卷曲乳杆菌益生特性及黏附机理研究

乳杆菌是人和动物肠道的正常菌群,具有维持肠道内菌群平衡,提高机体免疫力,促进营养物质吸收等生理功能。同时乳杆菌在生长过程中,能够产生乳酸、乙酸、过氧化氢、细菌素等抑菌物质,对肠道病原菌有抑制作用,因此,乳杆菌对机体具有重要的益生作用。益生菌的筛选标准包括黏附和定植能力、无致病性、对病原菌有拮抗作用、不携带可转移的抗生素基因等。其中,对消化道表面的黏附和定植能力是重要的筛选指标,其黏附能力决定于细胞表层蛋白的组成及疏水特性。另外,益生菌对病原菌具有抑制作用,作用机制包括竞争有限的资源限制病原菌的生长、竞争病原菌黏附位点、分泌抗菌物质及失活毒素等。因此临床上应用益生菌剂来预防或治疗因抗药性病原菌引起的肠道感染及腹泻等疾病。但是目前大部分的研究只停留在作用效果上,缺乏对益生菌作用机理的研究。为了深入研究益生菌的作用机理,筛选功能明确、安全性高的益生菌,本文主要围绕高黏附乳杆菌的筛选、乳杆菌对病原菌黏附上皮细胞的抑制作用、黏附蛋白的结构和功能鉴定、乳杆菌抗生素抗性评价、乳杆菌表达载体构建五个方面展开研究,其结果如下：1.高黏附乳杆菌的筛选及益生特性的体外评价目前乳酸菌的益生作用已被人们所接受。在畜禽养殖业,益生菌剂替代抗生素以促进动物的生长已得到认可,但目前仍存在菌株单一、特异性不强等问题,因此优良益生菌株的选育是开发益生菌制剂的关键。本文将分离自健康鸡肠道的42株乳杆菌与结肠癌细胞系HT-29共同温浴,结果6株乳杆菌具有较高的黏附能力,经16S rDNA基因序列分析,分别鉴定为Lactobacillus crispatus K313、Lactobacillus crispatus K243、Lactobacillus helveticus2020T、Lactobacillus johnsonnii H31、Lactobacillus salivarius Z4、Lactobacillus salivarius K233。其中,Lb. crispatus K313黏附能力最强,黏附量为73.2bacteria/cell。随后将这6株乳杆菌与黏蛋白(mucin)、胞外基质蛋白Ⅰ型胶原(CnⅠ)、Ⅳ型胶原(CnIV)和纤连蛋白(FN)共同温育,结果菌株Lb. crispatus K243和K313表现出较高的黏附能力,其中Lb. crispatus K243与CnIV结合能力最高,结合量为1239bacteria/field。菌株Lb. crispatus K243和K313对酸和胆盐表现出较强的耐受力,在pH3.5的酸性环境中仍能缓慢生长,在0.1%的牛胆盐中培养6h,活菌数基本不变。Lb. crispatus K313和K243还具有胆固醇降解能力,胆固醇脱出率分别为60.8%和51.4%。Lb. crispatus K313和K243对正十六烷、氯仿的亲和力高达90%,而对乙酸乙酯的结合能力较弱,说明它们都具有疏水性的、碱性的细胞表面。透射电镜观察菌株K243和K313的超微结构显示,这2株菌的细胞壁表面都存在S层蛋白,用LiCl提取菌株K243和K313的S层蛋白,SDS-PAGE分析显示其大小分别为45kDa和60kDa,将其命名为SlpA和SlpB。用LiCl、蛋白酶K、胃蛋白酶、胰蛋白酶处理菌体细胞,SDS-PAGE分析显示SlpB对胃蛋白酶敏感,对胰蛋白酶不敏感,而SlpA对胃蛋白酶和胰蛋白酶都不敏感。经过LiCl处理后,Lb. crispatus K243和K313对胶原蛋白和HT-29细胞的黏附能力都明显下降,并且纯化的S层蛋白也能阻碍Lb. crispatus K243和K313对CnⅣ和HT-29细胞的黏附,说明S层蛋白参与了黏附过程。用LiCl处理菌株后,Lb. crispatus的形态变为椭圆或不规则状,表明S层蛋白对维持细胞正常形态发挥重要作用。去除S层蛋白后,这2株菌的存活率及对人工胃肠液的耐受性降低,说明S层蛋白可以保护宿主菌免受不利环境的影响。通过以上特性的研究,说明这2株菌具有益生菌的优良特性,可作为益生菌展开后续工作的研究。2. Lb. crispatus调节病原菌感染HT-29细胞所引起的炎症反应益生菌能预防和治疗因病原菌感染造成的腹泻等疾病,其作用机制包括减少病原菌定植,调节肠道菌群平衡及促进宿主免疫反应。益生菌一般通过共絮凝、竞争排斥等抑制病原菌的定植,本文研究表明,Lb. crispatus K313与病原菌Salmonella, braenderup H9812和Escherichia coli ATCC25922的共絮凝率分别为57.7%和84.8%,而Lb. crispatus K243与它们的共絮凝率分别为28.7%和26.2%,明显低于Lb. crispatus K313。随后将菌株K313和K243与S. braenderup H9812和E.coli ATCC25922共同感染HT-29细胞,结果Lb. crispatus K243和K313都能降低S. braenderup H9812和E. coli ATCC25922对HT-29细胞的黏附能力,并且菌株K313比K243表现出更强的抑制能力,此结果与共絮凝作用相吻合。当S. braenderup H9812单独感染HT-29细胞时,促炎性细胞因子IL-8、CXCL1和CCL20的转录水平是上调的,而当Lb. crispatus与S. braenderup H9812共同感染时,K313和243下调了IL-8的转录水平,下调幅度分别为42%和37%。另外,K313还下调了CXCL1和CCL20的转录水平,下调幅度为63%和41%。ELISA分析进一步验证了IL-8的分泌水平,结果表明Lb. crispatus K243和K313都能抑制由S. braenderup H9812感染HT-29所诱导的IL-8的分泌,抑制量分别为32.8%和47.0%。上述实验结果说明,Lb. crispatus K243和K313具有消弱S。braenderup H9812感染上皮细胞产生炎症反应的能力,为益生菌在临床上的

Lactobacilli are dominant inhabitants of humans and animals. They can maintain a balanced intestinal flora, improve immunity and promote the absorption of the nutrients. Also Lactobacillus can produce lactic acid, acetic acid, hydrogen peroxide, bacteriocins and have an inhibitory effect on enteric pathogens. Therefore, Lactobacillus has an important role of prebiotics on human and animals. Probiotic screening criteria include the adhesion and colonization ability, antagonistic to pathogens and no transferable antibiotc resistance gene. Adhesion and colonization determined by the composition and hydrophobic characteristics of surface layer protens is an important screening index. Besides, probiotics have an inhibitory effect on pathogens. The mechanisms of action include the inhibition of pathogen growth by competition for nutritional sources and adhesion sites, secretion of antimicrobial substances and toxin inactivation. Consequently, Lactobacillus was used in clinical to prevent and treat gastrointestinal infections and antibiotic-associated diarrhea diseases. But most of the researches just study the effects of prebiotics and lack of the mechanism of action of Lactobacillus. For the development of probiotics with a clear feature and high security, we studied mainly on the screening of the high adhesive Lactobacillus, the inhibition of pathogens adhesion to epithelia cells, the structure and function of the adhesion proteins, antibiotic resistance evaluation of intestinal Lactobacillus and construction of expression vectors for Lactobacillus, the results were described as follows:1. The isolation and in vitro evaluation of high adhesive Lactobacillus strainsCurrently, there is an increasing interest in the use of probiotics as an alternative strategy to antimicrobial compounds. But there are still many problems such as single strains and poor specificity. Thus, the breedings of the excellent probiotic strains are the key to the development of probiotics. In this study, six Lactobacillus strains isolated from chicken intestinal tract had the adhsion ability to HT-29cells. They were Lactobacillus crispatus K313, Lactobacillus crispatus K243, Lactobacillus helveticus2020T, Lactobacillus johnsonnii H31, Lactobacillus salivarius Z4and Lactobacillus salivarius K233. Lb. crispatus K313exhibited the highest adhesion capacity to HT-29cells (73.2bacteria/cell). The six Lactobacillus strains adherences to mucin and individual proteins of the mammalian extracellular matrix were tested. the high attachment to collagen type IV (1239bacteria/field) were observed with the strain Lb. crispatus K243. Lb. crispatus K243and K313showed srong tolerance to acid and bile salts. The two strains also had a cholesterol degradation ability and the rate were60.8%and51.4%. Both of strains showed the very high percentages adhered to hexadecane demonstrated hydrophobic properties in the cell surface of Lb. crispatus. A strong affinity to chloroform and a low adherence to ethyl acetate indicated the basic characteristics in the two strain surfaces. SDS-PAGE analysis revealed the presence of the potential S-proteins Slp A and SlpB in Lb. crispatus K243and K313. SlpB of the strain K313was sensitive to pepsin, but not to trypsin, while SlpA was sensitive to neither pepsin nor trypsin. The treatment with LiCl greatly reduced the adhesiveness of the two Lb. crispatus strains compared to the untreated bacterial cells, subsequently, purified S-proteins also exhibited the inhibition of Lb. crispatus adhesion to collagens and HT-29cells, which suggested the S-layer proteins might be involved in the adhesion process. After LiCl treatment, Lb. crispatus K313and K243become oval or irregular shape. Consequently, the S-protein play an important role in maintaining the normal morphology of the cells. After removal of S-proteins, the viability and tolerance of the two Lb. crispatus strains to simulated gastric and small intestinal juice were reduced, indicating the protec