**4. Discussion**

In recent years, there has been a tendency to use LAB exopolysaccharides for the treatment of infections of the respiratory tract, gastrointestinal tract, and urinary system, as well as cancer, allergic diseases, and viral infections [26]. However, despite the wide range of applications of lactic acid bacteria exopolysaccharides, their role in living organisms and their biological activities are not fully understood. The production of exopolysaccharides (EPS) by lactic acid bacteria (LAB) has been intensively studied, and a significant amount of data describing their composition, structure, and properties have been accumulated. EPS producing strains were found among representatives of the genera *Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus,* and *Weissella*. LAB exopolysaccharides have unique physical and rheological properties. As a result, they are used in the food industry as binding, stabilizing, and gel-forming agents, specifically in the production of dairy products. In recent years, much attention has been paid to the study of the biological activity of EPSs. In particular, it was shown that they have immunostimulating, antitumor, and antioxidant qualities. Since the biological activity of EPSs is strain-specific, the search for new strain-producers among LAB representatives found in the natural microbiota of fermented products is of grea<sup>t</sup> interest. Strains producing EPSs, especially in high amounts, are important for improving the rheological properties of the product and may possess possible health-improving e ffects for the human body. Previously, searches for LAB strains producing exopolysaccharides were performed. The isolates of LAB present in traditional fermented milk products, fermented fruits and vegetables were analyzed. Most EPS producers were found among representatives of the genera *Lactobacillus, Leuconostoc* and *Pediococcus* isolated from pickled apples, pickled tomato juice, and sauerkraut. Therefore, the biological activity of these EPSs was analyzed in the current work. In the preclinical study of potential drugs, the first step is associated with the assessment of the compound's toxicity for cell culture (namely, the study of the e ffect of di fferent concentrations on the structural and functional properties of the cells). It should be noted that most toxic agents act on the cell by interfering with the molecular mechanisms of homeostasis. This action can be expressed in a whole set of e ffects, which include changes in the fundamental cellular reactions, leading to destructive e ffects. We have studied the impact of EPSs extracted from lactic acid bacteria of the genera *Pediococcus*, *Leuconostoc* and *Lactobacillus* on the growth and division of MDBK cells sensitive to adenovirus. It was identified that EPSs in the studied concentrations are not toxic to MDBK cells, since they reduce cell viability only up to 17%. Also, the analysis of the e ffect of the highest EPS concentration on the cell cycle of MDBK cells showed the absence of significant changes in the distribution of the cell population in the G1, S and G2/M phases of the cycle. The obtained data allowed the use of all EPSs for the study of their antiviral potential. It is known that bacterial exopolysaccharides show significant antiviral activity due to the degradation of the viral particles, a decrease in the titer of viruses, the blocking of viral DNA replication, and the release of the infectious virus particle. However, the antiviral potential of these drugs has not been studied su fficiently. As a result, to investigate and exhibit EPS-mediated anti-adenoviral activity and to determine the stage of viral reproduction inhibited by EPS, various EPS treatment schemes were used in the work. Based on three di fferent treatments, the results suggested that EPSs have a

specific inhibitory e ffect on HAdV-5. The EPSs showed low virucidal activity and reduced HAdV-5 infectivity up to 85%. In most cases, neither pre-treatment resulted in a significant inactivation of virion infectivity. The inhibitory e ffects were observed only when EPSs were added to cells at the end of the virus adsorption period. However, only EPS 26a (produced by *Lactobacillus* sp.) reduced the titer of the virus obtained de novo and inhibited HAdV-5 plaque formation by 100%.

Currently, several possible mechanisms of the influence of lactic acid bacteria and their metabolites on the development of viral infection have been identified: i) inhibition of virus adsorption and penetration into cells as a result of direct bacterium/metabolite-virus interaction; ii) inhibition of late stages of viral reproduction and reduction of their infectivity; and, iii) stimulation of the immune system [26]. As a result, it was shown that probiotics can "capture" the virus of vesicular stomatitis (VSV) by direct interaction between LAB cells (*L. paracasei A14, L. paracasei F19, L. paracasei*/*rhamnosus Q8, L. plantarum M1.1* and *L. reuteri DSM12246*) and the lipid envelope of the VSV, which leads to blocking of the virus adsorption on the cell. Similar data were shown for *E. faecium NCIMB 10415* against influenza viruses and *L. gasseri CMUL57* isolated from vaginal microbiota against the herpes simplex virus type 2 (HSV-2). The probiotics of lactic acid bacteria *Lactobacillus* and their exopolysaccharides can stimulate the synthesis and accumulation of interleukin 12 to enhance the activity of natural killer cells and the synthesis of IgA in the spleen. Such activation of Th1 immune responses and the production of IgA determine their action against influenza [27]. In addition to direct interaction between viruses and LAB, bacteria synthesize some metabolites with antiviral activity. As a result, hydrogen peroxide, which is produced by *Lactobacillus* sp., plays an essential role as a natural microbocide within the vaginal ecosystem and is toxic to human immunodeficiency virus type 1 (HIV-1) and HSV-2. Lactic acid, which is the final product of carbohydrate metabolism and is synthesized by all types of lactobacilli, is essential for maintaining the required pH of the genital organs, since it is known that the acidic pH of the environment inactivates HIV, and HSV-1 and -2. It has been revealed that lactobacilli can produce compounds that inhibit replication of the viruses. The *L. brevis* cell wall inhibits the replication of HSV-2 DNA in cell culture, while the acidic products of the *Lactobacillus* metabolism inhibit the activation of T-lymphocytes, leading to a decrease in the sensitivity of lymphocytes to HIV infection, which is especially important for mixed HIV–HSV infections [28]. It has been established that some types of bacteriocins and exopolysaccharides have an apparent virucidal e ffect on influenza, HSV-1 and -2, HIV and Newcastle disease.

The characteristic changes in DNA synthesis and content induced by HAdV-5 infection allow the use of flow cytometry to detect not only viral infection but also potential antiviral activities [25]. The influence of the EPSs on the cell cycle under a condition of adenovirus infection was studied using flow cytometric analysis of propidium iodide-stained cells. The use of EPSs did not lead to the normalization of the life cycle of HAdV-5 infected cells to the level of non-infected cells, as the number of cells in the S phase was not decreased significantly (except EPS 48a) and there was no transition of cells into the G1 phase, which indicates the blocking of the mitosis in infected cells.

Taken together, our data suggested that exopolysaccharides produced by LAB strains exhibited anti-HAdV-5 activity in vitro. Among the six used strains, one LAB strain (*Lactobacillus* sp.), which produced EPS 26a suppressing the yield of virus particles, was selected. Furthermore, this strain is interesting as potential probiotic or starter, whereas the in-depth investigation of the functional and technological properties, EPSs monosaccharide composition and structure are currently studied.
