Pinto Ana Luísa, Fernandes Melissa, Pinto Cristina, Albano Helena, Castilho Fernanda, Teixeira Paula, Gibbs Paul A
Escola Superior de Biotecnologia, Universidade Católica Portuguesa, R Dr António Bernardino de Almeida, Porto, Portugal.
Int J Food Microbiol. 2009 Jan 31;129(1):50-8. doi: 10.1016/j.ijfoodmicro.2008.11.005. Epub 2008 Nov 14.
This work had as main objectives to characterize two bacteriocins produced by lactic acid bacteria (LAB) previously isolated from non-fermented seafood, in order to evaluate their potential as new food protective agents. The two bacteriocinogenic isolates were identified by Polymerase Chain Reaction (PCR) using genus- and species-specific primers, and confirmed by 16S rDNA sequencing, as Enterococcus faecium and Pediococcus pentosaceus. The antimicrobial spectrum of each strain included several indicator microorganisms, some of them also isolated from seafood. Growth of Listeria innocua, L. monocytogenes, Staphylococcus aureus, Bacillus cereus and other LAB species were inhibited, although no inhibition of Gram-negative microorganisms was observed. Proteolytic, but not lipolytic or glycolytic enzymes, completely inactivated the antimicrobial effect of both cell-free supernatants confirming the proteinaceous nature of the inhibitors. The antimicrobial activity was maintained after treatment with NaCl, SDS, Triton X-100, Tween 20, Tween 80 and EDTA after 2 h or 5 h of exposure and both bacteriocins were stable over a wide range of pH and temperatures. Production of bacteriocin by E. faecium (bacALP7) was detected initially at exponential phase and reached a maximum activity of 25,600 AU/ml in the early stationary phase, whereas bacteriocin production by P. pentosaceus ALP57 (bacALP57) reached the maximum at exponential phase with 12,800 AU/ml. The bacteriocins did not kill L. monocytogenes ESB54 nor L. innocua 2030c however, cellular growth was reduced. The partially purified bacteriocins, bacALP7 and bacALP57, were below 6.5 kDa in size as determined by Tricine-SDS gel electrophoresis. E. faecium and P. pentosaceus contained DNA fragments corresponding in size to those recorded for enterocin B and pediocin PA-1, respectively. Sequencing of the fragments from both bacteriocins confirmed the homology. To our knowledge, for the first time two LAB producing bacteriocins similar to pediocin PA-1 and enterocin B, were isolated from non-fermented shellfish. The adaptation of the cultures to seafood matrices may be advantageous in terms of application as a biopreservation strategy for reduction of L. monocytogenes levels in seafood products.
这项工作的主要目标是对先前从非发酵海产品中分离出的乳酸菌（LAB）产生的两种细菌素进行表征，以评估它们作为新型食品保护剂的潜力。使用属特异性和种特异性引物通过聚合酶链反应（PCR）对两种产细菌素的分离株进行鉴定，并通过16S rDNA测序确认，它们分别为粪肠球菌和戊糖片球菌。每种菌株的抗菌谱包括几种指示微生物，其中一些也从海产品中分离得到。无害李斯特菌、单核细胞增生李斯特菌、金黄色葡萄球菌、蜡样芽孢杆菌和其他LAB菌种的生长受到抑制，尽管未观察到对革兰氏阴性微生物的抑制作用。蛋白水解酶而非脂解酶或糖解酶完全消除了两种无细胞上清液的抗菌作用，证实了抑制剂的蛋白质性质。在分别暴露2小时或5小时后，用NaCl、SDS、Triton X-100、吐温20、吐温80和EDTA处理后，抗菌活性得以维持，并且两种细菌素在很宽的pH和温度范围内都很稳定。粪肠球菌（bacALP7）产生细菌素最初在指数期被检测到，并在早期稳定期达到最大活性25600 AU/ml，而戊糖片球菌ALP57（bacALP57）产生细菌素在指数期达到最大值，为12800 AU/ml。然而，这些细菌素并未杀死单核细胞增生李斯特菌ESB54和无害李斯特菌2030c，只是细胞生长受到了抑制。通过Tricine-SDS凝胶电泳测定，部分纯化的细菌素bacALP7和bacALP57的大小均低于6.5 kDa。粪肠球菌和戊糖片球菌分别含有大小与肠球菌素B和片球菌素PA-1记录相对应的DNA片段。两种细菌素片段的测序证实了同源性。据我们所知，首次从非发酵贝类中分离出两种产生与片球菌素PA-1和肠球菌素B相似细菌素的LAB。就作为降低海产品中单核细胞增生李斯特菌水平的生物保存策略的应用而言，使培养物适应海产品基质可能具有优势。
