【作者】 许飞利；

【导师】 刘秀梅；

【作者基本信息】 江南大学 ， 食品营养与安全， 2011， 博士

【摘要】 自从益生菌在食品工业中应用以来,它的安全性问题就一直受人们关注。尤其是当今,随着越来越多食品安全问题的出现,人们对食品安全也有了更深的认识。各国科学家也对食品中益生菌的添加更加关注,投入的研究也在逐渐增多。而在益生菌的安全性研究中,益生菌基础性的研究则至关重要,只有如此,才能从根本上确保所添加食品的安全性。本研究以三种主要的益生乳酸菌乳酸杆菌、双歧杆菌、嗜热链球菌实验室保存菌株及其酸奶分离株为研究对象,就其生化分型及鉴定、16SrRNA基因PCR扩增及序列分析、耐药情况、PFGE分型及对Hela细胞的体外粘附等方面做了较为全面的研究,以期为我国食品用益生乳酸菌的安全性溯源、监测和分析等提供可靠的理论依据,同时综合所有研究结果建立一个益生乳酸菌菌种数据库,以便更好地保证益生乳酸菌在食品中使用的安全性。一、用API50CHL生化鉴定试剂盒鉴定了实验室保存菌株:55株乳酸杆菌、46株双歧杆菌、10株嗜热链球菌,及分离株:57株乳酸杆菌、10株双歧杆菌、42株嗜热链球菌,并对其生化谱进行聚类分析。结果显示:生化鉴定保存菌株鉴定结果与厂家所给菌名不完全相符。分离株生化鉴定结果与包装袋标示菌名也不完全相符。样品中双歧杆菌的检出率较低。各菌株分型结果显示生化谱相同或相似的菌被分为一类,乳酸杆菌、双歧杆菌保存菌株由于种类多生化谱较为复杂,其余则比较单一。生化分型可能无法进行准确分型。二、设计不同菌种的引物,采用16SrRNA基因PCR扩增及序列分析方法,对乳酸杆菌、双歧杆菌及嗜热链球菌进行了鉴定和分型。结果显示:乳酸杆菌、双歧杆菌保存菌株16SrRNA鉴定结果与API50CHL鉴定结果不完全相符。分离株两种方法鉴定结果完全一致。嗜热链球菌所有菌株两种方法鉴定结果完全一致。总的来看,几种菌的种引物特异性均较好,可用于种的鉴定。该鉴定方法可大大节约实验成本。16SrRNA基因序列分型效果不理想。三、采用脉冲场凝胶电泳(pulse field gel electrophoresis,PFGE)方法,对乳酸杆菌、双歧杆菌及嗜热链球菌实验室保存菌株及酸奶分离株进行了分子分型及分析,结果显示:确定了乳酸杆菌属的主要限制性内切酶为AscI及SmaI,双歧杆菌属的主要限制性内切酶为XbaI,嗜热链球菌的主要限制性内切酶为SmaI,初步建立了三种菌种水平PFGE分型鉴定方法。根据进化树图谱分析了保存菌株与酸奶分离株的同源性关系。PFGE分子分型方法可以在基因水平很好地对益生乳酸菌进行分型,可成为判定其他鉴定方法鉴定益生乳酸菌是否准确的一个判别标准。几种鉴定方法相比而言,API50CHL生化鉴定结果可能会出现不该有的误差。16SrRNA基因PCR扩增方法鉴定结果相对稳定,而且基本可以准确地鉴定菌株到种水平,PFGE型别的分析则可以证实鉴定结果的准确性。四、采用CLSI手册推荐的肉汤微量稀释法对乳酸杆菌、双歧杆菌、嗜热链球菌实验室保存菌株及酸奶分离株进行了耐药性检测和分析。结果显示:乳酸杆菌保存菌株对16种抗生素均表现出较强的耐药性,大部分菌株为多重耐药株,分离株耐药性略低,但大部分菌株也表现出多重耐药性。保存菌株耐药范围更广。双歧杆菌保存菌株大部分对2-3种抗生素耐药,少数几株对3种以上抗生素耐药;10株分离株中3株表现为多重耐药。保存菌株耐药范围更广。嗜热链球菌10株保存菌株2株没有耐药性,其余耐1-6种抗生素;分离株大部分耐2-3种抗生素。保存菌株与分离株耐药范围无明显差别。三种菌相比,乳酸杆菌耐药性最强,嗜热链球菌次之,最后是双歧杆菌。五、采用体外粘附实验,选取子宫颈癌细胞Hela细胞为体外细胞模型,以乳酸杆菌、双歧杆菌及嗜热链球菌为有益菌,鼠伤寒沙门氏菌AMCC50333为病原菌,对这几种菌对Hela细胞的粘附作用以及对鼠伤寒沙门氏菌粘附Hela细胞的影响进行了分析,结果显示:乳酸杆菌、双歧杆菌、嗜热链球菌的保存菌株与分离株均对Hela细胞表现出粘附特性,但差异不明显。同属内不同种的粘附性不同。三种菌保存菌株与分离株均对鼠伤寒沙门氏菌粘附Hela细胞有显著的抑制作用。但三种菌的抑制性没有明显差别。被鉴定为乳酸乳球菌的菌株对Hela细胞的粘附性较强,对鼠伤寒沙门氏菌粘附Hela细胞的抑制作用也较强。综合所有分型方法来看,PFGE分子分型方法是最佳的分型方法,其次为16SrRNA序列分型、生化分型。通过一系列的实验,建立了我国食品工业中常用益生乳酸菌菌种数据库。更多还原

【Abstract】 Since probiotic strains are used in the food industry its security problem has been attended. Especially today, as more and more food safety problems appeared, people have deep comprehension on food security. Scientists from various countries also focus on addition of probiotic to foods, and more research is gradually increasing. In the study of probiotic safety the basic research is essential, and only in this way can the safety of probiotic food be ensured.In this study, three main lactic acid bacteria strains, as follows, Lactobacillus, Bifidobac- terium, and Streptococcus thermophilus strains the laboratory saved and the isolates of yogurt were studied, and analyzed the biological classification and identification, 16SrRNA gene PCR amplification and sequence analysis, and resistance to antibiotics, and PFGE typing, and adhesion on Hela cells in vitro in order to provide a reliable theoretical basis of tracing to the source, and monitoring, and analyzing, while consolidating all results of probiotic bacteria to establish a database to better monitor the use of lactic acid bacteria in food safety.Firstly, the laboratory storage isolates: 55 Lactobacillus strains, 46 Bifidobacterium strains, and 10 Streptococcus thermophilus strains, and 57 Lactobacillus isolates, 10 Bifidobacterium isolates, 42 Streptococcus thermophilus isolates were identified with API50CHL, and we analyzed their biochemical spectrum by clustering. The results showed that:The results of biochemical identification of the storage strains did not exactly match with the one of the given name of the manufacturers. Biochemical identification of isolates were not entirely consistent with the names marked on the bags. Detection rate of Bifidobacterium isolates of samples was lower. The typing results of strains showed that the same or similar biochemical spectrum is divided into a class of bacteria, and the biochemical spectrum of Lactobacillus strains and Bifidobacterium strains were more complex because of they had many species. The rest were relatively simple. Biological classification can not be accurately classified.Secondly, different primers of the strains were designed, and then all the strains were identified and classified by 16SrRNA gene PCR amplification and sequence analysis. The results showed that:16SrRNA identification results of the storage strains are not entirely consistent with the results of API50CHL identification. Both methods have the same results of isolates. All Streptococcus thermophilus strains have the same identification results from the two ways. From the whole, the specificity of the primers was good, and these primers were available to identify species. The identification method can significantly save the cost. 16SrRNA gene sequence typing results were not ideal.Thirdly, Lactobacillus, Bifidobacterium and Streptococcus thermophilus storage strains and yoghurt isolates were genotyped with PFGE, and the results showed that:AscI and SmaI were selected as the major restriction enzyme of Lactobacillus genus, and XbaI was the major restriction enzyme of Bifidobacterium genus, SmaI was the major restriction enzyme of Streptococcus thermophilus. The PFGE genotyping methods of these strains were established. The homology relationship of the storage strains and isolates was analyzed according to the profile of genetic relationship. Lactic acid bacteria strains were typed by PFGE molecular typing method at the gene leve, and we could determine the accuracy of other identification methods of probiotics according to the PFGE method.Comparing with the several identification methods, API50CHL biochemical identifica- tion results may cause errors which should not exist. The method of 16SrRNA gene PCR amplification was relatively stable. And this method could identify the strains at spieces level. PFGE type analysis can confirm the accuracy of identification results.Fourth, the drug resistance of the all the strains was detected by broth microdilution method according to the manual of CLSI. The results showed that:The Lactobacillus storage strains to 16 antibiotics all showed strong resistance, and most of the strains were multiple drug-resistant strains, and the resistance to isolates was slightly lower, but most of the strains also showed multiple drug resistance. The storage strains showed wider resistance range. Bifidobacterium storage strains were mostly resistant to 2-3 antibiotics, a few more strains were resistant to more than 3 antibiotics; 3 of 10 isolates showed multiple drug resistance. Resistance range of the storage strains were wider. 2 of 10 storage Streptococcus thermophilus strains showed no resistance, and the remaining were resistant to 1-6 antibiotics; Most isolates were resistant to 2-3 antibiotics. The range of their resistance showed no significant difference. Among the three species, the Lactobacillus strains showed the strongest resistance, and followed by the Streptococcus thermophilus strains, and the Bifidobacterium strains was the last one.Fifth, adhesion assay in vitro was selected, and cervical cancer Hela cells were selected as cell model, and Lactobacillus, Bifidobacterium and Streptococcus thermophilus strains was selected as beneficial bacteria, and Salmonella typhimurium AMCC50333 was selected as pathogens, then adhesion of the bacteria to Hela was analyzed, and we analyzed the effect on adhesion of these beneficial bacteria to AMCC50333. The results showed:All the strains showed adhesion properties to Hela cells, but the difference is not obvious. Strains with different species in one genus showed different adhesion. Three spieces of bacteria storage strains and isolates all could significantly inhibit the Salmonella typhimurium adhering to Hela. However, there was no significant difference. The strains which were identified as Lactococcus lactis showed strong adhesion to Hela cell, and also showed strong inhibition of Salmonella typhimurium adhering to Hela.In a whole, PFGE molecular typing method is the best typing methods, followed by 16S rRNA sequence analysis, and biochemical typing. Through a series of experiments, this study established a database of lactic acid bacteria strains Chinese food industry commonly used.更多还原