EVALUATION OF THE EXOMETABOLITES CYTOTOXIC EFFECT OF LACTOBACILLUS PLANTARUM IN VITRO

Korotkykh O. O., Kalinichenko S. V.

MICROBIOLOGY

Scentific article

An exceptionally important role of lactobacilli in the microorganisms is determined by the diversity of their functions. They are participating in the regulation of optimal levels in metabolic processes, creating a high colonization resistance of mucosal, inhibit adhesion, penetrate and propagate pathogenic and opportunistic microorganisms, have a broad spectrum antimicrobial mechanisms, produce biologically active substances. One of the main criteria, which are now presented for evaluation of probiotics and probiotic properties of the functional food is to determine their toxicity. In recent years, attention is attracted to the cell cultures as a model for preclinical testing of drugs, biologically active substances, etc. This is due to the fact that the cell cultures are capable to specifically respond to impact of biotic factors. Specificity is typically manifested as a cytotoxic effect which is evaluated by the degree of morphological changes, disorders of morphological apparatus and proliferative properties of cells. Our next aim was staged proceeding from the above: evaluate the possibility of using cell culture to determine the cytotoxic effect of probiotic strains by the degree of morphological changes. The study involved the culture substrate dependent cell lines HeLa, Vero, Hep-2, which morphologically is an epithelial cell. For the experiments was prepared suspension of cells with the inoculum concentration 0.5 × 106 / ml. Cultures of cells were cultured in DMEM / F12 (Sigma, Germany) supplemented with 5% fetal serum of cattle embryos, solutions gentamicin and nystatin in 80% ethanol to a final concentration of 50 U / ml, in a sealed culture dish in a CO2 incubator with (37 ± 1)°C and constant humidity of 95% and a CO2 content of 5%. To the culture medium were added exometabolites L. plantarum in an amount of 0.3 ml 0.2 ml 0.1 ml 0.05 ml 0.025 ml ml 0.0125 0.00625 ml per 1.0 ml of medium, since it did not cause the amount of change pH of the growth medium (titration agent of influence). A suspension of the daily strain culture with an optical density of 1.0 МсF was prepared for exometabolites L. plantarum. 5.0 mL of microbial suspension was added to 45 ml of nutrient broth supplemented with 1% glucose (sugar broth). The resulting mixture was incubated at (37 ± 1)°C for 72 h. Then it was centrifuged twice at 3000 rev / min for 30 minutes and the supernatant was collected which contains exometabolites. As a result were obtained, while calculating the data, the absolute numbers, which express the descriptive and quantitative traits. The data which studied the cytotoxic effect of L. plantarum exometabolites by degree of morphological changes HeLa cell cultures, Vero, Hep-2 are given in. The first stage of the study was to investigate the influence of sugar broth on the adhesive properties and the presence / absence of compromising the integrity of the monolayer cell culture lines HeLa, Vero, Hep-2. The next step was to study the action of ekzomatebolites L. plantarum in the cell culture. It was determined that the culture Vero cell line was not sensitive to the action of specified agent. Hep-2 cells and HeLa lines were sensitive to the action of lactobacilli exometabolites. Dose-dependent cytotoxic effect of exometabolites was established experimentally: the degree of degeneration of the cell monolayer is decreased with a reduction of their concentration in the growth medium. Thus, after 72 hours of incubation in 50% HeLa cell lines that are cultured in growth medium supplemented with an agent of influence in an amount of 0.3 ml and 0.2 ml per 1.0 ml of medium destructive signs were noted. As for the cell line Hep-2, after 72 hours of incubation, the signs of degradation were detected in 100% of the cells that were incubated with the addition of exometabolites at 0.3 ml; 75% of the cells — in an amount of 0.2 ml; 50% of the cells — in an amount of 0.1 ml and 0.05 ml; 25% of the cells — in an amount 0,025ml; and unit — in an amount of 0.0125 ml. Experimental results on the use of cell cultures as a possible model for testing probiotic agents reveal prospects of their use in biotechnological processes during pre-clinical trials instead of research on laboratory animals.

Lactobacillus, cell culture, probiotics, cytotoxic effect

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«Bulletin of problems biology and medicine» Issue 1 part 2 (127), 2016 year, 158-162 pages, index UDK 615.277.3: 576.3: 579.262