L. G. Stoyanova

[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.

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.

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 culture 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 broth than that of the 194-D peptide. In comparision 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 of bacteriocin 194-D by the 194-K strain in these media occurred parallel to producer growth, and its maximal accumulation in the culture broth was observed at14–20 h of the strain’s growth.

Detection of Lactobacillus Species Using a Gene Fragment of the RNA Polymerase Beta Subunit rpoB

In this paper, we report on the comparative analysis of nucleotide sequences of the rpoB gene to assess its potential for use as a genetic marker for identification of Lactibacillus spp. recovered from acid milk products in different regions of Kazakhstan. The discriminatory power of the rpoB gene sequence was shown to be significantly higher for identification of closely related species of the Lactobacillus genus as compared to that of the 16S rRNA gene. The phylogeny based on the rpoB gene proved identical to that of the 16S rRNA gene and could be used as a supplement phylogenetic marker.

Establishment of taxonomic status of new prospective bacteriocin-synthesizing Lactococci strains of various origins

The taxonomic status of new prospective bacteriocin-synthesizing strains of mesophilic lactococci isolated from raw milk and milk products from different regions of Russia and also of strain F-119, obtained by protoplast fusion of two related strains with low bacteriocin-synthesizing activity, was established by classical methods of identification. The values of antibiotic activity displayed by the strains toward a test microorganism Bacillus coagulans were up to 4650 IU/ml, which is significantly higher than in natural lactococci strains. In spite of some differences in morphology, ability to ferment carbohydrates, requirements for nutrients, and antibiotic suspectability, the strains were identified as Lactococcus lactis subsp. lactis. The new strains differed from the classic nisin-producing strain L. lactis subsp. lactis MGU by a remarkably broad spectrum of bactericidal and fungicidal activity. Study of 16S rRNA gene sequences of new natural strains, fusants F-119 and another one obtained earlier, F-116, and their parental strains in comparison with reference strains confirmed the new strains’ taxonomic status as Lactococcus lactis subsp. lactis. The nucleotide sequences of 16S rRNA genes were deposited with GenBank under accession numbers EF100777-EF114305.

New Probiotic Culture of Lactococcus lactis ssp. lactis: Effective Opportunities and Prospects

We have isolated several new strains of lactococci from raw milk of Buryatia region of Russia near Lake Baikal with wide variety of climatic and ecological niches. Physiological and biochemical features of new strains were studied and compared to the nisin-producing strain Lactococcus lactis ssp. lactis MSU. According to morphological, cultural, physiological, biochemical properties and gene sequence of 16S rRNA a novel most effective strain 194 was identified as Lactococcus lactis ssp. lactis (GenBank database DQ 255954), which has status “GRAS” (absolutely harmless for human health and animals). The strain 194 had inhibitory activity against Gram-positive, Gram-negative pathogenic bacteria and also on fungi of genera Aspergillus, Fusarium and Candida. This is unique biological property for natural strains of Lactococcus lactis specie. We also studied the probiotic properties of strain as resistance to HCl and bile acids, sensitivity to antibiotics and show the therapeutic effect of strain as a food additive on model mice CBRB-Rb (8,17) 1Iem chronic dermatitis.

Dependence of growth characteristics of original strains of Lactococcus lactis subsp. lactis on the composition of agar media used for biomass growth

Data on the dependence of growth characteristics of Lactococcus lactis subsp. lactis 729 and TB2 extracted from milk and homemade curd on the content of casein peptone (CP) as the main source of nitrogen in the agar medium is presented. The number of colony-forming units (CFUs), cell division rate (CDR), and mean hourly doubling time (HDT) were compared between two time segments of cultivation: from 1 to 2.5 h, and from 2.5 to 3.5 h. In liquid media, logarithmic growth was observed for the 729 strain in all groups for 3.5 h at minimal pH of 6.5. The maximum biomass production rate (2.5 × 108 cells/mL) was observed in the media containing the meat extract; the minimal HDT was in the biomass transferred from the minimal MA4 medium to the enriched M7 medium. In contrast with the TB2 strain, the 729 strain does not form colonies on agar media at CP concentrations below 0.4%. The colonies of the TB2 strain increase in size from 2.85 mm2 to 5 mm2 when decreasing CP concentration from 1 to 0.4%. The yeast extract has a stimulating effect in liquid media at said concentration ratios, leading to an increase in the efficiency of cell division at lower rates of lactic acid production, to pH of 5.5 when oligopeptide transport is blocked. The meat extract provides adaptation of bacteria to the conditions of excessive acidity and has positive effect on cell division in the 729 strain at later stages of bacterial colony development. The smaller size of colonies of the strain might be evidence of active acid formation.

The Probiotical Potential of Lactobacilli from Therapeutic Preventive Beverage Kurunga

Introduction Kurunga is a dairy drink made of a mix of lactic acid and alcoholic fermentation, characterized by high biological value based on protein composition, amino acid spectrum, fatty acid composition of lipids, vitamin and mineral substances, and physiological activity of microbiota containing lactobacilli, lactococci, bifidobacteria, and yeast. Among the probiotic correctors of normal microbiota isolated from national products, lactobacilli was of particular interest, with regards to a therapeutic – preventive effect. The aim of the study was to examine the probiotic properties of lactobacilli from kurunga. Methods We isolated lactic acid bacteria strains from kurunga. The isolated cultures were identified using common microbiological methods and phylogenetic analysis. The antibiotic activities of these strains were determined by measuring the growth inhibition zone of test cultures. The probiotic properties were measured as levels of resistance to bile and hydrochloric acids, in addition to the presence of superoxide dismutase (SOD) activity using the xanthine oxidase-cytochrome method. Proteolitic activity was determined at the various levels of pH (3.0, 4.2, 5.3, and 7.0). Results According to the morphological, cultural, physiological, biochemical properties and the genotypic analysis of the oligonucleotides sequence of specific genes, the most effective strain was identified as Lactobacillus diolivorans KL-2 (GenBank database KC438372). The isolated strain suppressed the growth of Gram-positive bacteria, such as Bacillus, Staphylococcus, and Listeria sp., as well as Gram-negative bacteria, such as E.coli, Proteus, Salmonella sp. They also possessed fungicidal action (based on Penicillum, Aspergillus sp, and Candida sp.). The strain was resistant to the action of the bile acids at concentrations of 0.8% to 1.0% and hydrochloric acid. The strain KL-2 possessed a relatively high SOD activity (25.74 U/mg of protein), a low proteolytic activity at a pH 3.0 (4.74·10-3 PU/ml), and high proteolytic activity at pH 4.2 (294.74·10-3 PU/ml), pH 5.3 (330.52·10-3 PU/ml) and pH 7.0 (713.68·10-3 PU/ml). Conclusion The unique properties of this strain, such as stability in the gastrointestinal tract, the wide spectrum of bactericidal and fungicidal action to the pathogenic species, the relatively high superoxide dismutase and proteolytic activities, and the absence of toxicity, make it a prime candidate for probiotic culturing.