Object of study: probiotic strains of bifidobacteria and lactobacilli; reference strains and clinical isolates of pathobionts; toxicity, biochemical composition, antioxidant, bacteriotropic, immunotropic and probiotic properties of cell-free postbiotic products from disintegrates and cultures of Bifidobacterium bifidum and Lactobacillus reuteri. The purpose of the research: to theoretically substantiate and develop new approaches to the creation of metabiotics based on derivatives of Bifidobacterium bifidum 1 and Lactobacillus reuteri DSM 17938 industrial strains, to investigate their biological properties and the effectiveness of in vivo application in experimental modeling of the infectious process in animals against the background of antibiotic-associated dysbiosis. Methods of research: microscopic, bacteriological, physicochemical, biochemical, immunological, biological, analytical and medical-statistical. Equipment: densitometer «Densi-La-Meter»; pH meter millivoltmeter pH-410; microscopes: «Biolam», «Primo Star», MPI-5; flow cytometer FACS Calibur; analyzers: «Lisa Scan EM», «Elise microplate reader with PC software Utrao SM 600», «Stat-Fax 3000+», Sapphire-400, «AAA 339M»; fluorometer «Qubit 3.0 Fluorometer»; spectrophotometers: SF-56, «Specord UV VIS»; equipment for gel chromatography, HPLC system Milichrome A-02.
The dissertation is devoted to development of new theoretically substantiated approaches to creation of metabiotics based on derivatives of Bifidobacterium bifidum 1 and Lactobacillus reuteri DSM 17938 industrial strains. For the first time biologically active derivatives of bacteria of probiotic strains were obtained by their disintegration by thermal cycling and subsequent cultivation of probiotic cells in their own disintegrates. The freezing-thawing regime of suspensions of B. bifidum and L. reuteri cells for their effective disintegration is substantiated and postbiotic products containing both structural components and metabolites of probiotic bacteria are obtained. The level of quantitative and functional preservation of probiotic bacteria after thermal cycling is established. As a result of in vivo toxicological screening for the first time established the lack of toxicity obtained by the original method of cell-free extracts with single and repeated administration at a dose corresponding to the daily therapeutic dose of cellular probiotics and the possibility of their long-term oral administration under strict dosing. In vitro cytotoxicity studies revealed the ability of cell-free extracts to significantly affect the metabolic activity of eukaryotic cells. The dependence of the nature and direction of the effect on the concentration, method of obtaining cell-free extracts and the type of test cells is established. New data on the biochemical composition of cell-free extracts from B. bifidum and L. reuteri were obtained. Differences between extracts from disintegrates and extracts from cultures of probiotic bacteria cultured in own disintegrates on the content of biologically active components and antioxidant properties are revealed. For the first time screening of potential precursor compounds using probiotic cultures of B. bifidum and L. reuteri as biochemical modification systems revealed the potentiation effect of ascorbic acid on the inhibitory activity of cell-free derivative-containing postbiotic products (cell-free supernatants and extracts) against patobionts, in particular isolates, resistant to antimicrobials. The level of knowledge about the stimulatory properties of probiotic bacterial derivatives has been significantly deepened. The ability of most cell-free extracts from disintegrates and cultures of B. bifidum and L. reuteri to increase the adhesive properties, proliferation and biofilm formation of probiotic bacteria in vitro has been revealed. For the first time, the ability of cell-free extracts obtained by the original method to modulate the functional activity of innate immune cells under the influence of bacteria in vitro and in vivo was demonstrated. Also for the first time the ability of cell-free extracts to modulate lipopolysaccharide-induced cytokine production (TNFα, IL-6 and IL-10) by human blood mononuclear cells in vitro was shown. The ability of extracts to increase the survival of animals at the experimental modeling of generalized lethal infection, to provide anti-infective protection and to correct microecological disorders in vivo at the modeling of intestinal infection in mice on the background of antibiotic-associated dysbiosis was established. The degree of implementation: 3 patents, 3 acts of implementation and1 technology. Sphere (industry) of use: health care (medicine: correction of dysbiosis, treatment of infectious and inflammatory diseases).
