【作者】 李雪；

【导师】 孟祥晨；

【作者基本信息】 东北农业大学 ， 食品科学， 2015， 硕士

【摘要】 益生菌是指当摄入足够量时能对人体产生有益作用的活的微生物,部分植物乳杆菌具有益生作用,包括缓解乳糖不耐受、调节胃肠道菌群平衡、降低血液胆固醇水平、调节机体免疫力等。根据世卫组织(FAO/WHO)对益生菌的定义,只有满足如下要求的菌株才能称之为益生菌,包括:胃酸耐受性、胆汁酸抗性、对黏液或人上皮细胞的黏附能力、对潜在致病菌的抑菌能力、降低病原菌在表面黏附的能力、胆汁盐水解能力等。植物乳杆菌KLDS1.0391和KLDS1.0706分离自内蒙古传统发酵乳制品,前者能代谢合成细菌素,后者则不能,本研究的主要目的是评价这两株菌抗消化道逆环境的性质及一般性质在此基础上进一步体外评价它们的益生特性,最后评价其安全性,以便揭示细菌素合成能力与抗逆境能力、益生性质的关系。具体研究结果如下:细菌素合成能力。乳杆菌无菌上清液(CFSs)经除有机酸、过氧化氢、10倍浓缩后,植物乳杆菌KLDS1.0391浓缩CFSs对金黄色葡萄球菌和枯草芽孢杆菌两株菌的生长有明显的抑制效果,但植物乳杆菌KLDS1.0706和LGG没有。验证了植物乳杆菌KLDS1.0391是一株产细菌素植物乳杆菌,而植物乳杆菌KLDS1.0706和LGG是不产细菌素的乳杆菌。两株菌的抗逆境能力。在酸耐受实验中,植物乳杆菌KLDS1.0391与KLDS1.0706对p H2的酸环境能耐受性较差,而在p H 3的酸性环境中有较好的耐受性,在孵育3 h后菌落数仍能维持在109 CFU/m L以上。且植物乳杆菌KLDS1.0391表现出更好的酸耐受性。在胆盐耐受性试验中,两株植物乳杆菌在孵育8 h后菌落数均能维持在108 CFU/m L以上,表相出了良好的耐受性,且植物乳杆菌KLDS1.0391表现出更好的耐受性。同样的,在模拟人工胃液、胰液试验中有类似的实验结果。两株乳杆菌与商业菌LGG相比,植物乳杆菌KLDS1.0391表现出与之相仿或更好的耐受能力,而植物乳杆菌KLDS1.0706与之相比具有较差的耐受性。表面性质与黏附能力。在表面性质实验中,植物乳杆菌KLDS1.0391与KLDS1.0706相比具有较好的表面性质(包括表面疏水性、自动聚集能力、共聚能力),但差异不显著(P>0.05)。两株植物乳杆菌与商业菌株LGG相较,其表面疏水性及聚集能力均低于LGG。在黏附实验中,其黏附能力依次为:LGG>植物乳杆菌KLDS1.0391>植物乳杆菌KLDS1.0706。而在黏附抑制实验中,两株植物乳杆菌均表现出了比LGG更好的抑制能力,其中植物乳杆菌KLDS1.0391的黏附抑制能力最为明显。胆盐水解酶活性与体外脱除胆固醇能力。胆盐水解酶活性依次为植物乳杆菌KLDS1.0706>LGG>植物乳杆菌KLDS1.0391。而胆固醇脱出能力依次为植物乳杆菌KLDS1.0391>植物乳杆菌KLDS1.0706>LGG,两结果无相关性。体外免疫活性。植物乳杆菌KLDS1.0391与KLDS1.0706对小鼠脾细胞均有促进增殖的作用,且活性植物乳杆菌对脾细胞的增殖作用要好于热致死乳杆菌的增殖作用。而且,植物乳杆菌KLDS1.0706对小鼠脾细胞的体外增殖效果明显高于KLDS1.0391。两株菌与商业菌株LGG相比较,植物乳杆菌KLDS1.0391促进增殖作用与LGG相仿,而植物乳杆菌KLDS1.0706显示出最高的增殖作用。菌株安全性评价。将植物乳杆菌KLDS1.0391与KLDS1.0706接种在哥伦比亚血平板中均无溶血圈出现,证明无溶血性;在抗生素抗性试验中,两株菌对万古霉素与链霉素具有抗性。此外,植物乳杆菌KLDS1.0706还对庆大霉素具有抗性。研究结果表明:产细菌素植物乳杆菌KLDS1.0391在抗逆境能力、表面性质与黏附能力以及胆固醇脱出能力方面较不产细菌素乳杆菌KLDS1.0706有更好的性能。而植物乳杆菌KLDS1.0706在胆盐水解酶活性及体外免疫活性方面表现出更好的能力。两株植物乳杆菌与商业菌株LGG相较,除抗逆境能力、表面性质方面虽相对较差,其他方面均表现出相仿或更突出的性能,所以植物乳杆菌KLDS1.0391与KLDS1.0706具有作为益生菌的潜在能力。更多还原

【Abstract】 Probiotics are live microorganisms, which when administered in adequate amounts confer a health benefit in the host. As probiotics, characteristics commonly presumed to be essential, such as alleviation of lactose intolerance, regulation of immunity, the ability to improve the balance of the intestinal microbiota and to lower blood cholesterol levels. According to WHO working group report on drafting guidelines for the evaluation of probiotics in food, the experiments of probiotic properties included:(1) Resistance to gastric acidity,(2) Bile acid resistance,(3) Adherence to mucus and/or human epithelial cells and cell lines,(4) Antimicrobial activity against potentially pathogenic bacteria,(5) Ability to reduce pathogen adhesion to surfaces,(6) Bile salt hydrolase activity,(7) Resistance to spermicides(applicable to probiotics for vaginal use).Lactobacillus plantarum KLDS1.0391 and Lactobacillus plantarum KLDS1.0706 were isolated from ‘‘Jiaoke”, a traditional, naturally fermented cream from Inner Mongolia in China. The former is a bacteriocin-producing strain which can produce bacteriocin MG, and the later is a non-bacteriocin-producing strain. The aim of the study was to evaluate the the tolereance to the harsh environment of the digestive tract and then evaluate their in vitro probiotic properties and the safety of the strains, finally was to reveal the relationship between the ability of tolerance to harsh environment, in vitro probiotic properties and the bacteriocins.The antibiotic activity research results show that, the obvious inhibition zone was observed in the plate which contained the cell-free supernatants(CFSs) of Lactobacillus plantarum1.0391 and Lactobacillus plantarum KLDS1.0706 respectively. The same result were founded in the plate contained tenfold concentrated CFSs of Lactobacillus plantarum KLDS1.0391 which pretreated to remove the component of H2O2 and organic acid. The above results illustrated that the two Lactobacillus plantarum strains produced some antibacterial substances(like organic acid and H2O2 etc) after the fermentation so that could inhibit the growth of pathogens. While the pro-prosessing CFSs of Lactobacillus plantarum KLDS1.0391 had the sme result certified that Lactobacillus plantarum KLDS1.0391 is a bacteriocin-producing strain, and Lactobacillus plantarum KLDS1.0706 is a non- bacteriocin-producing strain.Both of lactobacilli strains showed bad performance to p H 2. However, both of the tested strains showed good tolerance to p H 3, at which the viable counts exceeded 109 CFU/ml after incubation for 3 h. Lactobacillus plantarum KLDS1.0391 possessed better tolerance at p H 2 than Lactobacillus plantarum KLDS1.0706(P<0.05), but it possessed slight lower tolerance than the reference strain LGG. Lactobacillus plantarum KLDS1.0391 and Lactobacillus plantarum KLDS1.0706 showed a good capability to grow in different concentration of bile salt. The viable counts for lactobacilli strains were above 108 CFU/ml, while Lactobacillus plantarum KLDS1.0391 showed better risistence wich was similar to the result of the tolerance to artificial gastric juice and artificial pancreatic juices. However, compaired with LGG, Lactobacillus plantarum KLDS1.0391 showed the similar or the better performance, but Lactobacillus plantarum KLDS1.0706 were not.Surface properties and adhesion abilities. Lactobacillus plantarum KLDS1.0391 possessed better performance in surface properties(including surface hydrophobicity, auto-aggregation assay and co-aggregation assay) compared with Lactobacillus plantarum KLDS1.076, but there was no significant differences(P>0.05). But LGG performed the strongest surface properties among the three strains. In the adhesion assay, the adhesion ability followed the order: LGG >Lactobacillus plantarum KLDS1.0391 > Lactobacillus plantarum KLDS1.0706. While in the assay of inhibition adhesion, both of the Lactobacillus plantarum strains showed better performance to LGG, and Lactobacillus plantarum showed the best effect.BSH activity and cholesterol removal capability. BSH activity was followed the order: Lactobacillus plantarum KLDS1.0706>LGG>Lactobacillus plantarum KLDS1.0391,while the cholesterol removal capability was followed the order: Lactobacillus plantarum KLDS1.0391>Lactobacillus plantarum KLDS1.0706 > LGG. There was no relationship between the two properties.In the preliminary evaluation of immunological activity, Lactobacillus plantarum KLDS1.0706 exhibited more effective than that of Lactobacillus plantarum KLDS1.0391(no matter active lactobacillus and the heat-killed ones). Compared with the commercial strain LGG, Lactobacillus plantarum KLDS1.0391 showed lower effective, but there were no significant differences between each other. The result showed that both Lactobacillus plantarum KLDS1.0391 and Lactobacillus plantarum KLDS1.0706 showed the potencial in immunomodulatory in vitro.In the hemolytic activity, there was no hemolysis ring appearing on the Columbia plate. It demonstrated that Lactobacillus plantarum KLDS1.0391 and Lactobacillus plantarum KLDS1.0706 were non-hemolytic strains; in antibiotic resistance assay, we found that the two Lactobacillus plantarum strains only had resistance to vancomycin and streptomycin which had the same resistance with LGG. In addition, Lactobacillus plantarum KLDS1.0706 also had resistance to gentamicin. Through the evaluation of the above two aspects, we prove that there was no security problems with Lactobacillus plantarum KLDS1.0391 and Lactobacillus plantarum KLDS1.0706.In summary, Lactobacillus plantarum KLDS1.0391 had better performance in the tolerance to harsh environment, surface properties, adhesion ability and cholesterol removal capability than Lactobacillus plantarum KLDS1.0706. While Lactobacillus plantarum KLDS1.0706 showed better performances in BSH activity and immunological activity than Lactobacillus plantarum KLDS1.0391. Compaired with LGG, both of the Lactobacillus plantarum strains showed the same or more prominent performance except the tolerance to harsh environment and surface properties. So Lactobacillus plantarum KLDS1.0391 and Lactobacillus plantarum KLDS1.0706 had the potential of being as probiotics.更多还原