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A promising area for improving probiotics is the search for new sources of strains and the development of complex preparations which would include different types of bacterial cultures that complement each other. Sources of selection may be traditional dairy products, in particular, cheeses made from raw milk. Wild strains can be endowed with antibacterial properties. The antagonistic action of lactic acid bacteria (LAB) has long attracted the attention of researchers and scientists. The aim of the study was to investigate the antagonistic activity against pathogenic and opportunistic microorganisms of LAB strains isolated from traditional Carpathian cheese. Three samples of cheese were selected for the research – one sample of brynza and budz (brynza before salting), made in the highlands of the Carpathians, and one sample of budz, made in the foothills. LAB were identified using classic microbiological and modern molecular genetic methods (RAPD-PCR, RFLP-PCR, 16S rRNA gene sequencing). The objects of our studies were five strains of LAB: Lactococcus lactis IMAU32258, L. garvieae JB2826472, Enterococcus durans FMA8, E. faecium L3-23, E. faecium IMAU9421. Technological parameters such as acid-forming activity of milk fermentation, resistance to high concentrations of NaCl and temperature optimums of cultivation were taken as the main criteria for assessing the suitability of LAB for inclusion in fermentation preparations. Antagonistic activity was determined by agar diffusion (agar well method) and optic density of test cultures using a Multiscan FC microplate reader (Thermo scientifiс, USA) at the wave of 620 nm. There were four reference strains of pathogenic and opportunistic microorganisms were test cultures: Listeria monocytogenes PCM 2191, Staphylococcus aureus PCM 458, Escherichia coli PCM 2208, Salmonella typhimurium PCM 2182. Strains of the test cultures were received from the collection of microorganisms of the Institute of Biology and Biotechnology the (University of Rzeszów, Poland). According to the ability of LAB strains to form lactic acid, L. lactis IMAU32258 was the best acid-forming agent with an acid-forming energy of 94 °T. E. faecium was characterized by moderate levels of active and titratable acidity. Less pronounced acid-forming ability was determined for the species E. durans and L. garvieae. Cultures of the genus E. faecium, L. garvieae and E. durans were the most resistant to high concentrations of NaCl (6.5%). Regarding temperature optimums, we found that strains of E. faecium and E. durans species grew both at temperatures of 10, 15 and 45 °C, whereas no growth of L. lactis IMAU32258 and L. garvieae JB282647 2 was observed at 45 °C. Among the studied bacteria, the strains of E. durans FMA8 and E. faecium L3-23 were characterized by the highest antagonistic activity in producing the largest zones of growth inhibition and optic density of pathogenic and opportunistic microorganisms. The strain L. garvieae JB282647 2 exhibited the lowest level of antagonistic activity against pathogenic and opportunistic microorganisms.

The article presents the results of determining the ability of enterococci extracted from traditional Carpathian cheese bryndza to produce biologically active substances, in particular, amino acids, B vitamins and cations (ammonium, potassium, sodium, magnesium, calcium). It was found that the studied strains of enterococci in different quantities synthesized both essential and essential amino acids. Thus, the essential amino acid lysine was found in the cultivation of strains of E. durans SB18, E. durans SB20, in particular, its concentration was significantly increased by 15.6 and 10.4 %, respectively (P < 0.05) compared to the control. A probable increase in the essential amino acid histidine by 20 and 53.3 % (P < 0.05) was detected in the cultivation of only E. faecium SB12 and E. durans SB18. In addition, it was found a probable increase in threonine synthesis by enterococci E. durans SB6 and E. durans SB18, respectively – 33.3 and 39.6 % (P < 0.05). The replacement amino acid serine was able to synthesize strains of E. faecium SB12, E. durans SB18 and E. durans SB20, while its concentration increased by 40.0 (P < 0.001), 30.0 and 35.0 %, respectively < 0.01), and strains of E. durans, SB6, and E. durans SB18 synthesized glycine, the concentration of which increased by – 10.2 and 16.2 %, respectively (P < 0.01). In addition, it was found that the studied strains in small quantities synthesized B vitamins, or not synthesized at all. In all experimental samples the most vitamin B1 was detected, its concentration increased from 8.5 to 10.0 times (P < 0.001). Riboflavin was synthesized by three strains of enterococci – E. durans SB6, E. durans SB18, E. durans SB20, so the concentration of vitamin B2 probably increased, respectively, 4.1, 2.0 and 2.0 times (P < 0.05). Enterococci E. durans SB6, E. faecium SB12, E. durans SB18 and E. durans SB20 synthesized in significant quantities only vitamin B3, in particular, its concentration probably increased by 1.5, 1.5 (P < 0.05), respectively, 1.5 (P < 0.01) and 1.6 (P < 0.001) times, and vitamin B5 was produced by E. faecium SB12, E. durans SB18 and E. durans SB20, the concentration of nicotinic acid increased, respectively, 2.9 (P < 0.05), 8.4 and 9.5 (P < 0.001) times. Analysis of the macroelement composition of the supernatant of enterococci showed that strains of E. durans, SB6, E. faecium SB12, E. durans SB18 and E. durans SB20 are able to produce only Calcium, in particular, found a probable increase, respectively, in 1.8, 2.4, 1.6 and 1.4 times (P < 0.05).

The article presents the results of determining the ability of enterococci isolated from traditional Carpathian cheese to affect the antibiotic sensitivity of opportunistic gram-positive and gram-negative microorganisms, particularly test strains of S. aureus and E. coli. It was found that enterococci had different effects on the sensitivity of opportunistic pathogens to different groups of antimicrobial drugs. In particular, co-cultivation of S. aureus with enterococci did not affect the change in sensitivity to amoxicillin and glycopeptides (vancomycin). In the study of aminoglycosides, it was found that the sensitivity of staphylococcus to gentamicin increased when co-cultured with strains of E. faecium SB12 and E. durans SB18 by 14.8 (P < 0.05) and 13.1 % (P < 0.01), respectively. The tendency to increase the sensitivity of staphylococcus to streptomycin was also observed in cultivation with E. faecium SB12 and E. durans SB18. Concerning macrolides, in particular erythromycin, only one strain of E. durans SB18 caused a 3.2 % increase in staphylococcal susceptibility, and a probable increase in staphylococcal susceptibility was found in fluoroquinolones. In particular, the sensitivity of staphylococci to enrofloxacin and norfloxacin increased by 7.6 and 5.3 %, respectively (P < 0.05). The antibiotic susceptibility of E. coli with enterococci found a slight increase in sensitivity to penicillins when co-cultured with strains of E. faecium SB12 and E. durans SB18. Thus, the antibiotic sensitivity of E. coli increased by 5.1 and 7.5 %, respectively. In addition, the sensitivity of E. coli to aminoglycosides (streptomycin) increased under co-cultivation with E. durans SB6 and SB18 strains by 7, 5, and 9.7 %, respectively (P < 0.05). In addition, when co-cultured with E. faecium SB12 and E. durans SB18 strain, the sensitivity of E. coli to enrofloxacin increased by 7.6 and 5.3 %, respectively (P < 0.05), and to norfloxacin – by 3, 8, and 4.9 %. Co-cultivation with E. durans SB6 and E. faecium SB12 strains increased the sensitivity of E. coli to ẞ-lactam antibiotics, in particular, cefazolin by 9.1 % (P < 0.05), and in co-cultivation with E. durans SB18 – at 14.1% (P < 0.01).

In order to give the product functional properties, promising for the industry strains of cultures of lactic acid bacteria L. lactis ssp. lactis IMAU 32258, Lb. plantarum KLDS 1.0728 and E. durans SB18, isolated from traditional Carpathian bryndza. It was found that under conditions of co-cultivation, the studied strains were compatible, ie did not show interspecific antagonism. Based on these probiotic strains, five compositions of L. lactis ssp. lactis IMAU 32258, Lb. plantarum KLDS 1.0728 and E. durans SB18 in the ratios,%: composition № 1 – 50:25:25; № 2 – 33:33:33; № 3 – 50:40:10; № 4 – 50:10:40 and № 5 – 70:15:15 respectively. When determining the rate of biomass accumulation, it was found that at a temperature of 32 ° C the highest growth intensity was registered in the cultivation of compositions № 1, № 2 and № 4, it exceeded the control by 19.5–20.1 times (P <0.001). At a temperature of 37 °C for 24 h, the highest change in the optical density of the nutrient medium was found in the cultivation of compositions № 1, № 2 and № 3, in particular, it increased, respectively, in 17.9, 17.8 and 18.1 (P < 0.001) times compared to control. When determining the ability of different compositions of the consortium of lactic acid bacteria to reduce the pH of the medium, it was found that for 24 h of cultivation at 32 ° C the best acid-forming properties showed compositions № 3, № 4 and № 5 because they reduced the pH of the medium by 32.8, 33.3 and 32.8 %, respectively, to 4.87–4.91 units. At a temperature of 37 °C formed by us compositions were able to reduce the pH of the medium to 5.03-5.12 units, while the best acid-forming properties showed compositions № 1, № 3 and № 5. In determining the antagonistic properties of the studied compositions of microorganisms when cultured at temperature 32 °C was found to have moderate antagonistic activity against Escherichia coli, Enterobacter aerogenes, Proteus mirabilis, Salmonella enteritidis, Staphylococcus aureus and Pseudomonas aeruginosa, ie against all cultures of pathogenic and opportunistic microorganisms. Compared with other compositions, composition № 3 showed the highest antagonistic activity against Staphylococcus aureus, as well as quite high against Salmonella enteritidis, Pseudomonas aeruginosa and Enterobacter aerogenes. When culturing the studied compositions of a consortium of microorganisms at a temperature of 37 °C, all compositions showed moderate antagonistic activity against all test cultures of pathogenic and opportunistic microorganisms. Composition № 3 showed slightly higher antagonistic activity against Staphylococcus aureus, Salmonella enteritidis, Enterobacter aerogenes, as well as at a temperature of 32 °C, as well as against Pseudomonas aeruginosa. Therefore, isolated from traditional Carpathian cheese L. lactis ssp. lactis IMAU 32258, Lb. plantarum KLDS 1.0728 and E. durans SB18 have a high degree of biocompatibility and are suitable in different ratios for use in one composition, as indicated by the ability to accumulate biomass, lower pH and antagonistic activity to pathogenic and opportunistic microflora.