Antilisterial activity of raw sheep milk from two native Epirus breeds: Culture-dependent identification, bacteriocin gene detection and primary safety evaluation of the antagonistic LAB biota

Abstract

Raw milk from native small ruminant breeds in Epirus, Greece, is a valuable natural source of autochthonous lactic acid bacteria (LAB) strains with superior biotechnological properties. In this study, two bulk milks (RM1, RM2) from two local sheep yards, intended for traditional Kefalotyri cheese production, were preselected for bacteriocin-like antilisterial activity by in vitro tests. Their antagonistic LAB biota was quantified followed by polyphasic (16S rRNA gene sequencing; IGS for Enterococcus; a multiplex-PCR for Leuconostoc) identification of 42 LAB (RM1/18; RM2/24) isolates further evaluated for bacteriocin encoding genes and primary safety traits. Representative isolates of the numerically dominant mesophilic LAB were Leuconostoc mesenteroides (10) in both RMs, Streptococcus parauberis (7) in RM2, and Lactococcus lactis (1) in RM1; the subdominant thermophilic LAB isolates were Enterococcus durans (8), E. faecium (6), E. faecalis (3), E. hirae (1), E. hermanniensis (1), Streptococcus lutetiensis (2), S. equinus (1) and S. gallolyticus (1). Based on their rpoB, araA, dsr and sorA profiles, six Ln. mesenteroides strains (8 isolates) were atypical lying between the subspecies mesenteroides and dextranicum, whereas two strains profiled with Ln. mesenteroides subsp. jonggajibkimchi that is first-time reported in Greek dairy food. Two RM1 E. faecium strain biotypes (3 isolates) showed strong, enterocin-mediated antilisterial activity due to entA/entB/entP possession. One E. durans from RM1 possessed entA and entP, while additional nine RM2 isolates of the E. faecium/durans group processed entA or entP singly. All showed direct (cell-associated) antilisterial activity only, as also both S. lutetiensis strains from RM2 did strongly. Desirably, no LAB isolate was β-hemolyrtic, or cytolysin-positive, or possessed vanA, vanB for vancomycin resistance, or agg, espA, hyl, and IS16 virulence genes. However, all three E. faecalis from RM2 possessed gelE and/or ace virulence genes. In conclusion, all Ln. mesenteroides strains, the two safe, enterocin A-B-P-producing E. faecium strains, and the two antilisterial S. lutetiensis strains should be validated further as potential costarter or adjunct cultures in Kefalotyri cheese. The prevalence of α-hemolytic pyogenic streptococci in raw milk, mainly S. parauberis in RM2, requires consideration in respect to subclinical mastitis in sheep and the farm hygiene overall.

Keywords: E. faecium; Enterocin A-B-P; Leuconostoc; Native sheep breeds; Raw milk; S. parauberis.

Graphical abstract

Fig. 1

Workflow chart followed for the isolation, culture-dependent identification, bacteriocin gene detection and primary safety evaluation of 42 LAB isolates from two batches (RM1 and RM2) of fresh raw sheep milk from two native Epirus breeds which were found to retain a strong to moderate in vitro antilisterial activity after a natural fermentation/culture enrichment step at 37 °C for 48 h, without or with added 4 % salt, followed by adjustment of their pH at 6.0-6.2 and heating at 80 °C for 15 min to inactivate viable cells.

Fig. 2

Multiplex PCR profiling of ten Leuconostoc mesenteroides raw sheep milk (KFM) isolates. Lane 1: Nippon Genetics ready-to-use DNA ladder, 100 to 3000 bp fragments; Lane 2: Negative control; Lane 3: Ln. mesenteroides subsp. dextranicum ACA-DC 0231; Lane 4: Ln. mesenteroides subsp. dextranicum ACA-DC 0493; Lane 5: Ln. mesenteroides subsp. mesenteroides ACA-DC 0750; Lanes 6 – 15: Ln. mesenteroides KFM isolates; Lane 16: negative control. At 925 bp is the rpoB gene band; at 774 bp is the araA gene band; at 549 bp is the dsr gene band; at 253 bp is the sorA gene band.

Fig. 3

Species identification of raw sheep Enterococcus spp. KFM isolates using the IGS method. A) Lane 1: Nippon Genetics ready-to-use DNA ladder, 100 to 3000 bp fragments; Lane 2: E. faecium KE82; Lane 3: E. durans KE100; Lane 4: E. faecalis GL322; Lane 5: KFM6; Lane 6: KFM17; Lane 7: KFM28; Lane 8: KFM29; Lane 9: Negative control B) Lane 1: Nippon Genetics ready-to-use DNA ladder, 100 to 3000 bp fragments; Lane 2: E. faecium KE82; Lane 3: E. durans KE100; Lane 4: E. faecalis GL322; Lanes 5 - 9: KFM46 – KFM50; Lanes 10 - 13: KFM56 – KFM59; Lane 14: negative control.