Approaches for enhancing in situ detection of enterocin genes in thermized milk, and selective isolation of enterocin-producing Enterococcus faecium from Baird-Parker agar

Enterococci are naturally selected for growth in thermized ewes'/goats' milk mixtures used for traditional cooked hard cheese processing in Greece. A culture-independent PCR-based approach was applied to detect the presence of enterocin-encoding genes in naturally culture-enriched thermized milk (TM). Portions of TM (63 °C, 30 s) collected from a commercial cheese plant before addition of starters were fermented at 37 °C for 48 h to facilitate growth of indigenous enterococci. The multiple enterocin-producing (m-Ent+) Enterococcus faecium KE82 and the nisin A-producing Lactococcus lactis subsp. cremoris M104 served as bacteriocin-positive inocula in separate TM treatments. The PCR results revealed a constant presence of the enterocin A, B and P genes in TM fermented naturally at 37 °C. Eleven out of 42 (26.2%) lactic isolates from the enriched TM cultures without inoculation were Ent+ E. faecium assigned to three biotypes. Biotype I (4 isolates) included single entA possessors, whereas biotype II (5 isolates) and biotype III (2 isolates) were m-Ent+ variants profiling entA-entB-entP and entA-entB genes, respectively. Biotype II displayed the strongest antilisterial activity in vitro. Surprisingly, 85.7% (6/7) of the m-Ent+ E. faecium were selectively isolated from Baird-Parker agar, reflecting their natural resistance to 0.01% tellurite contained in the egg yolk supplement. No cytolysin-positive E. faecalis or other Ent+ Enterococcus spp. were isolated. In conclusion, commercially thermized Greek milk is a natural pool or 'reservoir' of antagonistic Ent+ or m-Ent+ E. faecium strains that can be easily detected and recovered by applying this PCR-based approach to naturally fermented milks or cheese products.