Specific composition of indigenous microflora (Lactobacillus spp., Bifidobacterium spp., Lactococcus spp.) in farm animals

A.P. Paliy, S.O. Gujvinska, L.P. Livoshchenko, L.I. Nalivayko, Ye.M. Livoshchenko, V.I. Risovaniy, R.A. Dubin, N.V. Berezhna, A.P. Palii, R.V. Petrov National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine”, 83 Pushkinska St., Kharkiv, 61023, Ukraine Sumy National Agrarian University 160 Kondratieva St., Sumy, 40021, Ukraine Luhansk National Agrarian University 68 Slobojanska St., Starobilsk, 92703, Ukraine Kharkiv National Technical University of Agriculture named after Petro Vasylenko, 44 Alchevskih St., Kharkiv, 61002, Ukraine *Corresponding author E-mail: paliy.dok@gmail.com


Introduction
In recent years, much attention has been paid to probiotic microorganisms in the scientific literature to maintain a balance of normal microflora (de Vrese & Schrezenmeir, 2008;Ravinder et al., 2012;Dash et al., 2018). The interest to probiotics in the scientific community, in the food and processing industries is growing. However, there is a need for comprehensive and consistent approaches to assess the characteristics and efficacy and safety of probiotics and the products containing them (Rijkers et al., 2010;Reid, 2012). Lactic acid bacteria and bifidobacteria are introduced into probiotics, food supplements and functional foods (Reid, 2006). Improvement and development of new preparations based on living cultures of microorganisms is one of the urgent tasks of modern biotechnology. The search for strains of microorganisms that have the properties of the vitamins synthesis, amino acids, enzymes, antibiotic substances, is crucial in the development of innovative probiotic drugs and requires methodologically correct research using modern methods (Toomy et al., 2010;Didari et al., 2014;. Isolation of lactobacilli was performed from the children's faeces. At the same time, 20 cultures were isolated, which were able to survive at low pH and in the Probiotics have shown promise for the treatment of certain diseases in both human and veterinary medicine, but understanding the molecular mechanisms behind the direct and indirect effects on the immune response of the intestine will contribute to better and possibly more effective treatment of diseases (Vieira et al., 2013). Although the vast majority of probiotics used today are generally considered safe and useful, a scientifically sound approach is needed in the application of these agents on a case-by-case basis (Boyle et al., 2006;Rijkers et al., 2011;Fijan, 2014). Considering the relevance of this field of research and development of innovative probiotic agent, we carried out the indication and identification from the milk of healthy cows and gastrointestinal tract of healthy pigs and calves of bacteria the genus Lactobacillus, Bifidоbacterium, Lactococcus, as the most physiologically.

Materials and Methods
The objects of study were cultures of micro-organisms isolated from cows' milk (82), gastrointestinal tract of cattle (317) and pigs of different age groups (114). Experimental studies were conducted in the laboratory of veterinary sanitation and parasitology of the National Science Center "Institute of Experimental and Clinical Veterinary Medicine" (Kharkiv). Simple and selective nutrient media were used to isolate microorganisms from biological material and further determine their cultural properties (Basu et al., 2015). To detect and identify lactobacilli and bifidobacteria used MRS medium, Blaurok's medium, improved on their basis for 24-72 hours at 37°C (Süle et al., 2014;Yang et al., 2018;. The biological properties of lactic acid bacteria were studied by tinctorial, cultural-morphological and biochemical characteristics. The morphology of the cells and colonies of the studied cultures was determined by microscopy of Gram stained smears, the growth pattern by sowing the cultures on liquid and solid nutrient media. Identification of bacteria by species was performed on the spectrum of carbohydrate fermentation using the ARI 50 CH test systems and API 20Strep (Bio Mérieux, France). These systems determine the digestion possibility of 47 carbohydrates and their derivatives. The results were recorded after 24 and 48 hours. The results were interpreted using API-WEB (Professional). Species belonging to isolated microorganisms were determined by appropriate tests (van Teeseling et al., 2017). The data obtained were processed using a Microsoft Excel 7.0 software.

Results and Discussion
As a result of bacteriological examination of biological's samples material from the gastrointestinal tract of clinically healthy calves, 317 cultures of microorganisms were isolated and typed (Table 1). A total of 82 bacterial cultures were isolated in determining the species composition of the cisternal (16) and parenchymatous (21) milk microflora selected from clinically healthy cows (Table 2).
Ukrainian Journal of Ecology, 10(1), 2020 As a result of the work (Table 2), 82 cultures of microorganisms were isolated from 37 milk samples, represented by lactic acid streptococci -27 (32.93%), staphylococci -7 (8.54%), enterobacteria -2 (2.44%), lactobacilli -26 (31.70%), and bifidobacteria -20 (24.39%). Analysis of the results obtained in the study of cisternal and parenchymatous milk shows that there is no significant difference between the species composition of the microflora, except that enterobacteria in cisternal milk were isolated in 4.44% of cases, whereas they were absent in parenchymal milk. Based on the study of the cultural-morphological and biochemical properties of the bacteria isolated from milk, they were assigned to Lactococcus lactis (27), Staphylococcus epidermidis (7), Escherichia coli (1), Proteus vulgaris (1). The specificity of 153 cultures of Lactobacillus spp. and 124 cultures of Bifidobacterium spp. were determined using appropriate test systems (Table 3). The cultures of Lactobacillus spp. (153) were gram-positive, asparagenic long sticks and coccyx forms that were located in short chains. Lactobacilli grown on MRS-2 medium had a brushlike growth colonies in the middle and lower part of the tube, small transparent colonies were formed on MRS-4 agar medium and hydrolyzed milk agar. Colonies on agar medium had a diameter of 2-5 mm, convex, with entire edges, opaque, not pigmented. Fermentation type metabolism was observed in cultures, nitrates were not recovered, gelatin was not liquefied, catalase-negative, cytochromes were not contained. As a result of the identification of lactobacilli, the test cultures are classified as L. plantarum, L. casei, L. acidophilus, L. fermentum, L. delbrueckii, L. brevis. The microorganisms of Bifidobacterium spp. (124) were gram-positive sticks, variable in shape and arrangement. Usually they were curved, club-shaped and often branched at the ends, not movable. After 48 hours of culturing, the cells did not stain evenly. All tested microorganisms grew only under anaerobic conditions. On the surface of the solid medium MRS they formed cream-white, convex, round, with an equal colony margin, with a diameter of 1-2 mm, with a paste-like consistency.
In the semi-liquid environment, the studied strains formed colonies in the form of "comets", "nail" etc. In the liquid medium, without anaerobic conditions, bottom growth was observed in a high column of medium and in anaerobic conditions, there was a uniform growth throughout the volume of the medium. The strain had no activity of catalase and nitrate reductase, did not form glucose Based on the study of the cultural and morphological properties of the selected cultures, 69 isolates of the genus Lactococcus were isolated. The test bacteria had cells of spherical or oval shape, were gram-positive, non-motile, had no capsules, grew in pairs and short circuits in a liquid medium and did not form endospores. The strains were optional anaerobes, catalase and oxidase negative, chemorangotrophs, had fermentation-type metabolism, fermented carbohydrates with lactic acid, and did not produce gas.
According to the established characteristics, the culture is classified as Lactococcus lactis.
The next step was to study species belonging to 28 cultures of lactic acid bacteria and 26 cultures of bifidobacteria isolated from healthy pigs aged 1-15 days, as well as 40 cultures of lactic acid bacteria and 20 cultures of bifidobacteria from pigs aged 30-120 days (Table 4). The species composition of microorganisms of two different age groups of piglets revealed some differences. Thus, heteroenzymatic species of lactobacilli (L. fermentum, L. brevis, L. delbrueckii) were found only in 1-15 daily piglets, accounting for 35.71% (10) of the total number of identified microorganisms. L. casei var. rhamnosus, and L. plantarum, as well as Lactococcus lactis were isolated from pigs of both groups.
Significant differences in the species composition of microorganisms isolated from pigs of different ages were found among bifidobacteria. Among Bifidobacterium spp. only piglets 1-15 days old are characterized by the presence of B. bifidum, B. infantis, and B. suis, accounting for 76.92% (20) of the total number of detected bifidobacteria, and for animals 30-120 days old -B. lactentis and B. longum, which is 60.0%, respectively (12). B. adolescentis was a common culture found in piglets of both age groups. Thus, among the lactic acid bacteria, the most common and versatile species for all age groups of piglets was L. casei var. rhamnosus, and among bifidobacteria -B. adolescentis.
Our view is in line with the opinion of other researchers (Asmahan, 2011;Aloisio et al., 2012), which indicate that most often probiotics include microorganisms of the genus Lactobacillus and Bifidobacterium, which have variability in antagonistic activity.
Therefore, it was among these microorganisms that the most effective probiotic strains of microorganisms were searched.
According to the literature, lactobacilli of the genus Lactobacillus belong to the group of gram-positive asparagine sticks of the usual form (Felis & Dellaglio, 2007;. Lactobacilli grown on MRS-2 medium had a brushlike growth colonies in the middle and lower part of the tube, small transparent colonies were formed on MRS-4 agar medium and hydrolyzed milk agar. Colonies on agar medium convex, with entire edges, opaque, not pigmented (Hor et al., 2019). According to cultural and morphological characteristics, the cultures of lactobacilli selected from samples of biological material from farm animals were assigned to L. plantarum, L. casei, L. acidophilus, L. fermentum, L. delbrueckii, L. brevis. Microorganisms Bifidobacterium spp. belong to the group of gram-positive non-spore-forming sticks of irregular shape (Cheikhyoussef et al., 2008). Bifidobacteria grown on Blaurok medium have stem growth in the middle of the tube. In smears, the sticks are very variable in shape, 0.5-1.3 x 1.5-8 microns in size, slightly curved. Cells arranged individually, in pairs, in a V-shape, sometimes by a chain, palisade or slingshot, also have swollen coccyx forms (Margolles et al., 2003 Prospects for further studies include the selection of lactic acid bacteria to create probiotic drugs for the prevention and treatment of dysbiosis in farm animals.

Conclusion
In