具有益生菌特征的阴道乳酸杆菌的筛选及其生物学特性的研究

发布时间：2018-01-24 19:12

本文关键词： 乳酸杆菌阴道感染微生态益生菌拮抗黏附　出处：《南昌大学》2007年硕士论文　论文类型：学位论文

【摘要】： 妇女阴道是一个复杂的微生态系统，乳酸杆菌是其中占主导地位的细菌，对维持阴道微生态环境的平衡起到了关键作用。乳酸杆菌发挥作用的机制推测为：1)粘附上皮细胞，阻止病原菌初始粘附；2)分泌有机酸，起到阴道自净作用；3)产生细菌素；4)产生H_2O_2；5)产生生物表面活性蛋白物质；6)刺激宿主局部免疫。研究表明乳酸杆菌制剂不仅可以阻止病原菌对阴道的感染，而且对治疗阴道感染有显著的效果。目前国内外都有相关的商品化乳酸杆菌制剂，在预防治疗阴道感染中起到了重要的作用。本文采用改良MRS培养基对162份健康孕妇阴道样本中的嗜酸性细菌进行了定向筛选。通过革兰氏染色、细胞形态观察以及生理生化实验初步将分离的细菌确定为乳酸杆菌，共获得326株乳酸杆菌。通过TMB-HRP-MRS培养基对其产H_2O_2能力进行了半定量分析。获得H_2O_2阳性菌株244株。其中H_2O_2弱阳性菌株为89株，阳性116株，强阳性39株。统计分析结果表明：产H_2O_2菌株在孕妇中的检出率为87.59％，其分布与孕周以及孕妇年龄相关；产H_2O_2乳酸杆菌的检出率随着孕周的增加而升高，随着孕妇年龄的增长而降低。乳酸杆菌属涵盖了102种，德氏乳杆菌、植物乳杆菌、嗜酸乳杆菌、唾液乳杆菌等菌种在食品药品领域显示出良好的应用前景。传统的乳酸杆菌鉴定方法主要是依据形态学观察和简单生理生化反应。本研究除了运用API细菌学鉴定金标准进行生理生化鉴定之外，还运用现代分子生物学的技术对乳酸杆菌的鉴定进行了探讨，尝试采用两次PCR，即组间和组内PCR的方法将分离菌株鉴定到种。结果表明，在孕妇阴道中检出率较高的是嗜酸乳杆菌和卷曲乳杆菌，H_2O_2产量较高的也是嗜酸乳杆菌和卷曲乳杆菌。筛选具有较强拮抗病原菌能力的阴道乳酸杆菌，可以作为益生菌制剂用于治疗阴道细菌感染。本研究采用Hela细胞黏附实验模型考察了乳酸杆菌分离株的黏附能力，采用双层平板法考察各分离株对致病菌的拮抗能力，结果发现乳酸杆菌的黏附能力具有菌种的差异，同一种不同株的差异也很大。乳酸杆菌分离株中有53.24％的菌株对白色假丝酵母有抑制作用，13.65％的菌株有较强的拮抗作用。黏附拮抗实验表明乳酸杆菌可以阻止白色假丝酵母对于Hela细胞的黏附。以拮抗白色假丝酵母能力为主要指标，综合对其它三种致病菌(大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌)的拮抗作用以及对Hela细胞的黏附能力，从326株阴道乳酸杆菌分离株中筛选得到11株有益生效果的乳酸杆菌，对其分类学地位、益生的生物学特性进行了较全面的分析。首先测定了11株阴道乳酸杆菌的16S rRNA基因(即16S rDNA)全序列，在基因数据库(GenBank、EMBL、DDBJ、PDB)中进行了同源性搜索。取相似度大于99％的序列为参考，，对其进行了鉴定，并构建了系统发育进化树，确定了其在系统发育中的分支地位。通过拮抗致病菌实验发现，11株乳酸杆菌对致病菌有较强的拮抗作用，提示可以用于治疗致病菌感染，恢复乳酸杆菌的主导地位。通过对其生物学特性进行研究，初步掌握了其生长特性，并测定了其抗菌谱，分析了个别菌株的抑菌机理，为进一步研究和应用奠定了基础。本研究筛选的11株乳酸杆菌是正常妇女阴道的原籍菌，具有较强的粘附和拮抗病原菌能力，可以作为微生态制剂的候选菌株进行深入研究。
[Abstract]:The vagina is a complex micro ecosystem, lactobacillus is one of the dominant bacteria, to maintain vaginal micro ecological environment balance plays a key role in the mechanism of the role of lactic acid bacteria. That is: 1) adhesion to epithelial cells, prevent pathogen initial adhesion; 2) the secretion of organic acids, play vaginal self purification; 3) produce bacteriocin; 4) H_2O_2; 5) to produce bio surfactant protein; 6) stimulate the host local immunity. The research showed that lactobacillus can not only prevent the infection of the vagina, but also has a significant effect on the treatment of vaginal infection. At home and abroad have commercial lactic acid preparation of relevant, in the prevention and treatment of vaginal infection plays an important role.
This paper uses the modified MRS medium for directional screening of 162 vaginal samples of healthy pregnant women in the acidophilic bacteria. By Gram staining, observe cell morphology and physiological and biochemical experiments will be the separation of bacteria identified as Lactobacillus, a total of 326 strains of Lactobacillus. Medium was studied by semi quantitative analysis of the H_2O_2 production by TMB-HRP-MRS training. H_2O_2 244 positive isolates. The H_2O_2 weakly positive 89 strains, 116 strains were positive, 39 strong positive strains. The results showed that the H_2O_2 producing strains in pregnant women in the detection rate was 87.59%, and the distribution of gestational age and maternal age; H_2O_2 producing lactic acid bacteria detection rate increased with the increase of weeks of pregnancy, pregnant women decreased with age.
Lactobacillus covers 102 kinds of Deshi, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus salivarius and other strains showed good application prospect in the field of food and drug. The traditional methods of identification of lactic acid bacteria is mainly based on the morphological observation and simple physiological and biochemical reactions. In this study, in addition to the gold standard for bacteriological identification by API physiological and biochemical identification, identification of the application of modern molecular biology technology of lactic acid bacteria were discussed, using two PCR, namely groups and PCR methods isolates were identified to species. The results show that the higher detection rate of maternal vaginal is Lactobacillus acidophilus and Lactobacillus casei, H_2O_2 high yield is Lactobacillus acidophilus and Lactobacillus plantarum.
Screening of vaginal Lactobacillus bacteria with strong antagonistic pathogens, can be used as probiotics in treating vaginal bacterial infection. This study investigated the adhesion ability of Lactobacillus isolates by Hela cell adhesion test model, effects of Antagonistic Ability of the isolates of the pathogen by double-layer plate method, has found differences in adhesion of lactic acid bacteria coli, with a different strain is also a great difference. Lactobacillus isolates and 53.24% strains had inhibitory effect on Candida albicans, 13.65% strains with strong antagonistic effect. Antagonism experiments show that lactic acid bacteria can prevent adhesion of Candida albicans to Hela cells.
The antagonism of Candida albicans is the main index of comprehensive ability, other three kinds of pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa) and the antagonistic effect on Hela cell adhesion from 326 strains of vaginal lactobacilli isolates screened 11 strains of Lactobacillus probiotic effects on. The taxonomic status and biological characteristics of probiotic were comprehensively analyzed. Firstly, the 16S rRNA gene of 11 strains of lactobacilli were determined (16S rDNA) sequence in the gene database (GenBank, EMBL, DDBJ, PDB) by homology search. The sequence similarity is greater than 99% for reference to they were identified, and construct phylogenetic tree, determine its phylogenetic position. Through antagonistic bacteria branch experiments showed that 11 Lactobacillus strains had strong antagonistic effect on pathogenic bacteria, that can be used for the treatment of The infection of pathogenic bacteria restored the dominant position of Lactobacillus. By studying its biological characteristics, its growth characteristics were preliminarily mastered, its antibacterial spectrum was determined, and the bacteriostatic mechanism of some strains was analyzed, which laid a foundation for further research and application.
The 11 lactobacilli screened from this study are the original vaginal bacteria of normal women, and have strong adhesion and antagonistic ability to pathogenic bacteria. They can be used as candidate strains for further research.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R371

【引证文献】