Artisanal cheesemaking is still performed using practices and conditions derived from tradition. Feta and Kefalograviera cheeses are very popular in Greece and have met worldwide commercial success. However, there is a lack of knowledge regarding their lactic acid microecosystem composition and species dynamics during ripening. Thus, the aim of the present study was to assess the microecosystem as well as the autochthonous lactic acid microbiota during the ripening of artisanal Feta and Kefalograviera cheeses. For that purpose, raw sheep’s milk intended for cheesemaking, as well as Feta and Kefalograviera cheeses during early and late ripening were analyzed, and the lactic acid microbiota was identified using the classical phenotypic approach, clustering with PCR-RAPD and identification with sequencing of the 16S-rRNA gene, as well as with the Biolog GEN III microplates. In addition, the functional properties of the bacterial community were evaluated using the Biolog EcoPlates, which consists of 31 different carbon sources. In general, concordance between the techniques used was achieved. The most frequently isolated species from raw sheep’s milk were Enteroroccus faecium, Lactiplantibacillus plantarum and Pediococcus pentosaceus. The microecosystem of Feta cheese in the early ripening stage was dominated by Lp. plantarum and E. faecium, whereas, in late ripening, the microecosystem was dominated by Weissella paramesenteroides. The microecosystem of Kefalograviera cheese in the early ripening stage was dominated by Levilactobacillus brevis and E. faecium, and in late ripening by W. paramesenteroides and E. faecium. Finally, Carbohydrates was the main carbon source category that metabolized by all microbial communities, but the extent of their utilization was varied. Kefalograviera samples, especially at early ripening, demonstrated higher metabolic activity compared to Feta cheese. However, dominating species within microbial communities of the cheese samples were not significantly different.
The aim of the present study was to examine 189 LAB strains belonging to the species Enterococcus faecium, E. faecalis, Lactococcus lactis, Pediococcus pentosaceus, Leuconostoc mesenteroides, Lactiplantibacillus pentosus, Latilactobacillus curvatus, Lp. plantarum, Levilactobacillus brevis, and Weissella paramesenteroides isolated form sheep milk, Feta and Kefalograviera cheeses at different ripening stages, for their technological compatibility with dairy products manufacturing, their activities that may compromise safety of the dairy products as well as their capacity to survive in the human gastrointestinal tract. For that purpose, milk acidification and coagulation capacity, caseinolytic, lipolytic, hemolytic, gelatinolytic, and bile salt hydrolase activity, production of exopolysaccharides, antimicrobial compounds, and biogenic amines, as well as acid and bile salt tolerance and antibiotic susceptibility were examined. The faster acidifying strains were Lc. lactis DRD 2658 and P. pentosaceus DRD 2657 that reduced the pH value of skim milk, within 6 h to 5.97 and 5.92, respectively. Strains able to perform weak caseinolysis were detected in all species assessed. On the contrary, lipolytic activity, production of exopolysaccharides, amino acid decarboxylation, hemolytic, gelatinase, and bile salt hydrolase activity were not detected. Variable susceptibility to the antibiotics examined was detected among LAB strains. However, in the majority of the cases, resistance was evident. None of the strains assessed, managed to survive to exposure at pH value 1. On the contrary, 25.9 and 88.9% of the strains survived after exposure at pH values 2 and 3, respectively; the reduction of the population was larger in the first case. The strains survived well after exposure to bile salts. The strain-dependent character of the properties examined was verified. Many strains, belonging to different species, have presented very interesting properties; however, further examination is needed before their potential use as starter or adjunct cultures.
Enterococci are commensal organisms that have probiotic effects for their hosts and can be used as adjunct cultures in fermented dairy products. The dark side of Enterococci is manifested in E. faecium and E. faecalis, which are the causative agents of nosocomial infections, and thus Enterococci have not been granted a safety status as food additives. In this context, we aimed to assess the safety and functional profile of an Enterococci collection retrieved from traditional dairy products through a high-resolution genomic characterization and comparative genomic analysis. Analysis did not reveal major differences in the main cellular processes of Enterococci. Moreover, a diverse repertoire of resistance and virulence genes was present, though known hallmark pathogenicity factors were either absent or occurred rarely. The abundance of bacteriocins and CRISPR/Cas systems suggested the ability of the isolates to suppress pathogens and evade bacteriophages, respectively. Presence–absence patterns of genes suggested that dairy-originated E. faecium are not associated with pathogenicity factors, while those of human origin are strongly linked with notorious resistance and virulence determinants. Our comparative analysis provided some notable insights regarding the genomic composition of Enterococci in the context of their origin. However, their pathogenic lifestyle is likely to be explained by the interplay of multiple genomic factors.
Halitzia is a traditional white-brined cheese produced by a limited number of producers in Cyprus. During a survey of the microbiome of a number of different Halitzia samples, we identified a bacterial strain that exhibited enhanced proteolytic activity compared to the other isolates. The strain was further studied, and it was assigned as Enterococcus faecalis PK23. We proceeded with sequencing of its whole genome using Illumina technology. Initial sequencing and assembly produced 116 scaffolds with a length of 3,149,036 bp. Comparison with the available E. faecalis genomes revealed that the strain PK23 exhibited high levels of identity to the genome sequence of E. faecalis isolate 26975_2#180 deposited in GenBank as a single complete contig. From the 116 scaffolds 106 could be aligned to the genome of isolate 26975_2#180 leading to a chromosomal length of 3,132,784 bp with a GC content of 37.3%. From the remaining 10 scaffolds, five showed similarity to plasmid sequences. More specifically, scaffold 54 showed high identity with most part of plasmid pEF1071 of E. faecalis strain BFE 1071, which carries the gene cluster involved in the biosynthesis of enterocins 1071A and 1071B, while scaffold 77 showed high identity with the entire sequence of the unnamed_5 cryptic plasmid of Enterococcus faecium strain PR05720-3. The other three scaffolds were only short parts of larger plasmids. The remaining five scaffolds which could not be related to any plasmid sequence most probably constitute chromosomal sequences present in strain PK23 but absent from isolate 26975_2#180. Their total length was around 2.7 kb, which does not affect the sequence of the PK23 pseudochromosome in a major way. The whole-genome sequence annotation of strain PK23 identified 3161 coding sequences and 62 RNA sequences. The results from the Rapid Annotation using Subsystem Technology (RAST) version 2.0 server indicated the presence of seven putative genes which were related to the subsystem of Protein Degradation. This dataset provides a first overview of the proteolytic and bacteriocin producing properties of E. faecalis PK23. The dataset may also be used in future experiments which could shed light on the adaptation of the strain in the dairy environment and its role in cheese production.
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