抗腐败酵母乳酸菌的筛选及特性分析-screening and characterization of anti-corruption yeast lactic acid bacteria.docxVIP

摘要随着消费者食品安全意识的提高，选择不含化学添加剂的“天然”食品将成为一种 趋势，这已使得健康安全的生物保鲜成为食品安全研究的热点。乳酸菌作为食品安全级（generally recognized as safe, GRAS）微生物，已有众多报道称其具有抑制真菌的作用， 因而成为生物防腐剂的研究重点之一。本文采用溶钙圈法，并经革兰氏染色及过氧化氢酶试验，从四川传统发酵食品中进行乳酸菌的分离筛选，得到 45 株 G+、过氧化氢酶阴性的乳酸菌株。使用双层平板法和 微量孔板法，以白色念珠菌、光滑假丝酵母等腐败酵母作为指示菌，以上述乳酸菌培养 液上清进行抑菌实验，得到 6 株具有较好抗菌作用的乳酸菌。对该 6 菌株进行生理生化试验及分子生物学鉴定。鉴定菌株 AT6 和 AT22 为食窦魏 斯氏菌（Weissella cibaria）；AT4、AT28、AT36 均为植物乳杆菌（Lactobacillus plantarum）； AT15 为屎肠球菌（Enterococcus faecium）。对具有最佳抑菌作用的菌株 AT6 的培养条件进行优化。通过单因素试验，确定对 其培养液上清抑菌率影响较为显著的因素为培养温度、培养时间和培养基初始 pH 值。 以此三因素为自变量设计响应曲面优化，结果表明在 34℃、培养基初始 pH 5.5、培养时间为40h 时，结果得抑菌率为 90.51±0.87%，较优化前提高了 16.36%。 对食窦魏斯氏菌 AT6 的抑菌特性进行初步分析发现，其抑菌效果是多种抑菌物质的协同作用产生。在相同 pH 条件下，AT6 的抑菌作用强于使用乳酸和醋酸调整的 MRS 肉汤培养基，且其抑菌效果受 pH 影响较大，但经蛋白酶处理后的变化并不显著。通过 HPLC 测得 AT6 发酵液中苯乳酸含量为 31.228±0.24μg/mL，使用 GC-MS 分析发现 AT6 菌液中有 4 种环二肽的存在，分别是 cyclo(Phe–Pro)、cyclo( Gly-Pro)、cyclo( Leu-Pro)、 cyclo(Gly-Leu)。分析 AT6 培养上清抑制腐败酵母的特性是由多种抑菌物质的综合作用引起。本研究筛选到了具有抑制腐败真菌作用的乳酸菌 6 株，其中食窦魏斯氏菌 AT6 培 养上清液对腐败酵母等抑制率可高达 90.51%，本研究为生物防腐剂、发酵菌剂等生物保鲜方法提供了候选菌株。关键词：乳酸菌；抗腐败酵母；食窦魏斯氏菌；生物保鲜AbstractWith the enhancement of food safety awareness of consumers, natural food with no chemical additives has become the trend in food consumption, which makes biopreservation the hot issue of food safty study. Various researches have reported that lactic acid bacteria (LAB), which were generally recognized as safe (GRAS), showed antifungal activities and became the potential candidates of biopreservation.Forty-six Gram positive and catalase negative LAB were isolated from several traditional fermented food in Sichuan by calcium-dissolving method. Several spoilage yeast indicators such as Canidia albicans and Canidia. glabrata were used for the screening of anti-yeast LAB by the double-plate method and microtitre plate well assay. Cell-free culture supertanents (CFSs) of the isolated LAB were collected and six of them showed anti-yeast activities.According to physiological and biochemical tests, as well as 16S rDNA sequencing analysis, isolates AT6