results have been reported on the use of goats' milk in cows' milk allergy. In this study the hypothesis was tested, using a guinea pig model of cows' milk allergy, that these discrepancies could be due to the high genetic polymorphism of goats' milk proteins. Forty guinea pigs were fed over a 20 d period with pelleted diets containing one of the following: soyabean proteins (group S), cows' milk proteins (group CM), goats' milk proteins with high (group GM1) or low (group GM2) alpha(s1)-casein content. Parenteral sensitization to GM1 and GM2 proteins as also assessed. The sensitization was measured (1) by systemic IgG1 antibodies directed against bovine or caprine beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and whole caseins, and (2) by intestinal anaphylaxis measured in vitro in Ussing chambers, by the rise in short-circuit current (delta Isc) in response to milk proteins. Guinea pigs fed on CM and GM1 developed high titres (> 1500) of anti-beta-lg IgG1, with an important cross reactivity between goat and cow beta-lg. However, in guinea pigs fed on GM2, anti-goat beta-lg IgG1 antibodies were significantly decreased compared with GM1 guinea pigs (mean IgG1 titres were 546 and 2046 respectively), and the intestinal anaphylaxis was significantly decreased (3.5+/-4.5 microA/cm2) compared with that observed in GM1 guinea pigs (8.3+/-7.6 microA/cm2). Animals receiving GM1 or GM2 proteins via the parenteral route developed a marked sensitization. These results suggest that the discrepancies observed in the use of goats milk in cows' milk allergy could be due, at least in part, to the high genetic polymorphism of goats' milk proteins.