Vandera Elpiniki, Lianou Alexandra, Kakouri Athanasia, Feng Jinbo, Koukkou Anna-Irini, Samelis John
Dairy Research Institute, General Directorate of Agricultural Research, Hellenic Agricultural Organization DEMETER, Katsikas, 45221 Ioannina, Greece.
Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, 45100 Ioannina, Greece.
J Food Prot. 2017 Jan;80(1):74-85. doi: 10.4315/0362-028X.JFP-16-082.
Enterococcus faecium KE82, isolated from traditional Greek Graviera cheese, was identified in pure broth cultures in vitro as a multiple enterocin-producing bacterial strain possessing the structural entA, entB, and entP enterocin genes. E. faecium KE82 was further assessed for in situ antilisterial activity in raw milk (RM) and commercially thermized milk (TM; 63°C for 30 s) in the presence of the indigenous microbiota and in sterile raw milk (SRM; 121°C for 5 min) with or without the addition of two commercial starter culture (CSC) strains Streptococcus thermophilus and Lactococcus lactis . Growth of Listeria monocytogenes was completely inhibited in RM incubated at 37°C for 6 h, whereas the pathogen was significantly inactivated in RM+KE82 samples during further incubation at 18°C for 66 h. In contrast, L. monocytogenes levels increased by approximately 2 log CFU/ml in TM, but in TM+KE82 samples, pathogen growth was retarded during the first 6 h at 37°C followed by growth cessation and partial inactivation at 18°C. After 48 to 72 h, growth of L. monocytogenes in SRM+CSC samples decreased by 4 to 5 log CFU/ml compared with the SRM control, whereas additional 10-fold decreases in the pathogen were observed in SRM+CSC+KE82 samples. Reverse transcription PCR analysis of SRM+KE82 and SRM+CSC+KE82 samples confirmed that the entA and entB genes were transcribed, but entP gene transcription was not detected. All RM and SRM samples inoculated with E. faecium KE82 displayed strong in situ inhibitory activity against L. monocytogenes in well diffusion bioassays, whereas activity was weaker to undetectable in comparable or additional TM+KE82 samples; no milk sample without E. faecium KE82 had activity against L. monocytogenes . The findings of this study indicate that E. faecium KE82 is an antilisterial agent that could be used in traditional dairy foods because it concomitantly produces enterocins A and B in situ in milk.
从希腊传统格拉维拉奶酪中分离出的屎肠球菌KE82，在体外纯肉汤培养物中被鉴定为一种产生多种肠球菌素的细菌菌株，拥有结构肠球菌素基因entA、entB和entP。屎肠球菌KE82在原料乳（RM）和商业热杀菌乳（TM；63°C处理30秒）中，在本地微生物群存在的情况下，以及在添加或不添加两种商业发酵剂菌株嗜热链球菌和乳酸乳球菌的无菌原料乳（SRM；121°C处理5分钟）中，进一步评估其原位抗李斯特菌活性。在37°C孵育6小时的RM中，单核细胞增生李斯特菌的生长被完全抑制，而在18°C进一步孵育66小时期间，RM+KE82样品中的病原体被显著灭活。相比之下，在TM中，单核细胞增生李斯特菌水平增加了约2 log CFU/ml，但在TM+KE82样品中，病原体在37°C的前6小时生长受到抑制，随后在18°C生长停止并部分灭活。48至72小时后，与SRM对照相比，SRM+CSC样品中单核细胞增生李斯特菌的生长减少了4至5 log CFU/ml，而在SRM+CSC+KE82样品中观察到病原体数量额外减少了10倍。对SRM+KE82和SRM+CSC+KE82样品的逆转录PCR分析证实entA和entB基因被转录，但未检测到entP基因的转录。所有接种屎肠球菌KE82的RM和SRM样品在扩散平板生物测定中对单核细胞增生李斯特菌显示出很强的原位抑制活性，而在可比的或额外的TM+KE82样品中活性较弱或无法检测到；没有屎肠球菌KE82的牛奶样品对单核细胞增生李斯特菌没有活性。本研究结果表明，屎肠球菌KE82是一种可用于传统乳制品的抗李斯特菌剂，因为它能在牛奶中原位产生肠球菌素A和B。
