Chromatographic characterization of ovine kappa-casein macropeptide

Ovine casein macropeptide (CMP) was characterized by anion-exchange FPLC and reversed-phase (RP) HPLC. To study heterogeneity (the degree of glycosylation and phosphorylation), CMP was desialylated with neuraminidase and dephosphorylated with acid phosphatase. Following RP-HPLC, the main CMP components were identified using either on-line or off-line mass spectrometry. The most abundant ovine CMP component was a diphosphorylated carbohydrate-free form, followed by one or two monophosphorylated and a non-phosphorylated asialo-aglyco species. Aglyco non-phosphorylated, monophosphorylated and diphosphorylated forms were in the ratio 3:20:77. Only approximately 30% of ovine CMP was glycosylated. Assuming that the monosaccharide fraction of ovine CMP is composed of N-acetylgalactosamine, galactose and N-glycolylneuraminic acid, molecular masses consistent with the presence of CMP containing tetra-, tri-, di- and monosaccharide were identified.     

Heterogeneity of caprine kappa-casein macropeptide

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine kappa-casein due to the silent substitution Ile/Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8-14% and 86-92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.     

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.     

Primary structure of kappa-casein isolated from mares' milk

In this work the purification and the complete primary structure of kappa-casein from equine milk are reported for the first time. Mares' milk casein was separated by RP-HPLC into four fractions. Complete primary sequence was obtained by sequence analysis of the protein in the fastest eluting peak isolated by chromatography. This sequence was 95% identical to that reported for the C-terminal portion of the zebras' kappa-casein and showed high similarity with kappa-caseins from sources other than Equidae, confirming that this protein was indeed kappa-casein in equine milk. The presence of post-translational modifications in equine kappa-casein was investigated by mass spectroscopy, after enzymic dephosphorylation. Two main components were found, the smaller component being more abundant. Equine kappa-casein was recognized by a lectin specific for one of the glucosidic bonds in the saccharide moiety of bovine kappa-casein. Sequence comparison with prevision studies showed that the distribution of charged and hydrophobic regions in equine kappa-casein was similar, but not identical, to that found in the bovine protein; these regions are associated with the role of kappa-casein in the formation and stabilization of the micellar structure of casein in milk.     

Proteolytic activity among psychrotrophic bacteria isolated from refrigerated raw milk

Psychrotrophic bacteria were isolated from refrigerated raw milk from a processing plant in Southern Brazil. Psychrotrophic counts were between 4.9 and 7.8 log cfu/mL, and 5.3 to 7.2 log cfu/mL, for samples collected at the truck and the milk storage silo, respectively. Among the bacterial isolates, 90% were Gram-negative. Most strains presented low proteolytic activity, but strains of Burkholderia cepacia, Klebsiella oxytoca and Aeromonas sp. showed higher than 20 U/mL on azocasein as substrate. Crude proteases from selected strains were resistant to conventional heat treatments and caused coagulation of UHT milk after 5 days storage at room temperature.     

Proteolytic activity of Lactobacillus strains isolated from dryfermented sausages on muscle sarcoplasmic proteins

The proteolytic activity of seven strains of Lactobacillus from two species isolated from dry cured sausages was assayed using a soluble muscle extract as a source of proteins, at a temperature of 30 °C. The results indicated that the strains of Lactobacillus plantarum tested had the more active proteolytic system, showing the highest amino acid release in the medium after 72 hr of incubation (L. plantarum CRL 681) when the microorganism was in the stationary phase of growth. The strains of L. casei showed a continued hydrolytic activity with a lower amino acids concentration along the studied period. The SDS-PAGE profiles showed that the major changes in sarcoplasmic proteins were produced in the 13 kDa and 36-40 kDa molecular weights region.     

Proteolytic activity in UHT-sterilized milk.

The proteolytic activity in UHT-sterilized milk of a proteinase-containing fraction, prepared from the milk, is reported. The proteinase fraction was isolated from casein micelles as described by Reimerdes & Klostermeyer (1974). The fraction was added to UHT milks and the proteolysis in the samples, measured as the liberation of non-protein nitrogen, compared with that in control samples without added proteinases. The increase in proteolytic activity and deterioration in samples containing added proteinases indicates that an enzyme process occurred in UHT-sterilized milk which could influence gel formation.     

Evidence of cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation

Abstract: Cell‐associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation were extracted and characterized. Proteinases were effectively released when washed cells were incubated in 0.3 mg/mL lysozyme in 50 mM Tris‐maleate (pH 7) at 37 °C for 2 h. Major cell‐associated proteinases exhibited molecular mass of 17, 32, and 65 kDa, but only a 32‐kDa proteinase showed strong amidolytic activity toward Suc‐Ala‐Ala‐Pro‐Phe‐AMC. Activity of all cell‐associated proteinases was completely inhibited by phenylmethanesulfonyl fluoride, indicating a characteristic of serine proteinase. In addition, a 65‐kDa serine proteinase was also inhibited by ethylenediaminetetraacetic acid, implying a metal‐dependent characteristic. Optimum activity toward a synthetic peptide substrate was at 50 °C and pH 8 and 11. Proteinases with molecular mass of 17 and 32 kDa exhibited caseinolytic activity at 25% NaCl and activity based on a synthetic peptide substrate increased with NaCl concentrations up to 25%, suggesting their role in hydrolyzing proteins at high salt concentrations. This is the first report of liberated cell‐associated proteinases from a moderate halophile, Virgibacillus sp. Practical Application: The cell‐associated proteinases could be extracted from Virgibacillus sp. SK 33 using lysozyme. The extracted enzyme could be applied to hydrolyze food proteins at NaCl content as high as 25%. In addition, this study demonstrated that not only extracellular but also cell‐associated proteinases are key factors contributing to protein‐degrading ability at high salt environment of Virgibacillus sp. SK 33.     

Action of rennin on kappa-casein.

The action of rennin on kappa-casein was studied as a function of time using several methods to measure activity. The first indication of rennin cleavage of kappa-casein is precipitability in .1 M acetate buffer at pH 5.2 and 5 C. A longer exposure to rennin is required to alter kappa-casein so that it forms a precipitate with calcium ions and loses its ability to stabilize alpha s-casein. The least sensitive indication of rennin activity is measurement of nitrogen soluble in 2% trichloroacetic acid. Electrophoresis experiments showed that these methods detect various stages in the conversion of kappa-casein para-kappa-casein.     

Proteolytic, lipolytic and molecular characterisation of Yarrowia lipolytica isolated from cheese

This work studied the qualitative and quantitative proteolytic and lipolytic activities of Yarrowia lipolytica strains isolated from two cheese types. Randomly amplified polymorphic DNA-PCR (RAPD-PCR) analysis was used to compare the cheese strains of Y. lipolytica with strains isolated from other food products and with the type strain of the species in order to investigate the genetic diversity and occurrence of specific environmental groups. Diversity of proteolytic and especially lipolytic activity within Y. lipolytica strains isolated from dairy products was observed. In particular, the degree of specificity for saturated or unsaturated fatty acids as well as for even- or odd-numbered carbon free fatty acids (FFAs) varied among the strains. The RAPD-PCR profiles showed low genetic relatedness between many of the food isolates and the type strain of the species. Such genetic variability needs to be further evaluated. Most of the Y. lipolytica strains appeared to be specific to the particular environment from which they were isolated. However, phenotypic characteristics having technological importance in dairy products and, particularly, lipolytic activities did not correspond to the genetic differences observed by RAPD-PCR analysis.     

Identification of antibacterial peptides from bovine kappa-casein

The objective of the present study was to identify antimicrobial peptides present in several digests of commercial caseins with gastric enzymes. The most active hydrolysate against Escherichia coli ATCC 25922 and Listeria innocua CECT 910T corresponded to a pepsin digest of bovine kappa-casein. The protein digest was first separated by semipreparative high-performance liquid chromatography (HPLC), and the most active fractions were again subjected to a second chromatographic step. Finally, identification of the active peptides was carried out by online and offline HPLC-electrospray ionization-tandem mass spectrometry. By means of this technique, 21 peptides were identified in the active HPLC fractions. Although most were derived from bovine kappa-casein, some of the identified fragments corresponded to beta-casein and alpha(s)-casein fragments, a result of the presence of small amounts of these proteins in the preparation of kappa-casein. Some of the peptides identified were chemically synthesized and showed antibacterial effects against several gram-positive and gram-negative bacteria. Among the synthesized peptides, kappa-casein f(18-24), f(30-32), and f(139-146) were most effective against all bacteria tested. The antibacterial effect of these peptides is discussed in relation to their amino acid sequences.     

Proteolytic and lipolytic activities of mould strains isolated from Spanish dry fermented sausages

Proteolytic and lipolytic activities of ninePenicillium strain and oneMucor strain previously isolated from Spanish dry fermented sausages have been studied.P. camemberti STCC 2267 andAspergillus oryzae ATCC 9362 were also used. Myofibrillar and sarcoplasmic proteins as well as fat extracted from pork meat and -naphthyl acetate, -naphthyl caprilate and -naphthyl laurate were used as substrates. Mould strains were grown in an enrichment medium prepared at pH 5.5 and 7.0 and malt extract broth. Strains were incubated at 18°C and 22°C. Myofibrillar proteins and -naphthyl caprilate were the most hydrolysed substrates. The greater enzymatic activities were observed at a pH 7.0 and 22°C. However, five mould strains showed considerable enzymatic activities under conditions similar to those used for ripening dry fermented sausages. These strains can be assayed as potential starter cultures for industrial sausage production.     

Heterogeneity of the kappa-casein glycomacropeptide and physiological activity of its fractions

A number of fractions with differing intensity of the inhibitory action on the gastric secretion were obtained by gel filtration in a column with biogels P-30 and P-2 of cappa-casein glycomacropeptide. A low-molecular fraction displays the greatest capacity for inhibiting gastric secretion. Desialism, heating and partial hydrolysis of glycomacropeptide with pepsin, trypsin and chemotrypsin used separately have no effect on its inhibitory activity. The resulting data suggest that the inhibitory action of glycomacropeptide is caused by a certain part rather than by entire molecule.     

Extracellular proteinases ofBacillus spp. isolated from fermented African locust bean,Iru

Extracellular proteinase production by seven strains ofBacillus subtilis group isolated from fermented African locust bean was compared. The seven strains, which were designated BS1, BS2, BS3, BL1, BL2, BL4 and BP2, showed significant differences (α = 0·05) in extracellular proteinases production. The order of proteolytic activity (in descending order) of the strains in nutrient broth medium containing African locust bean was: BL2 > BP2 > BS2 > BL4 > BS3 > BL1 > BS1. The proteinases of strains BL2 were purified and characterized by ammonium sulphate precipitation, ion-exchange chromatography. Three proteinases (serine proteinase, neutral proteinase and an esterase) were identified with MW of 18·2–19·7, 22·6 and 33·5 kDa, respectively. The serine proteinase was highly hydrophobic while the esterase was characterized by low specific activity.     

Proteolytic activity of isolated lamb calpains on myofibrils under the conditions of pH, Ca2+ concentration and temperature existing in postmortem muscle.

The two calcium-activated neutral proteinases (calpains I and II) and their specific inhibitor were isolated by ion exchange chromatography in DEAE-Sephacel from lamb skeletal muscle (longissimus dorsi). Their proteolytic activities were then determined using myofibrils as substrate. The Ca2+ requirements were different for each form of the enzyme: calpain I needed only 50 mumol Ca2+ for half-maximal activity, while the other isoenzyme, calpain II, needed 1,000 mumol Ca2+ for reaching 50% of its maximum activity. Both calpains showed a relevant activity in the pH range 5.5-6.5 (over 40% of maximum activity found at pH 7.5). With regard to the effect of temperature, both isoenzymes retained about 25% of their activity at 25 degrees C with a temperature reduction down to 4 degrees C. It is concluded that calpain I is an active protease under conditions similar to that prevalent in lamb meat during postmortem storage.     

Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages.

The occurrence of amino acid-decarboxylase activity in 92 strains of lactic acid bacteria, coagulase-negative staphylococci, and Enterobacteriaceae isolated from Spanish fermented pork sausages was investigated. The presence of biogenic amines in a decarboxylase synthetic broth was determined by ion-pair high performance liquid chromatography with o-phtalaldehyde post-column derivatization. Among the 66 lactic acid bacteria strains tested, 21 lactobacilli (in particular, Lactobacillus curvatus) and all 16 enterococci were amine producers. Tyramine was the main amine produced by these bacteria, although they also produced phenylethylamine, tryptamine, and/or the diamines putrescine and cadaverine. None of the lactic acid bacteria produced histamine. Coagulase-negative staphylococci were found to be negative amine-producers. Aromatic monoamines, apart from histamine, were not formed by Enterobacteriaceae. This family was responsible for cadaverine and putrescine production. The results obtained for biogenic amine production by bacteria in a synthetic medium suggest that amino acid-decarboxylase activity is strain dependent rather than being related to specific species.     

Effect of rabbit kappa-casein expression on the properties of milk from transgenic mice

Transgenic mice were produced carrying the coding region of the rabbit kappa-casein gene linked to the upstream region of the rabbit whey acidic protein gene. Mice from the highest-expressing line produced 2.5 mg rabbit kappa-casein/ml in their milk. The foreign protein was associated with the casein micelles and altered micelle size, though in the high-expressing line rabbit kappa-casein also segregated into the whey fraction obtained after centrifuging the milk samples. Milk from transgenic mice had the same overall protein content as that from non-transgenic mice, except for the transgene product. However, litters fed with this transgenic mouse milk grew less well than litters given milk from non-transgenic mice. This reduction in growth was not related to changes in mammary gland structure or mammary cell morphology. Preliminary results indicated that milk from the transgenic mice had a higher viscosity.     

Three-dimensional molecular modeling of bovine caseins: kappa-casein.

Three-dimensional structures derived from X-ray crystallography are extremely important in elucidating relationships between structure and function for many proteins. However, not all proteins can be crystallized. The caseins of bovine milk are one class of noncrystallizable proteins. The complete primary and partial secondary structures of these proteins are known, but homologous proteins with known crystallographic structure are not available. In this report, sequence-based predictions of secondary structure were made and adjusted to conform with global secondary structures derived from Fourier transform infrared spectroscopy. With this information, a three-dimensional structure for kappa-casein was constructed using molecular modeling computer programs. The constructed model contains two unstranded beta-sheets; both are predominantly hydrophobic and capable of forming quaternary structural interaction sites with alpha s1-casein. This unrefined structure is in good agreement with much of the biochemical information available for kappa-casein.