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.

Although fresh whey cheeses are prone to rapid deterioration, mainly by psychrotrophic Gram-negative bacteria and lactic acid bacteria (LAB), data on the specific spoilage species in traditional Greek whey cheeses are scarce. Therefore, this study quantified growth and characterized the primary spoilage bacteria in fresh Anthotyros whey cheeses stored at 4 °C in a vacuum for 40 days, without or with an added 5% (v/w) of an enterocin A-B-P crude extract (CEntE). Psychrotrophic Pseudomonas spp., Aeromonas spp., Hafnia spp. and Serratia spp. grew faster than LAB during early storage. However, LAB outgrew the Gram-negative bacteria and prevailed by mid to late storage in all cheese batches, causing a strong or milder batch-dependent natural acidification. Two major non-slime-producing and two minor biotypes of Leuconostoc-like bacteria, all identified as Leuconostoc mesenteroides by 16S rRNA sequencing, dominated the LAB association (76.7%), which also included four subdominant Carnobacterium maltaromaticum biotypes (10.9%), one Leuconostoc lactis biotype (3.3%) and few Lactococcus (1.6%), mesophilic Lactobacillus (0.8%) and Enterococcus (0.8%). Growth and distribution of LAB and Gram-negative species were strongly batch-dependent and plant-dependent. The CEntE neither retarded growth nor altered the whey cheese spoilage association but enhanced LAB growth and the declines of Gram-negative bacteria by late storage.

The ability of the enterocin-A-B-P-producing Enterococcus faecium KE82 adjunct strain to inactivate Listeria monocytogenes during Galotyri PDO cheese processing was evaluated. Three artisan cheese trials from traditionally ‘boiled’ (85oC) ewe’s milk were processed. The milk cooled at 42oC was divided in two parts: A1 was inoculated with Streptococcus thermophilus ST1 and Lactococcus lactis subsp. cremoris M78, and A2 with the basic starter ST1+M78 plus the KE82 adjunct (step 1). All milks were fermented at 20-22oC for 24 h (step 2); the curds were drained at 12oC for 72 h (step 3) and then salted with 1.5-1.8% salt to obtain the fresh Galotyri cheeses (step 4), which were ripened at 4oC for 30 days (step 5). Because an artificial listerial contamination in the dairy plant was prohibited, A1 and A2 cheese milk (200-mL) or curd (200-g) portions were taken after steps 1 to 5, inoculated (3-4 log CFU/mL or g) with L. monocytogenes no.10, incubated at 37, 22, 12, and 4oC for predefined periods, and analyzed microbiologically and for pH. L. monocytogenes declined without growth in all cheese curd portions contaminated after steps 2 to 5 (pH 4.36 to 4.84), when stored at 4 or 12oC for 15 days. The final net reductions of Listeria populations were by 2.00, 1.07, 0.54 and 0.61 log units higher in the A2 than A1 curd portions after steps 2, 3, 4 and 5, respectively. As regards step 1 conducted in simulation of the whole cheese milk fermentation process, L. monocytogenes declined by 1.47 log units more in the A2 than A1 milk portions after 72 h at 22oC; however, a slight (0.6-log) growth was preceded during the first 6 h at 37oC. In conclusion, E. faecium KE82 showed growth compatibility with the starter and enhanced inactivation of L. monocytogenes across Galotyri cheese processing. Combined acid-enterocin antilisterial effects were the weakest in the fermenting milks, turned to the strongest in the unsalted fermented curds, and reduced in the salted fresh cheeses.

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