Microbiome and Metabiotic Properties of Kefir Grains and Kefirs Based on Them.

The analysis of the literature on the microbiome composition and metabolic properties of kefir available at the RSCI and Web of Science was carried out. Kefir has been used by humans for centuries. It is a useful product of mixed lactic and alcoholic fermentation, produced using evolutionally established associative cultures, collected in an aggregated state termed kefir grains. General characterization of kefir grains from the territorial zones of different continents (Russia, Europe, Asia, and America) is provided. The methods for differentiation and identification of individual species are described, as well as their interactions within the community. The diversity of microbial composition of kefir grains depending on local cultivation conditions and storage processes is shown. The microorganisms present in kefir have a number of properties that determine their metabolism, interaction in the community, beneficial effects on human health and immune system, which is important for the prevention and control of bacterial and viral infections, especially during the COVID-19 pandemic.

Analysis of Kefir Grains from Different Regions of the Planet Using High-Throughput Sequencing.

The taxonomic composition and spatial localization of yeast and bacteria in kefir grains (KG) obtained for study from different regions of the planet were investigated. The diversity of their microbiome has been demonstrated by high-throughput sequencing of bacterial 16S rRNA genes and the ITS1 region of the 18S-ITS1-5.8S-ITS2-28S complex of yeast rRNA. It has been established that the main representatives of the complex community of KG from different regions are lactic acid bacteria (LAB; lactobacilli, lactococci, and Leuconostoc spp. in different ratios) and different types of yeast of the genus Kazachstania (family Saccharomycetaceae). Acetic acid bacteria and a small percentage of yeast Kluyveromyces marxianus were detected in the KG from Tibet, and yeast Pichia kluyveri was detected in the KG from Ossetia.

Colonization strategy of the endophytic plant growth-promoting strains of Pseudomonas fluorescens and Klebsiella oxytoca on the seeds, seedlings and roots of the epiphytic orchid, Dendrobium nobile Lindl.

AIMS: Orchids form strong mycorrhizal associations, but their interactions with bacteria are poorly understood. We aimed to investigate the distribution of plant growth promoting rhizobacteria (PGPR) at different stages of orchid development and to study if there is any selective specificity in choosing PGPR partners. METHODS AND RESULTS: Colonization patterns of gfp-tagged Pseudomonas fluorescens and Klebsiella oxytoca were studied on roots, seeds, and seedlings of Dendrobium nobile. Endophytic rhizobacteria rapidly colonized velamen and core parenchyma entering through exodermis and the passage cells, whereas at the early stages, they stayed restricted to the surface and the outer layers of the protocorms and rhizoids. The highest amounts of auxin (indole-3-acetic acid) were produced by K. oxytoca and P. fluorescens in the nitrogen-limiting and NO3 -containing media respectively. Bacterization of D. nobile seeds resulted in promotion of their in vitro germination. The plant showed no selective specificity to the tested strains. Klebsiella oxytoca demonstrated more intense colonization activity and more efficient growth promoting impact under tryptophan supplementation, while P. fluorescens revealed its growth-promoting capacity without tryptophan. CONCLUSIONS: Both strategies are regarded as complementary, improving adaptive potentials of the orchid when different microbial populations colonize the plant. SIGNIFICANCE AND IMPACT OF THE STUDY: This study enlarges our knowledge on orchid-microbial interactions, and provides new features on application of the nonorchid PGPR in orchid seed germination and conservation.

Screening and Identifying Antioxidative Components in Ginkgo biloba Pollen by DPPH-HPLC-PAD Coupled with HPLC-ESI-MS2.

The Ginkgo biloba is one of ancient trees that exists from billions of years ago, its leaf and nut are used as herbs and foods in China, while so far its pollen does not have any application except pollination. In order to evaluate the antioxidant activity of Ginkgo biloba pollen, and rapidly screen its antioxidative components, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging ability, total flavonoid, total phenol, and proanthocyanidin of Ginkgo biloba pollen were determined and compared with those of Ginkgo biloba leaf and nut, and the off-line DPPH-HPLC-PAD and HPLC-ESI-MS2 were applied for screening and identifying the antioxidant flavonoids in Ginkgo biloba pollen. The results showed that the DPPH scavenging ability of Ginkgo biloba pollen was much higher than Ginkgo biloba nut, but lower than Ginkgo biloba leaf, while the total content of flavonoid in Ginkgo biloba pollen was approximately 4.37 times higher than in Ginkgo biloba leaf. Further studies found that the major flavonol aglycone in Ginkgo biloba pollen was kaempferol, which accounted for 96.71% of the total aglycones (includes quercetin, kaempferol and isorhamnetin), and the main flavonoid components in Ginkgo biloba pollen were flavonoid glycosides. Finally, ten antioxidant peaks were screened and identified to be flavonoids (including kaempferol and nine flavonoid glycosides), so flavonoids were likely to be the main antioxidant components in GP, and among them, three novel kaempferol glycosides (peaks 1, 2, and 3) were found in Ginkgo biloba pollen for the first time, which had never been found in Ginkgo biloba.

[Production of Antimicrobial Polypeptides by Propionibacterium freudenreichii RVS-4-irf].

Propionibacterium freudenreichii RVS-4-irf is a probiotic bacterium producing antimicrobial exometabolites applicable for foodstuff protection. Production of antimicrobial factors other than lowmolecular propionates was observed in media with and without trypton. A method was developed for production of the fraction of high-molecular mass exopolymers from the culture liquid. Their polypeptide nature was confirmed using proteinase K. The preparation of extracellular polypeptides from P. freudenreichii RVS-4-irf exhibited species-specific activity in suppression of fungal and bacterial growth.

[Development of Lyophilization Procedure Ensuring Survival of Bifidobacteria and Preservation of Their Probiotic Potential upon Long-Term Storage].

Survival of bifidobacteria and preservation of their morphological characteristics after 12-month storage of lyophilized cells was studied for the strains of Bifidobacterium bifidum and B. animalis isolated and maintained in the microbial collection of the Department of Microbiology, Moscow State University. A combined approach to pre-lyophilization treatment of microorganisms and subsequent storage was developed in order to improve cell survival. Compared to the standard cryoprotector concentrations, sucrose and glucose (5% and higher) in skim milk, as well as freezing at ‒70°C with subsequent storage at the same temperature resulted in improved survival of bifidobacteria. Under such conditions, the number of viable cells (CFU) after 12 months of storage was two to three orders of magnitude higher than in the case of the standard lyophilization procedure. Investigation of dynamics of resistance of reactivated clones to such gastrointestinal stress factors as gastric juice and bile acids revealed preservation of these properties at all storage modes. However, since the number of surviving cells decreased during storage according to the standard procedure, the number of stress-affected cells was correspondingly lower. Reactivated cultures exhibited high resistance to oxygen, with survival decreasing to 35% of the initial level.

Russian Kefir Grains Microbial Composition and Its Changes during Production Process.

By combining DGGE-PCR method, classical microbiological analysis and light- and electron microscopic observations, it was found that the composition of microbial communities of central Russia regions kefir grains, starter and kefir drink include bacteria of the genera Lactobacillus, Leuconostoc and Lactococcus, and yeast anamorphs of the genera Saccharomyces, Kazachstania and Gibellulopsis. Fifteen prokaryotic and four eukaryotic pure cultures of microorganisms were isolated and identified from kefir grains. It has been shown that members of the genus Lactobacillus prevailed in kefir grains, whereas strains Leuconostoc pseudomesenteroides and Lactococcus lactis dominated in the final product - kefir drink. Yeasts contained in kefir grains in small amounts have reached a significant number of cells in the process of development of this dairy product. The possibility of reverse cell aggregation has been attempted in a mixed cultivation of all isolated pure cultures, but full formation kefir grains is not yet observed after 1.5 years of observation and reinoculations.

[Biodegradation of Cellulose-Containing Substrates by Micromycetes Followed by Bioconversion into Biogas].

The ability of micromycetes Trichoderma viride and Aspergillus terreus to decompose the cellulose-containing substrates was studied. Office paper and cardboard, as well as a paper mixture, were found to be the most hydrolyzable. The cellulolytic activity of T. viride was 2-3 times higher than that of A. terreus; the highest values of 0.80 and 0.73 U/mLwere obtained from office paper and the paper mixture, respectively. The micromycete cultivation conditions (composition of culture medium, sucrose cosubstrate addition, seeding method) and the conditions of the fungus biomass treatment for its subsequent bioconversion into biogas by anaerobic microbial communities were optimized. It was shown that pretreatment improves the efficiency of biogas production from lignocellulosic materials under seeding with microbial community of bovine animal manure. After pretreatment of the Jerusalem artichoke phytomass (stems and leaves) and its subsequent bioconversion into biogas by methanogenic community, the biogas yield was increased by 1.5 times.

Characterization of Extracellular Yeast Peptide Factors and Their Stress-Protective Effect on Probiotic Lactic Acid Bacteria.

Protective effect of the extracellular peptide fraction (reactivating factors, RF) produced by yeasts of various taxonomic groups (Saccharomyces cerevisiae, Kluyveromyces lactis, Candida utilis, and Yarrowia li- polytica) on probiotic lactic acid bacteria (LAB) Lactobacillus casei, L. acidophilus,'and L. reuteri under bile salt (BS)-induced stress was shown. RF of all yeasts were shown to be of peptide nature; the active component of the S. cerevisiae RF was identified as a combination of low-molecular polypeptides with molecular masses of 0.6 to 1.5 kDa. The protective and reactivating effects of the yeast factors were not species-specific and were similar to those of the Luteococcusjaponicus subsp. casei R. In BS-treated cells of the tester bacteria, a pro- tective effect was observed after 10-min preincubation of the LAB cell suspension with yeast RE: the number of surviving cells (CFU) was 2 to 4.5 times higher than in the control. The reactivating effect was observed when RF was added to LAB cell suspensions not later than 15 min after stress treatment. It was less pro- nounced than the protector effect, with the CFU number I to 3 times that of the control. Both the protector and the reactivating effects were most pronounced in the S. cerevisiae and decreased in the row: C. utilis > K. lactis > Y lipolytica. The efficiency of protective action of yeast RF was found to depend on the properties of recepient LAB cells, with the L. casei strain being most sensitive to BS treatment. In both variants, the highest protective effect of RF (increase in the CFU number) was observed for L. acidophilus, while the least pronounced one, for L. casei. The reasons for application of the LAB strains combining high stress resistance and high response to stress-protecting metabolites, including RF factors, as probiotics, is discussed.

[New Approaches for the Isolation of Bifidobacterial Strains, Their Molecular Characterization, and Assessment of Their Probiotic Potential].

Six stably growing strains of bifidobacteria possessing probiotic properties were isolated from the feces of newborn children and animals. According to the results of molecular analysis, one strain was classified as Bifidobacterium bifidum, while five belonged to Bifidobacterium animalis. Initial identification of the strains was carried out using the primer pairs for the 16S rRNA gene (g-Bifid-F/R, Bif164/662) and for the xfp gene specific for bifidobacteria. Subsequent sequencing of complete genes encoding 16S rRNA synthesis in the isolates confirmed their species affiliation. The cultures exhibited high resistance to gastroenterological stress (5 ≤ RD ≤ 10) and may be therefore recommended as:potential probiotics.

[Biogas production from cellulose-containing substrates: a review].

Anaerobic microbial conversion of organic substrates to various biofuels is one of the alternative energy sources attracting the greatest attention of scientists. The advantages of biogas production over other technologies are the ability of methanogenic communities to degrade a broad range of substrates and concomitant benefits: neutralization of organic waste, reduction of greenhouse gas emission, and fertilizer production. Cellulose-containing materials are a good substrate, but their full-scale utilization encounters a number of problems, including improvement of the quality and amount ofbiogas produced and maintenance of the stability and high efficiency of microbial communities. We review data on microorganisms that form methanogenic cellulolytic communities, enzyme complexes of anaerobes essential for cellulose fiber degradation, and feedstock pretreatment, as biodegradation is hindered in the presence of lignin. Methods for improving biogas production by optimization of microbial growth conditions are considered on the examples of biogas formation from various types of plant and paper materials: writing paper and cardboard.

[Biogas production by microbial communities via decomposition of cellulose and food waste].

Several active microbial communities that form biogas via decomposition of cellulose and domestic food waste were identified among 24 samples isolated from different natural and anthropogenic sources. The methane yield was 90-260 ml CH4/g from microbial communities grown on cellulose substrates, office paper, and cardboard at 37 degrees C without preprocessing. Under mesophilic conditions, bioconversion of paper waste yields biogas with a methane content from 47 to 63%; however, the rate of biogas production was 1.5-2.0 times lower than under thermophilic conditions. When microbial communities were grown on DFW under thermophilic conditions, the most stable and effective of them produced 230-353 ml CH4/g, and the methane content in biogas was 54-58%. These results demonstrated the significance of our studies for the development of a technology for the biotransformation of paper waste into biogas and for the need of selection of microbial communities to improve the efficiency of the process.

[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.

Moorella humiferrea sp. nov., a thermophilic, anaerobic bacterium capable of growth via electron shuttling between humic acid and Fe(III).

An anaerobic, thermophilic, spore-forming bacterium (strain 64-FGQ(T)) was isolated from a terrestrial hydrothermal spring from the Kamchatka peninsula, Russia. This strain utilized lactate as an electron donor, insoluble poorly crystalline Fe(III) oxide incorporated into alginate beads as a potential electron acceptor and 9,10-anthraquinone-2,6-disulfonate (AQDS) as an electron-shuttling compound. Vegetative cells of strain 64-FGQ(T) were Gram-stain-positive, peritrichously flagellated, motile, straight rods, 0.3-0.5 µm in diameter and 2.0-5.0 µm long, growing singly or forming short chains. Cells formed round refractive endospores in terminal swollen sporangia. The temperature range for growth was 46-70 °C, with an optimum at 65 °C. The pH range for growth was 5.5-8.5, with an optimum at pH 7.0. The substrates utilized by strain 64-FGQ(T) in the presence of AQDS as an electron acceptor included lactate, malate, succinate, glycerol and yeast extract. The strain fermented galactose, fructose, maltose, sucrose, pyruvate and peptone. Strain 64-FGQ(T) used AQDS, humic acid, thiosulfate, nitrate and perchlorate as electron acceptors for growth. Fe(III) was not directly reduced, but strain 64-FGQ(T) was able to grow and reduce Fe(III) oxide in the presence of small amounts of AQDS or humic acid as electron-shuttling compounds. The G+C content of the DNA of strain 64-FGQ(T) was 51 mol%. 16S rRNA gene sequence analysis placed the isolate in the genus Moorella, with the type strain of Moorella glycerini as its closest relative (97.2% similarity). Based on phylogenetic analysis and physiological characteristics, strain 64-FGQ(T) is considered to represent a novel species of the genus Moorella, for which the name Moorella humiferrea sp. nov. is proposed; the type strain is 64-FGQ(T) (=DSM 23265(T)=VKM B-2603(T)).

[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.

[Methanogenic destruction of (amino)aromatic compounds by anaerobic microbial communities].

Destruction of a number of aromatic substrates by anaerobic microbial communities was studied. Active methanogenic microbial communities decomposing aminoaromatic acids and azo dyes into CH4 and CO2 were isolated. Products of primary conversion were found to be 2-hydroxybenzyl and benzyl alcohols gradually transforming into benzoate. It was shown that isolated microbial communities are capable of converting the initial substrates--benzyl alcohol, benzoate, salicylic acid, and golden yellow azo dye--into biogas without a lag-phase but with different velocities. Aromatic and linear intermediates of biodestruction of aromatic amines by obtained enrichment cultures were determined for the first time. Selective effect of aromatic substrates on a microbial community that was expressed in decrease in diversity and gradual change of dominant morphotypes was revealed.

[Special traits of decomposition of azo dies by anaerobic microbial communities].

We present the results of an investigation into the special traits of conversion of azo dies golden yellow, acid orange, methyl orange, and methyl red under anaerobic conditions in comparison to aerobic conditions. In the presence of oxygen, only methyl red underwent decomposition, while under oxygen-free conditions, all remaining substances were fully discolored under the action of a methanogenous consortium of organisms. The products of reduction of the azo bond are determined in the case of each die. Introduction of additional acceptors of electrons (sulfate and nitrate) had a negative influence on the discoloration of azo dies. Addition of ethanol as an available organic cosubstrate accelerated decomposition of azo dies both under methanogenous and sulfate- and nitrate-reducing conditions. There is no direct correlation between the rates of conversion of azo dies under anaerobic conditions or their toxicity to acetoclastic methanogens. Changes in the morphological composition of the community discoloring an azo die depended on the duration of its impact on microorganisms. The mechanism of the reduction of the azo bond under the action of substances acting as mediators is explained. These substances are products of the metabolism of the microbial community in anaerobic conditions. It is shown that the supposed mediators NADH and sulfide efficiently discolor azo dies in a cell-free system, while riboflavin significantly increased the rate of conversion of substrates in recurrent cycles of discoloration only in the presence of an anaerobic microbial consortium.

[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.

[Base for use a new liquid accumulation growth medium for the isolation of Salmonella from water objects].

Base for use of new luquid mild inhibitory accumulation medium for the isolation of Salmonella from water objects has been proposed. High sensitivity and efficacy of the new medium compared with routinely used magnesium and selenitic media were shown in experimental conditions. During examination of water from surface reservoirs the new accumulation medium allowed to isolate Salmonella with higher index and various serologic patterns.