Bacteriocin-Producing Strain Lactiplantibacillus plantarum LP17L/1 Isolated from Traditional Stored Ewe's Milk Cheese and Its Beneficial Potential.

Stored ewe's milk lump cheese is a local product that can be a source of autochthonous beneficial microbiota, especially lactic acid bacteria. The aim of this study was to show the antimicrobial potential of Lactiplantibacillus plantarum LP17L/1 isolated from stored ewe's milk lump cheese. Lpb. plantarum LP17L/1 is a non-hemolytic, non-biofilm-forming strain, susceptible to antibiotics. It contains genes for 10 bacteriocins-plantaricins and exerted active bacteriocin with in vitro anti-staphylococcal and anti-listerial effect. It does not produce damaging enzymes, but it produces ß-galactosidase. It also sufficiently survives in Balb/c mice without side effects which indicate its safety. Moreover, a reduction in coliforms in mice jejunum was noted. LP17L/1 is supposed to be a promising additive for Slovak local dairy products.

Slovak Local Ewe's Milk Lump Cheese, a Source of Beneficial Enterococcus durans Strain.

Slovak ewe's milk lump cheese is produced from unpasteurized ewe's milk without any added culture. Because of the traditional processing and shaping by hand into a lump, this cheese was given the traditional specialty guaranteed (TSG) label. Up till now, there have existed only limited detailed studies of individual microbiota and their benefits in ewe's milk lump cheese. Therefore, this study has been focused on the beneficial properties and safety of Enterococcus durans strains with the aim to contribute to basic dairy microbiology but also for further application potential and strategy. The total enterococcal count in cheeses reached 3.93 CFU/g (log 10) ± 1.98 on average. Based on a MALDI-TOF mass spectrometry evaluation, the strains were allotted to the species E. durans (score, 1.781-2.245). The strains were gelatinase and hemolysis-negative (γ-hemolysis) and were mostly susceptible to commercial antibiotics. Among the strains, E. durans ED26E/7 produced the highest value of lactase enzyme ß-galactosidase (10 nmoL). ED26E/7 was absent of virulence factor genes such as Hyl (hyaluronidase), IS 16 element and gelatinase (GelE). To test safety, ED26E/7 did not cause mortality in Balb/c mice. Its partially purified bacteriocin substance showed the highest inhibition activity/bioactivity against Gram-positive indicator bacteria: the principal indicator Enterococcus avium EA5 (102,400 AU/mL), Staphylococcus aureus SA5 and listeriae (25,600 AU/mL). Moreover, 16 staphylococci (out of 22) were inhibited (100 AU/mL), and the growth of 36 (out of 51) enterococcal indicators was as well. After further technological tests, E. durans ED26E/7, with its bacteriocin substance, can be supposed as a promising additive to dairy products.

Susceptibility to Bacteriocins in Biofilm-Forming, Variable Staphylococci Isolated from Local Slovak Ewes' Milk Lump Cheeses.

Seventeen staphylococci isolated from 54 Slovak local lump cheeses made from ewes' milk were taxonomically allotted to five species and three clusters/groups involving the following species: Staphylococcus aureus (5 strains), Staphylococcus xylosus (3 strains), Staphylococcus equorum (one strain) Staphylococcus succinus (5 strains) and Staphylococcus simulans (3 strains). Five different species were determined. The aim of the study follows two lines: basic research in connection with staphylococci, and further possible application of the bacteriocins. Identified staphylococci were mostly susceptible to antibiotics (10 out of 14 antibiotics). Strains showed γ-hemolysis (meaning they did not form hemolysis) except for S. aureus SAOS1/1 strain, which formed ß-hemolysis. S. aureus SAOS1/1 strain was also DNase positive as did S. aureus SAOS5/2 and SAOS51/3. The other staphylococci were DNase negative. S. aureus SAOS1/1 and SAOS51/3 showed biofilm formation on Congo red agar. However, using quantitative plate assay, 12 strains out of 17 showed low-grade biofilm formation (0.1 ≤ A570 < 1), while five strains did not form biofilm (A570 < 0.1). The growth of all strains, including those strains resistant to enterocins, was inhibited by nisin and gallidermin, with high inhibition activity resulting in the inhibition zone in size from 1600 up to 102,400 AU/mL (arbitrary unit per milliliter). This study contributes to microbiota colonization associated with raw ewe's milk lump cheeses; it also indicates bacteriocin treatment benefit, which can be used in prevention and/or elimination of staphylococci.

Some safety aspects of enterococci isolated from Slovak lactic acid dairy product "zincica".

In Slovakia, dairy products made from ewes' milk have a long tradition. These products include the lactic acid product called "zincica" which is a by-product occurring during the preparation of ewes' lump cheese. There is no information in the literature regarding the special properties of the microbiota, especially lactic acid Firmicutes, which can survive in "zincica." From the safety aspect, enterococci are a controversial group of bacteria, and those from "zincica" have never been tested for their properties. The "zincica" used in our study was supplied by several different agrofarms producing ewes' lump cheese in central Slovakia. The species Enterococcus faecium (strains EF30E1, EF32E1, EF34E1, EF34E5) and Enterococcus faecalis (strains EE30E4, EE35E1, E31E2, altogether 7) were detected in samples from "zincica" identified using MALDI-TOF spectrometry with secure genus identification/probable species identification and then confirmed by means of PCR. Enterococci were hemolysis-negative and the genes of the typical enterococcal virulence factors were mostly absent; the gelE gene was found in two E. faecium strains (EF30E1 and EF32E1), the agg gene was detected in E. faecalis EE35E1, and the esp gene was found in two E. faecalis strains (EE30E4 and EE31E2). No strains harbored the cytolysin A gene. Biofilm formation was detected in four strains (EF30E1, EF32E1, EF34E1, and EF34E5), indicating highly positive and low-grade positive biofilm formation. Enterococci were mostly susceptible to antibiotics tested for their phenotype. This is the first study to analyze enterococci in "zincica."

Isolation and characterization of bifidobacteria from ovine cheese.

Animal products are one of the niches of bifidobacteria, a fact probably attributable to secondary contamination. In this study, 2 species of the genus Bifidobacterium were isolated by culture-dependent methods from ovine cheeses that were made from unpasteurized milk without addition of starter cultures. The isolates were identified as Bifidobacterium crudilactis and Bifidobacterium animalis subsp. lactis using matrix-assisted laser desorption/ionization time-of-flight analysis and sequencing of phylogenetic markers (16S rRNA, hsp60, and fusA).

Genome Sequence of Lactobacillus plantarum 19L3, a Strain Proposed as a Starter Culture for Slovenska Bryndza Ovine Cheese.

The genome sequence of Lactobacillus plantarum isolated from ovine cheese is presented here. This bacterium is proposed as a starter strain, named 19L3, for Slovenská bryndza cheese, a traditional Slovak cheese fulfilling European Food Safety Authority (EFSA) requirements.