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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.
摘要：
来自Epirus的本地小反刍动物品种的原料奶，希腊,是具有优越生物技术特性的自生乳酸菌（LAB）菌株的宝贵天然来源。在这项研究中,来自两个当地牧羊场的两个散装牛奶（RM1，RM2），用于传统的Kefalotyri奶酪生产，通过体外测试预先选择细菌素样抗李斯特菌活性。对其拮抗性LAB生物群进行定量，然后进行多相（16SrRNA基因测序；肠球菌IGS；明串珠菌的多重PCR）鉴定42个LAB（RM1/18；RM2/24）分离株，进一步评估了细菌素编码基因和主要安全性状。在两个RM中，数字上占优势的嗜温LAB的代表性分离株是肠列明串珠菌（10），RM2中的副链球菌（7）和RM1中的乳酸乳球菌（1）；主要的嗜热LAB分离株是durans肠球菌（8），E.屎肠(6)，E.粪肠(3)，E.hirae(1),E.hermanniensis（1），黄体链球菌（2），马蹄链球菌（1）和溶胆囊链球菌（1）。根据他们的rpoB，araA,dsr和sorA配置文件，六Ln.中肠菌株（8个分离株）位于中肠亚种和右旋糖酐之间，而两个菌株的轮廓为Ln。中肠亚种。jonggajibkimchi首次在希腊乳制品中报道。两个RM1屎肠球菌菌株生物型(3个分离株)表现出强烈的，由于entA/entB/entP拥有，enterocin介导的抗李斯特菌活性。来自RM1的一个E.Durans拥有entA和entP，而屎肠球菌/durans组的另外9个RM2分离株单独处理entA或entP。全部仅显示直接（细胞相关）抗物质活性，来自RM2的两种黄体链球菌菌株也表现强劲。可取的是,没有LAB分离物是β-溶血，或细胞溶素阳性，或者拥有vana,vanB用于万古霉素抗性，或者agg，espA,hyl,和IS16毒力基因。然而,来自RM2的所有三个粪肠球菌均具有gelE和/或ace毒力基因。总之,所有Ln。肠系膜菌株，两个保险箱,产肠动力素A-B-P的屎肠球菌菌株，应进一步验证两种抗叶枯病菌菌株作为凯法洛替利奶酪中潜在的辅助培养物或辅助培养物。原料乳中α-溶血性化脓性链球菌的患病率，RM2中主要是副胆碱酯酶，需要考虑绵羊的亚临床乳腺炎和整个农场卫生。
