Bryndza is a traditional Slovak natural white spreadable ripened cheese with Protected geographical indication status (Commission Regulation (EC) No. 676/2008). Bryndza is made from ewes' or a mixture of ewes' and cows' milk and is a rich source of protein, vitamins, and minerals (Toth et al., 2016). Bryndza is a dairy product that naturally contains a broad spectrum of microorganisms that has crucial importance on cheese properties, flavor, and aroma. Microorganisms are present during the whole process of cheese production, they are important in coagulation and ripening (Andrade et al., 2008; Tilocca et al., 2020). Also, beneficial strains help to inhibit the growth of the pathogens and reduce the spoilage of the dairy products (Arqués et al., 2015).

In previous research Gram-positive, Gram-negative bacteria, and yeasts were found and identified in bryndza (Kačániová et al., 2019). The dominant group of bacteria in bryndza was lactic acid bacteria (LAB), mainly Lactobacillus species (Kačániová et al., 2020). Lactococcus, Pediococcus, Enterococcus, Streptococcus were abundant in bryndza from different Slovak regions also (Berta et al., 2009; Šaková et al., 2015; Sádecká et al., 2019). The probiotic properties of bacteria isolated from bryndza were observed in Lactobacillus plantarum, Enterococcus faecium, and Enterococcus faecalis strains. Researches claimed that these potentially probiotic strains can inhibit the growth of pathogenic bacteria, and some of them can survive in the acidic gastrointestinal environment, which is necessary for reaching the intestine of the host (Belicová et al., 2011; Belicová et al., 2013).

Probiotic bacteria as an important part of intestinal microbiota helps regulate the immune responses, relieve the gastrointestinal tract dysfunction, alleviate the allergies, or lower cholesterol levels (Dicks and Botes, 2010; Plaza-Diaz et al., 2019). Moreover, the anti-carcinogenic properties of probiotic bacteria have been described (Zhong et al., 2014).

Our study aimed to evaluate lactic acid bacteria isolated from ewes´ bryndza and select the most active probiotic bacterial strain against pathogens and opportunistic pathogens.

LAB antimicrobial products, such as bacteriocins, are very important in bioconservation of various foods. The diverse use of LAB bacteriocins, individually or as biopreservative combinations, may help to improve food safety, especially of traditional products (Jamuna and Jeevaratnam, 2004; Mojgani and Amimia, 2007).

In our study 130, lactic acid bacteria isolated from ewes´ cheese bryndza (Table 1) were tested for antimicrobial activity and antagonistic effect against pathogenic gram-positive and Gram-negative bacteria.

In agar test, lactic acid bacteria isolated from ewes´ cheese bryndza demonstrated different antimicrobial activity with the inhibition the zone ranged from ˂1 to ˃5 mm. Altogether, 92% of LAB showed activity against Yersinia enterocolitica, 91% against Klebsiella pneumoniae, 88% against Escherichia coli, 84% against Listeria monocytogenes. Antimicrobial activity lower than 84% was observed against Salmonella enterica subsp. enterica, Pseudomonas aeruginosa, Bacillus thuringiensis, Micrococcus luteus, Staphylococcus aureus subsp. aureus, and Streptococcus pneumoniae (Table 2). Belicová et al. (2013) tested 125 acid resistant presumptive lactobacilli isolated from bryndza against Listeria monocytogenes CCM 4699, Staphylococcus lentus CCM 3472, Acinetobacter calcoaceticus CCM 4503, Sphingomonas paucimobilis CCM 3293, and Salmonella enterica subsp. enterica, serovar Typhimurium.

LAB produces metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, ethanol, diacetyl, acetaldehyde, acetoin, carbon dioxide, and bacteriocins that were classified as antimicrobial agents. Moreover, the production of organic acids resulted in low pH, which also inhibits the activity of pathogenic microorganisms (Ponce et al., 2008; Šušković et al., 2010). Antibacterial activity of organic acids and bacteriocins was confirmed against various pathogenic Gram-positive and Gram-negative microorganisms (Maragkoudakis et al., 2009).

A total of 20 LAB isolates showed strong inhibition zones (more than 5 mm) against P. aeruginosa and M. luteus. Nineteen of LAB isolates showed strong antimicrobial activity against S. aureus. Cell suspension of Lactobacillus plantarum inhibited L. monocytogenes growth (Ennahar et al., 1998). Lactococcus lactis reduced levels of L. monocytogenes in Cheddar cheese (Buyong et al., 1998). Lactococci developed by Reviriego et al. (2005) and Reviriego et al. (2007) reduced the number of L. innocua, L. monocytogenes, S. aureus, and E. coli in cheese (Rodríguez et al., 2005). The intermediate inhibitory effect against Gram-negative and Gram-positive bacteria (1 – 5 mm) is shown in Figure 1. The most effective strains were Lactobacillus paracasei ssp. paracasei against all tested pathogens. This bacteria exhibited inhibitory activity against all 10 bacteria tested with 2 to 8 isolated were active against particular bacteria.

In total, inhibitory activity was expressed 64 times by 10 L. paracasei spp. paracasei isolates.

The most effective bacterial strain was L. lactis subsp. lactis against followed by L. paraplantarum against S. enterica subsp. enterica. The lowest antagonistic effect of lactic acid bacteria was found in Enterococcus hirae against Bacillus thuringiensis (Table 3).

Foodborne pathogens have become an important social topic and have received much attention from consumers and food safety regulatory agencies around the world because of frequent foodborne outbreaks. In previous studies, LAB showed a wide range of antimicrobial effects against many foodborne pathogens (Soerjadi et al., 1981; Ennahar and Deschamps, 2000; Messens and De Vuyst, 2002; Dodd, 2012). Studies of interaction between LAB and L. monocytogenes in various media and with various LAB strains have been performed, and in all cases LAB inhibited the growth of L. monocytogenes (Tharrington and Sorrells, 1992; Zhang et al., 2011; Zhu et al., 2014).

However, Kao and Frazier (1966) obtained a mixed result when LAB was cocultured with S. aureus. Many researchers have found that LAB can inhibit Salmonella (Keersmaecker et al., 2006; Zhang et al., 2016; Yang et al., 2017) and that the bacteriostatic substances produced by LAB are thermally stable. The research focused on the inhibition of E. coli by LAB is more extensive compared to inhibition of the other bacteria. Du et al. (2016) found three strains of Lactobacillus acidophilus which could inhibit E. coli ATCC 25922. Other investigations on E. coli O157:H7 proliferation control showed that LAB could effectively inhibit the growth of E. coli O157:H7 (Brashears et al., 1998; Fooladi et al., 2014). Klebsiella pneumonia, Bacillus cereus, Shigella flexneri (Sharma et al., 2017), Staphylococcus epidermidis (Diepers et al., 2016), and Candida albicans (Yu et al., 2006) also can be inhibited by LAB.

In conclusion, the present study of lactic acid bacteria strains isolated from ewes´ cheese bryndza confirmed antagonistic effect against Gram-positive and Gram-negative bacteria.

In our study, the best results were found for Lactococcus lactis subsp. lactis against Salmonella enterica subsp. enterica. In vitro screening of LAB from Slovak bryndza ewes´ cheese have good potential for use as probiotic cultures.