Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.     

Study on Protease of Dairy Bacteria

The action of intracellular proteases of lactic acid bacteria (IPLB) at pH 7 on various paracaseins was studied. Paracaseins prepared by releasing of 3~7% non casein type nitrogen (NCN) were hydrolyzed by IPLB with more difficulty than native or other paracaseins prepared by releasing of less or above 3~7% NCN. This phenomenon was not found in a case of a neutral protease of Bacillus subtilis. Hydrolyzed casein by rennin or IPLB of S. cremoris were studied by DEAE-cellulose column chromatography, starch-gel or agar-gel electrophoresis. It was estimated that not only some part of α-casein but also β-casein were hydrolyzed by IPLB of S. cremoris.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

Intracellular protease (IPLB) of Streptococcus cremoris was extracted from the cells, which were cultivated in liquid media, by momentarily disrupting between two disks by high pressure.

The hydrolyzing modes of α_s-, crude k-, β-, and whole casein by IPLB of Str. cremoris or rennet were observed through the released amounts of tyrosine, sialic acid, NPN, and calcium insensitive substance. Relative specific turbidity of casein solution and dissymmetry coefficient of casein were measured. Particle weight and UV absorption spectrum of each high molecular hydrolyzate of whole casein were also determined.

Among four kinds of casein fractions, α_s- or crude k-casein was most easily hydrolyzed by IPLB of Str. cremoris or rennet. Relative specific turbidity of crude k-casein solution was remarkably, but those of α_s-, β-, and whole casein slightly increased by the action of IPLB of Str. cremoris or of rennet. Changes of dissymmetry coefficients were negligibly induced by these two enzymes. Absorption spectrum of IPLB-Str. cremoris-casein showed some conformational change.

It was recognized that intracellular protease (IPLB) of L. bulgaricus, L. helveticus or Str. lactis, all together, more easily hydrolyzed α_s-casein than crude k-, β-, and whole casein. By the actions of three IPLBs, relative specific turbidity of crude k-casein solution remarkably but those of α_s-, β-, and whole casein slightly increased, and dissymmetry coefficients of these casein fractions changed negligibly.

Particle weight of whole casein hydrolyzed by each IPLB for five days was larger than that of control casein. UV absorption of each whole casein hydrolyzed by a IPLB increased at the wave length range of 280~250 mμ.     

Acid Protease of Bovine Milk

Occurrence of milk acid protease in bovine casein in addition to alkaline protease was found and purification of this enzyme was achieved. The enzyme had a pH optimum at 4.0 and was most stable at pH 3.5. The molecular weight of the enzyme was 36,000 and no inhibition was observed by diisopropyl-fluorophosphate, EDTA etc. This enzyme is considered to be similar to cathepsin D.

Milk acid protease mainly hydrolyzed α_s-casein and similar change was observed in autolysis of casein at pH 5.5. It is suggested that milk acid protease may have some significance in cheese ripening.     

Studies on the Changes of the Milk Casein by Various Treatments

A study has been made with the changes occurred in the milk casein during frozen storage. The flocculated protein formed during storage of frozen milk was resolved in 4 ~ 5 peaks in free-boundary electrophoretic pattern, using veronal buffer containing 7 m urea.

The flocculated protein was mainly casein. It may be considered that portions of casein have undergone some modification or denaturation during the destabilization process.

Significant increase has occurred in the relative proportion of β-casein in the casein remaining in supernatant after removal of flocculate, indicating that the partial fractionation of casein component occurred as a result of flocculation.     

Enrichment of n-Alkane Assimilation-Deficient Mutants of Candida Yeast by Synergistic Effect of Nystatin and Pyrrolnitrin

In order to clarify further the relationship between the heat stability of casein micelles and the formation of soluble casein upon heating concentrated milk, the effect of formaldehyde was examined. The addition of formaldehyde up to 20 mM markedly increased the heat stability of both concentrated skim milk and concentrated whey protein-free (WPF) milk. The stabilizing effect of formaldehyde was greater for concentrated skim milk than for concentrated WPF milk. The addition of formaldehyde depressed the formation of soluble casein upon heating concentrated milk. No soluble casein was formed on the addition of 20 mM formaldehyde. It was confirmed by Sephadex G-200 gel filtration in the presence of 6.6 M urea that cross-links among the casein components were formed in heated concentrated WPF milk containing formaldehyde. These facts suggest that formaldehyde may introduce cross-links among the casein components and prevent the formation of soluble casein accompanying the release of K-casein from micelles, thus stabilizing the casein micelles.     

Isolation of Tremerogen a-13, a Peptidal Sex Hormone of Tremella mesenterica

Bovine casein micelles were fractionated on controlled pore granule (CPG-10/3000) chromatography by size and the chemical properties of the fractionated micelles were compared. The results indicated the presence of two types of micelles distinguishable as large and small micelles. In skim milk, 72.7% of casein was calculated to be in the form of small micelles, 13.6% in the form of large micelles and 13.8% in non-micellar casein form.

The α_s1-casein content decreased, but β- and κ-casein content increased as the micelle size became smaller. κ-Casein in large micelles had a much higher sialic acid content than in small micelles. It was found that this difference in sialic acid content was due to the presence of non-glycosylated κ-casein in small micelles. In large micelles, non-glycosylated κ-casein was almost undetectable.

The addition of wheat germ lectin to micelles resulted in the formation of aggregates through intermicellar bridges between the carbohydrate chains of κ-casein located on the surface of the micelles. Both large and small micelles formed aggregates after the addition of wheat germ lectin. Large micelles were more sensitive to wheat germ lectin than small ones.     

Calcium-Binding Property of Dephosphorylated Caseins

Whole casein, α_s-casein and k-casein were dephosphorylated with a phosphoprotein phosphatase prepared from beef spleen and their calcium-binding capacities were compared with those of respective native caseins by a ultracentrifugal method.

The bindings of the calcium to 94% dephosphorylated whole casein and to 97 % dephosphorylated α_s-casein at neutral pH were approximately one third of those to respective native caseins. The decrease of calcium-binding capacity of k-casein due to dephosphorylation was also significant.

The effect of pH on the state and the calcium-binding capacity of dephosphorylated caseins was also examined and the role of organic phosphate groups of casein as calcium-binding sites was discussed.     

Studies on the Proteolytic Action of Dairy Lactic Acicl Bacteria

Aseptic rennet curd prepared under the aseptic conditions and Str. cremoris- and L. helveticus-cheese prepared by sandwiching the cell pellets of Str. cremoris and L. helveticus between aseptic rennet curd, respectively, were ripened at 10°C for desired period.

Water soluble nitrogen (WSN) contents of both aseptic rennet curd and two kinds of cheese were determined. Gradual increase of WSN content of aseptic rennet curd was recognized all through the ripening preiod. WSN contents of both Str. cremoris- and L. helveticus-cheese were remarkably higher than those of aseptic rennet curd after 12 days ripening. This tendency was more remarkably recognized after 60 or 70 days ripening. α^s-Casein was mainly hydrolyzed by these lactic acid bacteria during ripening. α^s-Casein in two kinds of the cheese was more easily degradated by these lactic acid bacteria than that in aseptic rennet curd by rennet.

Judging from the results in previous and present reports, it was estimated that lactic acid bacteria used as a starter began to autolyze after 12 days ripening and that intracellular proteases released from their cells mainly hydrolyzed α^s-casein contained in Ca-paracaseinate of aseptic rennet curd to water soluble substances. This hydrolysis was also estimated from the viscous texture observed by scanning electron micrography.     

Purification and Properties of Intracellular Proteinase from Streptococcus cremoris

Proteolytic activity in the extract from the cells of Streptococcus cremoris increased in the presence of casein, lactose, glucose, and CaCl₂ in the media but was negligibly detectable in the extract of the cells harvested from the culture containing succinate or citrate. The intracellular proteinase from S. cremoris harvested from tomato medium was purified 150-fold in this experiment. The enzyme had a molecular weight of 140,000, optimum pH at 6.5 to 7.0, and maximum activity at 30 C. The proteinase was activated by Ca²⁺ and inhibited by Zn²⁺, Cu²⁺, Hg²⁺, Fe²⁺, ethylenediaminetetraacetate, and sodium lauryl sulfate. The K_m value of the enzyme towards each casein fraction was almost the same, and the V_max of the enzyme towards α_s-casein was smaller than those towards the other casein fractions.     

Studies on Destabilization of Caseinate Complex during Frozen Storage of Milk

Unpasteurized skim milk was storaged in a frozen state at ?7°C or ?20°C for up to several months. There was no increase of non casein and non protein nitrogens, but a slight increase of free tyrosine and a slight decrease of alkaline phosphatase activity were detected when storage period was prolonged. Destabilization occurred solely in caseinate complex, but non micellar casein appeared to be stable.

The contents of calcium and inorganic phosphate in the caseinate complex separated by ultracentrifugation were increased appreciably after frozen storage. The viscosity characteristics of frozen storaged skim milk was also investigated.

Caseinate complex was ultracentrifugally separated from skim milk before and after frozen storage, and then lyophilized. Skim milk itself was also lyophilized before and after frozen storage. Dispersibility was examined on the reconstituted suspension of the lyophilized samples.

The lyophilized sample from frozen storaged milk was much less dispersible than the lyophilized control sample prepared before frozen storage. However, when lyophilized samples were once resolved with reagents such as urea and potassium oxalate and then dialyzed against fresh milk, stable micelle resulted in both samples prepared before and after frozen storage.

Some reduction of dispersibility occurred during lyophilization and subsequent storage in a dried state in the caseinate complex prepared before frozen storage. This reduction was small when skim milk was lyophilized and stored.     

Purification and Characterization of UDP-N-Acetylgalactosamine: κ-Casein Polypeptide N-Acetylgalactosaminyltransferase from Mammary Gland of Lactating Cow

UDP-N-acetyl-d-galactosamine: κ-casein polypeptide N-acetylgalactosaminyltransferase was purified from a crude Golgi apparatus of lactating bovine mammary gland after solubilization with Triton X-100. Through chromatography on DEAE-Sephadex A-50, apomucin-Sepharose 4B, FPLC mono S, and Sephacryl S-200, and then electrofocusing, the enzyme was purified up to 7500-fold from the homogenate.

The molecular weight of the enzyme was estimated at 200,000 from gel filtration. The pI value of the enzyme was 6.4 on electrofocusing. The purified enzyme transferred GalNAc from UDP-GalNAc, not to the carbohydrate chains but to the polypeptide chains of the substrates, κ-casein and mucin. The enzyme required Mn²⁺, DTT, and Triton X-100 for maximal activity. The Km value for UDP-GalNAc was 16.2μm. Km values for K-subcomponents 1 and 7, and apomucin were 1.15, 5.10, and 0.192mg/ml, and V_max values were 254, 259, and 581 nmol/hr/mg, respectively. Thermal stability and the effects of pH, milk components, lectins, and nucleotides were examined.

α_s1-Casein strongly inhibited GalNAc transfer to κ-casein. The inhibitory effect of α_s1-casein was canceled by the addition of Ca²⁺, which causes casein micelle formation. This means that the glycosylation of κ-casein occurs after casein micelle formation triggered by the accumulation of Ca²⁺ in vivo.     

Role of Tyrosine Residues in Interactions between Casein Components

It was indicated from ultraviolet difference spectra and ultracentrifugal experiments that associations occurred between two casein components (α_s- and κ-caseins, β- and κ-caseins and α_s- and β-caseins) at lower CaCl₂ concentrations (2~3 mm) and that aromatic amino acid residues participated in the associations. Chemical modification studies with 2-hydroxy-5-nitrobenzylbromide indicated that tryptophane residues of each casein component were not essential for these associations. It was also demonstrated by nitration of tyrosine residues with tetranitromethane that tyrosine residues of κ-casein were essential for α_s·κ-association and for β·κ-association and that tyrosine residues of α_s-casein were important to α_s·β-association.

Interactions between casein components were also studied at higher CaCl₂ concentration (10 mm) which is enough for micelle formation. It was found that tyrosine residues of κ- casein played an important role for the stabilization of α_s- and β-caseins. Properties of the nitrated-β-casein were almost the same as that of the native β-casein except the absorption spectrum. α_s·β-Interaction in the presence of 10 mm CaCl₂ was investigated by use of the nitrated-β-casein instead of the native β-casein. It was proved that α_s-casein was stabilized by the nitrated-β-casein and that precipitation of the nitrated-β-casein increased in the presence of α_s-casein.

The mechanism of interactions between casein components at higher CaCl₂ concentration (10 mm) are discussed in connection with the associations at lower CaCl₂ concentrations (2~3 mm).     

Changes of Casein Complex during Storage of Sterilized Skim Milk

Two types of sterilized skim milk were prepared; one was HTS–1 milk which was heated at 130°C flashly and the other was HTS–2 milk which was heated at 130~135°C for 75 sec. The changes of casein complex during storage of HTS–1 and HTS–2 milks were examined and compared with those of AUT milk which was heated at 120°C for 15 min. The results obtained are summarized as follows.

(1) Visible sediment was formed in HTS–1 and HTS–2 milks after 8 and 14 months of storage, respectively, while no sediment was observed in AUT miik throughout 15 months of storage. (2) The amount of calcium in the ultracentrifugal wheys of HTS–1 and HTS–2 milks decreased gradually with prolonged storage, while that in the ultracentrifugal whey of AUT milk was kept constant after 1 month of storage. (3) Almost no differences among the three samples were observed in the increments of Ca/N ratio of ultracentrifuged casein complex during storage. (4) The amount of soluble casein increased in AUT milk during storage, but decreased in HTS–1 and HTS–2 milks.

On the basis of the above results, the destabilization of casein complex during storage was discussed.     

Selection of Protease-Positive and Protease-Negative Variants of Streptococcus cremoris

Protease-negative variants were shown to outcompete the wild-type strains of Streptococcus cremoris E₈, HP, and Wg₂ at pH values higher than 6.0 in milk. For S. cremoris E₈ this process was studied in more detail. At lower pH values the wild type had a selective advantage. This pH-dependent selection was not found in all media tested. The poor growth of the protease-negative variant at low pH was not due to lower internal pH values. By growing S. cremoris E₈ and Wg₂ in acidified milk (pH 5.9) the proteolytic activity of the cultures could be stabilized. In continuous cultures under amino acid limitation the wild type S. cremoris E₈ and HP strains had a selective advantage over the protease-negative variants at low dilution rates (D < 0.2) at all pH values of the medium. This was apparently due to a lower affinity-constant (K_s) of the protease-positive variants for amino acids. Finally, a high fraction of protease-positive variants could be maintained in continuous cultures by using a growth medium with low concentrations of casein as a nitrogen source. At high dilution rates nearly all cells were protease positive.     

A Simple Device for Assuring High Reproducibility in Fluorometric Measurement of Deoxyribonucleic Acid in Yeast Cells

The heterogeneity and chemical composition were investigated in κ-casein from colostrum. The acid casein was obtained from four different Holstein cow colostra. The yield of acid casein from colostrum was higher than that from normal milk. κ-Casein from colostrum was prepared by the gel filtration method of Yaguchi et al. The gel filtration profiles differed among the four colostrum acid caseins.

Colostrum κ-casein was fractionated on a DEAE-cellulose column into one nonadsorbed and six adsorbed fractions with increasing salt concentration. Six adsorbed fractions had the same molecular weight and stabilizing ability for α_s1-casein in the presence of calcium ion. The amino acid composition and the phosphorus content of the adsorbed fractions were identical, but fractions eluted with high salt concentrations had more carbohydrates (galactose, sialic acid, glucosamine, galactosamine). Colostrum κ-casein was characterized by a higher content of carbohydrate moiety in comparison with normal κ-casein. Also glucosamine which has not been found in normal κ-casein was detected in colostrum κ-casein. The κ-casein component from colostrum contained at least one molecule of carbohydrate, though the carbo hydrate-free component was detected in normal κ-casein.     

Direct Effect of Sodium Nitrite on Extracted Histones

Irpex lacteus milk-clotting enzyme hydrolyzed the Phe(105)-Met(106) bond of κ-casein, causing the precipitation of para-κ-casein along with other casein fractions in the presence of calcium ions, with a mechanism similar to other milk-clotting enzymes. Furhtermore, Irpex enzyme hydrolyzed at the positions Leu(79)-Ser(80) and Tyr(30)-Val(31) of para-κ-casein.

Degradation patterns of β-casein by Irpex and Mucor miehei enzymes were almost the same by polyacrylamide gel electrophoresis, but Endothia parasitica enzyme showed a different degradation pattern. Under the conditions employed, β-casein appeared to be scarcely hydrolyzed by chymosin.

Comparing the specificity of Irpex enzyme on β-casein with that of chymosin, the common cleaving points were Leu(165)-Ser(166), Ala(189)-Phe(190), and Tyr(192)-Glu(193). The difference in the specificity between the enzymes was exhibited in the cleavage at the Leu(139)-Leu(140) bond by chymosin and of the Ser(142)-Trp(143) bond by Irpex enzyme. Although the cleaving points of β-casein by both enzymes resembled each other, each enzyme exhibited different degradation patterns of β-casein because of thier different order of cleavage.     

Interaction between Casein and Carboxymethyl Cellulose in the Acidic Condition

The stabilizing action of carboxymethyl cellulose (CMC-1 and CMC-2) on caseins was studied in the acidic pH region. CMC-1 stabilized 1% whole, α-, α_S- and β-casein at pH 4.6 and 5.0, and at 5°C. But CMC-2 could not completely stabilize these caseins at pH 5.0. Interaction between κ-casein and CMC-1 commenced when pH was adjusted to 6.3, but CMC-2 interacted with κ-casein below pH 5.6. An α_S- and κ-casein mixture (4 : 1) with CMC-2 was destabilized by the addition of 0.02 m NaCl or NaH₂PO₄ at pH 5.0. The α_S/κ ratio of the precipitated casein was about 10. But the same system with CMC-1 was not destabilized by the salts.     

Partial Isolation and Degradation of Caseins by Cell Wall Proteinase(s) of Streptococcus cremoris HP

The cell wall proteinase fraction of Streptococcus cremoris HP has been isolated. This preparation did not exhibit any activity due to either specific peptidases known to be located near the outside surface of and in the membrane or intracellular proteolytic enzymes. By using thin-layer chromatography for the detection of relatively small hydrolysis products which remain soluble at pH 4.6, it was shown that β-casein is preferentially attacked by the cell wall proteinase. This was also the case when whole casein or micelles were used as the substrate. κ-casein hydrolysis is a relatively slow process, and α_s-casein degradation appeared to proceed at an extremely low rate. These results could be confirmed by using ¹⁴CH₃-labeled caseins. A relatively fast and linear initial progress of ¹⁴CH₃-labeled β-casein degradation is not inhibited by α_s-casein and only slightly by κ-casein at concentrations of these components which reflect their stoichiometry in the micelles. Possible implications of β-casein degradation for growth of the organism in milk are discussed.     

Adsorption of Staphylococcal Bacteriophage by Milk Proteins

Propagation of homologous bacteriophage in a culture of Staphylococcus aureus (1:1 ratio of phage to bacteria) in Trypticase Soy Broth (TSB) and in skim milk indicated more activity of phage in TSB. Early lysis of bacteria in skim milk followed by a pronounced rise in bacterial population suggested that staphylococcal phages were being inactivated by milk. Titration of phages from skim milk, whey, and TSB indicated about 90% adsorption of phages by acid- and heat-precipitable proteins of skim milk, whereas numbers recovered from whey were quite comparable to those recovered from TSB. Reducing the pH from 6.5 to 4.0 increased the percentage of phages recoverable from skim milk from 10 to 56%. Apparently, the changes in electrical charges on the casein micelles at this low pH were responsible for release of many phages from their complex with casein.