【作者】 李平兰；

【导师】 马长伟；

【作者基本信息】 中国农业大学 ， 食品科学， 2004， 博士

【摘要】 目前，在我国乃至世界范围内，优良乳酸菌资源的开发和利用已成为热点。本课题针对国内外双歧杆菌的研究现状、存在的问题等，以世界第四长寿区—广西巴马百岁以上长寿老人源双歧杆菌为试材，从优良菌种的筛选、生物学特性研究、生理功能探讨与验证等方面进行了较为系统、全面的研究，以期为我国安全、优质、高效的食品用双歧杆菌发酵剂及活菌制剂的研制与开发提供理论依据、微生物资源及技术支持。具体从以下几个方面展开研究工作： 1．以人结肠腺癌细胞系HT-29细胞为试材，对来源于长寿老人肠道的24株双歧杆菌进行了体外黏附试验。结果发现，双歧杆菌均具有一定的黏附能力，其中的TTF、Z2、TZ5、J-1菌株具有较高的黏附能力。进一步对4株初筛双歧杆菌耐胃酸、胆汁酸和合成B族维生素能力的试验发现，双歧杆菌TTF菌株不仅能合成较高的B₁、B₂、B₆、B₁₂等多种B族维生素，而且在pH3.0的条件下处理120min存活率达93.11％，同时在2％胆盐浓度下处理24h有较好的存活，具有显著的综合优势。通过形态学、生理生化等相关指标的鉴定该菌株为长双歧杆菌。 2．以筛选出的长双歧杆菌TTF菌株为试验菌对其生长规律、最佳生长培养基、最适生长温度、起始DH值、促生长因子等生物学特性进行了研究。结果发现： 1) 长双歧杆菌TTF菌株在MRS、改良MRS和PTYG三种培养基中的生长以改奥MRS培养基为最佳。通常培养6h进入对数期，12h进入稳定期，最适生长温度为37℃，最适起始pH值为7.5，活菌数对数值最高可达9.72。 2) 某些食品原料可作为双歧因子选择性促进双歧杆菌的生长。本试验条件下的牛奶（M）、胡萝卜汁（CJ）和香菇浸提物（LE）三种食品原料对长双歧杆菌TTF具有较好体外促生长作用的组合分别为：CJ-2％，M-2％，LE-2％。其中，LE对双歧杆菌的体外促生长作用很大，同时，CJ与LE的最适添加量均与M的添加量有关。 3．对长双歧杆菌TTF与HT-29细胞黏附的相关因素、与常见抗生素、中草药的药敏性、体外清除活性氧自由基能力、增强机体免疫功能、润肠通便功能、维持和修复机体肠道正常菌群平衡作用等生理功能进行了较为全面的研究。研究结果为： 1) 以金黄色葡萄球菌ATCC 25923为质控菌，长双歧杆菌TTF对8类30种常用抗生素的药敏性为对大环内酯类、头孢素类、青霉素类、β-内酰胺酶抑制剂的复合物及其他类的氯霉素、呋喃妥因、利福平抗生素均敏感；对氨基糖苷类、糖肽类抗生素均耐药；对四环素类抗生素表现出不同的药敏性。长双歧杆菌TTF菌株经质粒DNA的检测发现不含有质粒，说明其对药物的耐受性不为质粒编码，该菌不存在耐药因子传递的危险，可作为发酵食品或活菌制剂用安全菌种使用。 2) 三类不同药效的中草药对长双歧杆菌TTF体外生长的影响是不同的，除补益类中药可显著促进其生长外（P＜0.05），清热解毒类中药在1％、5％和10％三个试验浓度下随着浓度的增加抑制作用增强但均未达到显著性差异（P＞0.05），而泻下类中药对受试菌均有显著的抑制作用（P＜0.05）且大黄和芒硝达到极显著水平（P＜0.01）。说明在临床使用清热解毒类、泻下类中药时，在保证疗效的前提下尽量使用低浓度并配伍补益类中药为佳。同时补益类中药均能显著促进双歧杆菌生长的结果也说明，在采用抗生素或其他药物治疗时，适当配合使用补益类中药，可起到减弱药物对有益菌的抑制和杀死作用，从而起到修复和维持肠道菌群平衡的作用。 3)长双歧杆菌TTF对HT一29细胞具有很强的薪附能力，其戮附作用受细菌浓度、细菌与细胞相互作用时间、温度和PH值等的影响，并在一定范围内具有依赖性。胰蛋白酶处理双歧杆菌竹F表面结构可导致其薪附的降低，说明长双歧杆菌竹F菌体表面的蛋白质可能是细菌砧附素的主要组成成分。 4)在模型组成立的前提下，三组受试物组的小鼠首次排黑便时间、排便重量二项指标均极显著优于模型对照组和阴性对照组（P<0 .01），但排便粒数没有明显的差异。三组受试物组小鼠的小肠推进率极显著高于模型对照组（P<0.01），表明三种受试物对便秘小鼠均具有明显的润肠通便功效。就长双歧杆菌竹F的三种受试物来讲，SCS的效果高于IE和LB。 5)长双歧杆菌竹F不同处理物灌胃盐酸林可霉素（LH）造成的菌群失调小鼠后可使肠道内双歧杆菌和乳杆菌极显著增加（P<0 .01），产气英膜梭菌显著降低（P<0 .05），肠杆菌、拟杆菌、肠球菌极显著降低（P<0 .01），说明该双歧杆菌无论LB、IE还是SCS均可起到修复与调节机体肠道微生态平衡的作用。同时长双歧杆菌竹F不同处理物灌胃正常小鼠后可使肠道内双歧杆菌和乳杆菌极显著增加（P<0 .01），肠杆菌极显著减少（P<0 .01），拟杆菌、肠球菌显著增加（P<0 .05），但增加的幅度不及双歧杆菌和乳酸菌。说明该双歧杆菌的三种处理物均具有维持机体肠道内正常微生态平衡的作用。 6)长双歧杆菌竹F三种受试物对环磷酞胺（Cy）造成的ID枷三项免疫指标均有显著（P<0 .05）或极显著（P<0 .01）影响，在本试验选取的三个剂量范围内基本呈量效关系，且表现为SCS的效果高于IE和LB。而对ID枷的非特异性免疫功能的影响普遍大于对小鼠的细胞免疫和体?更多还原

【Abstract】 Lactic acid bacteria, referred to a group of bacteria that can ferment hydrocarbons and produce lactic acid, have long been found and used in fermented food, such as fermented milk and meat products. At first, lactic acid bacteria were used in food industry only for technological purpose. As more knowledge about this group of microbes are obtained, people found lactic acid bacteria, especial Bifidobacteria, have some healthy function and ingestion of these bacteria can bring about some beneficial effects to people. Thus more and more scholars are becoming interested in lactic acid bacteria. Nowadays development of new source of lactic acid bacteria and their application in food industry have been gain worldwide interest.In this study, new strains of lactic acid bacteria are isolated from the faeces of old people aged one hundred years in Bama, Guangxi province. Their biological characteristics and physiological functions are also preliminarily investigated in the hope of obtain some valuable proinciples or basic knowledge that can be referred to in the the development of food-grade bifidobacteria starter and microecologics. My research work mainly focused on the following aspects:1. Using colon adenocarcinoma cell line HT-29 as testing material, adhesion test was carried out for 24 strains of Bifidobacteria isolated from dejecta of longevous people aged one hundred years. The results showed that all strains could adhere to colon adenocarcinoma cells HT-29, among which the strains of TTF, Z2, TZ5 and J-1 showed higher adhesive capability. Thus these four strains of Bifidobacteria were further tested for their endurance to stomach acid and bile acid. The production of vitamin B by these bacterial were also tested. All these tests indicated that the strain of TTF has high ability to produce Vitamin B1, B2, B6, B12, The survival rate after the treatment under pH 3.0 for 120 min was 93.11%, at the same time, the strain TTF can survive after being treated with 2% bile acid for 24h. According to its morphology, together with physiologic and biochemical tests, this strain was identified as Bifidobacteria longum.2. The strain of TTF was further characterized in terms of its optimal growth media, temperature, initial pH and growth promoting factors. The results as follows:1) B. longum TTF grows better in modified MRS broth than MRS and PTYG broth. It usually enters logarithm stage after 6 hours of inoculation and enters stability stage after 12 hours. Optimal temperature and initial pH for its growth are 37β and 7.5, respectively.2) Some food materials, used as BF, can promote its growth. According to our study, the best combination of three food materials that can considerably promote the growth of TTF in vitro was 2% CJ, 2% M and 2% LE. Extract of Lentinus edodes can significantly promote the growth of TTF. However, the optimal concentrations of the extracts of carrot and are related to the addition of milk.3. The factors influencing B. long TTF’s ability to adhere to colon adenocarcinoma cell HT-29, its sensitivity to antibiotic and herbs, its capability to eliminate active oxygenous free radicals in vitro, its effects on human immune function and intestine microftora were also studied. The main results are:1) With Staphylococcus aureus ATCC 25923 as indicator bacteria, B. longum TTF showed its antibiotic susceptibility to 30 antibiotics of 8 groups: sensitive to macrolide group, cephalosporin group, penicillin group, β-lactamases inhibitors, chloramphenicol, nitrofurantoin and Rifampin, and resistant to aminoglycoside group and peptide group, whileshowing different susceptibility to tetracyciine group. DNA identification of TTF showed it does not possess the drug resistant plasmids, it means that its resistance to the antibiotics wasn’t encoded by plasmids and the possibility of intercellular transferring of resistance genes was excluded. Strain TTF can be safely used in fermented foods and microecologics.2) The influence of herbs, including Fructus Lycii, Fructus Schizan更多还原