Autochthonous single (Ent+) or multiple (m-Ent+) enterocin-producing strains of dairy enterococci show promise for use as bioprotective adjunct cultures in traditional cheese technologies, provided they possess no pathogenic traits. This study evaluated safety, decarboxylase activity, and enzymatic (API ZYM) activity profiles of nine Ent+ or m-Ent+ Greek cheese isolates previously assigned to four distinct E. faecium (represented by the isolates KE64 (entA), GL31 (entA), KE82 (entA-entB-entP) and KE77 (entA-entB-entP-bac31)) and two E. durans (represented by the isolates KE100 (entP) and KE108 (entP-bac31-cyl)) strain genotypes. No strain was β-hemolytic or harbored vanA and vanB or the virulence genes agg, ace, espA, IS16, hyl, or gelE. All strains were of moderate to high sensitivity to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, penicillin, tetracycline, and vancomycin, except for the E. faecium KE64 and KE82 strains, which were resistant to erythromycin and penicillin. All cheese strains showed moderate to strong esterase-lipase and aminopeptidase activities and formed tyramine, but none formed histamine in vitro. In conclusion, all Ent+ or m-Ent+ strain genotypes of the E. faecium/durans group, except for the cyl-positive E. durans KE108, were safe for use as adjunct cultures in traditional Greek cheeses. Further in situ biotechnological evaluations of the strains in real cheese-making trials are required.

When developing protective starter cultures for application in cheese technologies, monitoring growth interactions between starter and adjunct LAB species and in situ expression of bacteriocin genes in the mixtures, are crucial. This study firstly aimed to monitor growth of mixed LAB strain populations during milk model fermentations by microbial counts and real-time quantitative PCR (qPCR). The primary starter Streptococcus thermophilus ST1 and costarter Lactococcus lactis subsp. cremoris M78 strains served as the basic starter composite co-inoculated in all milk treatments. Adjunct bacteriocinogenic Enterococcus faecium strains KE82 and GL31 and a ripening Lactiplantibacillus plantarum H25 strain were added separately to the starter composite resulting in four LAB combination treatments. The second aim was to quantify gene transcripts of nisin and enterocins B and A synthesized by M78, KE82 and GL31, respectively, by real-time reverse transcription PCR (RT-qPCR) and to detect the in situ antilisterial effects of the cocultures. Adjunct LAB strains showed growth compatibility with the starter, since all of them exhibited a 2 to 3 log units increase in their population levels, compared to their initial inoculation levels, with ST1 prevailing in all treatments. KE82 grew more competitively than GL31 whereas cocultures with KE82 displayed the strongest in situ antilisterial activity. Nisin gene expression levels were higher at the exponential phase of microbial growth in all treatments. Finally, expression of nisin and enterocins A and B genes was interrelated indicating an antagonistic activity.

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