BRYNDZA CHEESE OF SLOVAK ORIGIN AS POTENTIAL RESOURCES OF PROBIOTIC BACTERIA

Bryndza cheese includes several predominant lactic acid bacteria. The aim of our study was the antagonistic effect of lactic acid bacteria supernatant isolated from ewes ́ cheese bryndza against ten Gram-positive and Gram-negative bacteria. Isolated strains of bacteria were obtained from bryndza which were produced in five different regions of Slovakia. Isolated strains of lactic acid bacteria were identified with mass spectrometry MALDI-TOF MS Biotyper. A total of one hundred and thirty lactic bacteria include Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Lactobacillus brevis, Lactobacillus harbinensis, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus paracasei ssp. paracasei, Lactobacillus paraplantarum, Lactobacillus suebicus, Lactococcus lactis ssp. lactis, Lactococcus lactis, and Pediococcus acidilactici were tested in this study against Gram-negative bacteria: Escherichia coli CCM 3988, Klebsiella pneumoniae CCM 2318, Salmonella enterica subsp. enterica CCM 3807, Shigella sonnei CCM 1373, Yersinia enterocolitica CCM 5671 and Gram-positive bacteria: Bacillus thuringiensis CCM 19, Enterococcus faecalis CCM 4224, Listeria monocytogenes CCM 4699, Staphylococcus aureus subsp. aureus CCM 2461, Streptococcus pneumonia CCM 4501 with agar diffusion method. Lactic acid bacteria showed activity 92% against Yersinia enterocolitica, 91% against Klebsiella pneumoniae, 88% against Escherichia coli, 84% against Listeria monocytogenes. The most effective bacteria against Gram-positive and Gram-negative bacteria tested was Lactobacillus paracasei ssp. paracasei.


INTRODUCTION
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 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 anticarcinogenic 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.

Scientific hypothesis
Bryndza isolates possess probiotic activity. LAB can inhibit antagonistic activity against pathogens.

MATERIAL AND METHODOLOGY Isolation of lactic acid bacteria
A total of 130 lactic acid bacteria were isolated from Slovak ewes´ bryndza. The bryndza samples were obtained from five producers in Slovakia. Before identification, the lactic acid bacteria colonies were subcultured on 90% of Trypton Soya agar and 10% of Main Rogosa (MRS) agar (Oxoid) at 30 °C for 18 -24 h microaerobically. One colony of each bacterial isolate was selected for screening. Subsequently, an analysis of the bacteria identification was performed using the MALDI-TOF MS Biotyper.

Antibacterial activity of LAB isolate
The culture of lactic acid bacteria after 24 h of incubation in MRS broth (Oxoid, Basingstoke, UK) was centrifuged at 5500 g for 10 min at 4 °C and 2 mL of the supernatant was used for detection of antibacterial activity.
The well diffusion assay was used. Bacteria were spread on Petri dishes with MRS agar. LAB isolates were added into 6 mm diameter wells were created into the agar. The amounts of LAB and indicator bacteria were the same (100 µl, 10 8 CFU/mL) prepared from the broth culture of bacteria according to the 0.5 McFarland standard. After 48 h incubation at 37 °C in an aerophilically chamber, the inhibition zone diameter was measured for detection of the antagonistic effect of the LAB isolate against pathogenic bacteria.

Statistical analyses
The mean and standard deviation of inhibition zones was calculated for the detection of antagonistic effect against tested Gram-positive and Gram-negative bacteria.

RESULTS AND DISCUSSION
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 grampositive 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. 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 ( 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. Volume 14 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