[Features of dynamics of growth and formation of uncultivable forms in Lactococcus lactis].

AIM: Study strain differences in dynamics of viability and formation of uncultivable forms of Lactococcus lactis. MATERIALS AND METHODS: 3 strains of L. lactis--MSU, 729 and F116 were used in the study. Uncultivable forms were obtained by prolonged incubation of cultures of lactococcus in synthetic medium under conditions of carbohydrate deprivation. Medium was inoculated by cultures grown in Elliker broth, washed by saline and without washing. The populations obtained were incubated at 30 degrees C without mixing for 4 months. Samples of cultures were studied periodically for viability and cultivability. RESULTS: In cultures obtained by using unwashed inoculate active growth of quantity of bacteria in the first days after seeding was noted. Speed of formation of uncultivable forms is the faster the higher the level of metabolic activity of cells in the population. A fact of phenotypic dissociation in lactococcus culture under stress was detected. CONCLUSION: The speed of formation of uncultivable forms of L. lactis, as well as stability of their existence may probably depend on the strain, cultivation conditions and metabolic activity of cells in the population.

[Antibacterial metabolites of lactic acid bacteria: their diversity and properties].

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.

[Characteristics and identification of bacteriocins produced by Lactococcus lactis subsp. lactis 194-K].

The Lactococcus lactis subsp. lactis 194-K strain has been established to be able to produce two bacteriocins, one of which was identified as the known lantibiotic nisin A, and the other 194-D bacteriocin represents a polypeptide with a 2589-Da molecular mass and comprises 20 amino acid residues. Both bacteriocins were produced in varying proportions in all of the studied nutrient media, which support the growth of the producer. Depending on the cultivation medium, the nisin A content was 380- to 1123-fold lower in the 194-K stain culture fluid than that of the 194-D peptide. In comparision to to nisin A Bacteriocin 194-D possessed a wide range of antibacterial activity and suppressed the growth of both Gram-positive and Gram-negative bacteria. An optimal medium for 194-D bacteriocin synthesis was shown to be a fermentation medium which contained yeast extract, casein hydrolysate, and potassium phosphate. The biosynthesis ofbacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture fluid was observed at 14-20 h of the strain's growth.

[Microbiological characterization of a new strain of Lactococcus lactis subsp. lactis K-205].

Bacteriocin-producing strain Lactococcus lactis K-205 with antibacterial activity up to 2,700 IU/ml (calculated on nisine-producing activity) was isolated from Buryat beverage kurunga. Using genotypic analysis of oligonucleotide sequence of 16S rRNA gene, the strain was identified as L. lactis subsp. lactis. 16S rRNA gene nucleotide sequence of K-205 strain was deposed in GenBankdatabase under the number EF 114305. New K-205 strain as compared with museum nisine-producing strain L. lactis subsp. lactis had wider spectrum of bactericidal as well as fungicidal activity which is a rare characteristic for the natural isolates of this microorganism.

[A comparison of the properties of bacteriocins formed by Lactococcus lactis subsp. lactis strains of diverse origin].

Bacteriocins formed by four strains of Lactococcus lactis subsp. lactis have been studied and compared: 729 (a natural strain isolated from milk), 1605 (a mutant of strain 729), F-116 (a recombinant obtained by fusing of protoplasts of the two related strain 729 and 1605), and a nisin-forming strain obtained by adaptive selection at Moscow State University. Antimicrobial activity studies revealed differences between the strains in the effects on individual groups of microorganisms; the activities of the strains were also distinct from that of Nisaplin (a commercial preparation of the bacteriocin nisin). Methods for isolation and purification of bacteriocins have been developed, making it possible to obtain individual components of antibiotic complexes as chromatographically pure preparations. Bacteriocins formed by the strains of Lactococcus lactis subsp. lactis have been identified and differences in their biological and physicochemical properties, established. A novel potent broad-spectrum antibiotic substance distinct from nisin has been isolated from the recombinant strain F-116.

[Isolation and identification of new nisin-producing Lactococcus lactis subsp. lactis from milk].

A method for isolating active nisin-producing strains of mesophilic lactococci was developed. Overall, 55 strains of mesophilic lactic acid bacteria were isolated from fresh cow's milk obtained from milk farms in various regions throughout Russia; of them, 36 displayed nisin-synthesizing activity. The three most active strains were studied according to morphological, cultural, physiological, and biochemical characteristics and identified as Lactococcus lactis subsp. lactis. The species attribution of the strains studied was confirmed by the similarity of the nucleotide sequences of the 16S rRNA gene. The nucleotide sequences of the 16S rRNA genes were deposited with the GenBank under accession numbers DQ255951-DQ255954. The distinctions between these strains in physiological and biochemical characteristics and the ranges of their bactericide action on the microorganisms capable of developing in agricultural materials and food products were determined. The isolated strains displayed considerably wider ranges of action, which differed from the nisin-producing strain MGU and the commercial nisin preparation (Nisaplin), used as a biological preserving agent.

[Components of fermentation medium regulate bacteriocin synthesis by the recombinant strain Lactococcus lactis subsp. lactis F-116].

The regulation of the synthesis of bacteriocin produced by the recombinant strain Lactococcus lactis subsp. lactis F-116 has been studied. The synthesis is regulated by the components of the fermentation medium, the content of inorganic phosphate (KH2PO4), yeast autolysate (source of amine nitrogen), and changes in carbohydrates and amino acids. The strain was obtained by fusion of protoplasts derived from two related L. lactis subsp. lactis strains, both exhibiting a weak ability to synthesize the bacteriocin nisin. Decreasing the content of KH2PO4 from 2.0 to 1.0 or 0.5% caused bacteriocin production to go down from 4100 to 2800 or 1150 IU/ml, respectively; the base fermentation medium contained 1.0% glucose, 0.2% NaCl, 0.02% MgSO4, and yeast autolysate (an amount corresponding to 35 mg % ammonium nitrogen). The substitution of sucrose for glucose (as the source of carbon) increased the antibiotic activity by 26%, and the addition of isoleucine, by 28.5%. Elevation of the concentration of yeast autolysate in the low-phosphate fermentation medium stimulated both the growth of the lactococci and the synthesis of bacteriocin. Introduction of 1% KH2PO4, yeast autolysate (in an amount corresponding to 70 mg % ammonium nitrogen), 2.0% sucrose, and 0.1% isoleucine increased the bacteriocin-producing activity of the strain by 2.4 times.

[Variability of nisin-synthesizing wild strain Lactococcus lactis subsp. lactis 119 under the effect of ultraviolet light].

To increase the nisin synthesizing activity of the natural strain Lactococcus lactis subsp. lactis 119 isolated from sour milk, UV irradiation in different doses was used followed by isolation of productive clones. The highest mutation effect was observed with the dose of 76,000 erg/mm2, when 11% of the cells increased their productivity by 12.8% at the minimum survival rate. Two-step UV irradiation and adaptive selection on the nisin-contaning medium provided isolation of a strain with the activity 42.6% higher than that of the initial strain (3850 IU/ml). Natural and UV-induced variability of the strain by the nisin synthesis, growth rate, carbohydrate consumption and sensitivity to antibiotics of various groups were studied.

[Comparison of methods of storing lactic acid bacteria]. / Sravnenie sposobov khraneniia molochnokislykh bakterii.

The study was undertaken to elucidate how different methods of storage (immersing in mineral oil, lyophilization, and subculturing) of lactic acid bacteria belonging to the genera Lactobacillus and Lactococcus affect their viability, antibiotic activity, and ability to accumulate organic acids. Storage of the lactic acid bacterium L. lactis subsp. lactis by immersion in mineral oil proved to be ineffective. Lyophilization allowed the survival of a sufficiently large number of cells, although their antibiotic activity somewhat decreased. The resuscitation of lyophilized bacteria by subculturing them in rich nutrient media, such as skim milk, led to the restoration of their physiological activity, including the effective antimicrobial spectrum.

[The development of a nutrient medium for nisin-producing streptococci utilizing the waste products from a number of biotechnology plants]. / Razrabotka pitatel'noi sredy dlia nizinobrazuiushchikh streptokokkov s ispol'zovaniem otkhodov riada biotekhnologicheskikh proizvodstv.

The possibility of using some waste products of the dairy and agricultural industries as nutrient substrates for the cultivation of nisin-producing streptococci was studied. Yeast production wastes as well as potato liquor, whey and dried whey as waste products were shown suitable for the growth of Streptococcus lactis and the biosynthesis of nisin. The level of the nisin production increased when corn steep liquor and glucose were added to whey and dried whey respectively.

[Various aspects of protoplast production in Streptococcus lactis]. / Nekotorye aspekty polucheniia protoplastov u Streptococcus lactis]

The authors studied the effect of some factors, including the conditions of preincubation, the action of 2-mercaptoethanol, EDTA, alpha-amylase, on protoplast production in four strains of Streptococcus lactis caused by lysozyme. The strains differed in the nisin-producing activity and in the structure of the cell walls that were not affected with lysozyme without either preincubation in 2-mercaptoethanol or in a salt medium with minimal inhibitory concentrations of DL-threonine. EDTA and alpha-amylase increased the lysozyme effect. Among seven buffer systems studied the most favourable for protoplast production in S. lactis is the ammonia-citric buffer with EDTA, and the best regeneration medium is the agar salt medium to which, depending on the strain, either glucose or sucrose should be added as a stabilizer.

[Sublimation-dried mare's milk and the possibility of its use in creating infant and dietary food products]. / Kobyl'e moloko sublimatsionnoi sushki i vozmozhnost' ego ispol'zovaniia pri sozdanii produktov detskogo i lechebnogo pitaniia.

The main biological parameters were studied in sublimated mare's milk: protein fraction composition, amino-acid spectrum, fatty-acid composition of lipids and the content of vitamins and mineral substances. A high biological value of the sublimated mare's milk has been proved, as well as significant proximity of its composition to human milk. Preliminary data have evidenced expediency of mare's milk use for the development of adapted milk mixtures for baby foods. However, the fatty component of the mixtures needs certain correction by addition of vegetable oil. The sublimated mare's milk can be used for the production of kumiss, that would extend its application in dietotherapy by removing season and territory limitations.

[Use of the method of protoplast fusion in the selection of a nisin producer]. / Ispol'zovanie metoda sliianiia protoplastov v selektsii produtsenta nizina.

Experimental data on selection of Streptococcus lactis producing the polypeptide antibiotic nisin with the method of protoplast fusing, one of the modern methods of cell engineering are presented. Four strains of Streptococcus lactis differing in their nisin-producing levels and difficult for protoplasting were used in the study. It was shown possible to transfer them to the protoplast form when respective conditions for their preliminary cultivation and regeneration are provided. Distinctive features of these strains with respect to the antibiotic resistance, sugar fermentation and growth component requirements were revealed. The protoplast fusing yielded hybrids differing from the parent strains by a number of phenotypical features and nisin-synthesizing activity.

[Physiologo-biochemical features of the growth and development of Propionibacterium shermanii in egg albumin]. / Fiziologo-biokhimicheskie osobennosti rosta i razvitiia Propionibacterium shermanii v iaichnom belke.

Propionibacterium shermanii was shown to be able of growing in such an unusual substrate as egg albumen. Incubation of the bacterium in egg albumen results in complete utilization of free carbohydrates in this substrate and accumulation in it of such bacterial metabolites as vitamins B2 and B12, free amino acids, bacterial protein, acetic and propionic acids which favour conservation of egg albumen. The bacterium does not possess proteolytic activity and does not utilize albumen; in the conditions of nitrogen deficiency, it can utilize free amino acids in the substrate. The growth of P. shermanii in egg albumen is stimulated by the addition of ammonium sulfate in combination with potassium phosphate or ammonium phosphate to the substrate. These substances change the direction of propionic fermentation toward the accumulation of propionic acid; at the same time, the content of glutamine, asparagine, alanine, methionine, cysteine and other free amino acids increases in the substrate.

[Desugarization of egg white by microorganisms]. / Obessakharivanie iaichnogo belka mikroorganizmami

Glucose was eliminated from egg whites, using microorganisms, to prevent melanoidin formation which may damage the product. Desugarization was achieved by means of Acetobacter xylinum, Streptococcus lactis, Propionibacterium shermanii, Pr. petersonii and propionicacid cocci. Optimal conditions of desugarization were found, depending on the physiological characteristics of the above microorganisms. Propionibacterium shermanii may be well used to ferment a liquid egg white. These bacteria have no proteolytic properties and make no use of the egg white. They enrich the egg white with vitamin B12 and propionate, a preserving agent, during fermentation.