【作者】 肖琳琳；

【导师】 董明盛；

【作者基本信息】 南京农业大学 ， 食品科学， 2003， 硕士

【摘要】 随着生活水平的提高，胆固醇摄入过量带来的高脂血症、冠心病、动脉硬化等心血管疾病已经严重危害到人体健康。食物胆固醇被认为是继农药残留和激素之后又一个令消费者恐慌的危险因子。如何减少胆固醇摄入量，降低血液胆固醇水平的研究越来越受到广泛关注。本研究旨在筛选高效降胆固醇的乳酸菌，为进一步研制和开发机能性食品发酵剂打下基础。 藏灵菇是一种源于我国西藏等地的传统发酵奶制品，它是以新鲜牛奶为原料加入天然发酵剂(藏灵菇粒)发酵而成，制作方法与流传于东欧高加索地区的传统发酵奶制品克菲尔(Kefir)极为相似。据饮用者介绍，长期饮用藏灵菇酸奶对增强人体免疫力，抗肿瘤，降低血脂黏度、减少血清胆固醇水平等均有积极作用，但未见这方面的研究报导。本文对藏灵菇粒中的微生物分布进行分析，围绕降胆固醇乳酸菌的筛选及其降解特性和机理展开系列研究，得到如下主要结果： 1．以藏灵菇粒为发酵剂发酵蛋黄，发现藏灵菇粒对蛋黄胆固醇有明显的脱除效果，脱除率为13.3％。经过长时间的富集驯化，藏灵菇粒降胆固醇能力有大幅度提高，发酵后胆固醇的脱除率上升到26.1％，脱除量从0.43mg／mL增加到0.83mg／mL。首次证明藏灵菇粒具有明确的降低胆固醇作用，可望作为降胆固醇机能性食品发酵剂加以开发利用。 2．从富集培养的藏灵菇粒中分离到11株乳杆菌(Lactobacillus spp.)、4株乳球菌(Lactococcus spp.)和4株酵母菌(yeast)。对这19株菌降胆固醇能力进行测定，发现乳球菌、酵母菌降胆固醇活性不明显，而11株乳杆菌中有10株对蛋黄胆固醇有明显的脱除效果，其中FMLA-1，FML-16的脱胆固醇效果最为显著，脱除率分别为47.5％和51.8％。此外，本实验室驯化选育的耐氧长双歧杆菌BL-1192(Bifidobacterium longum)也被证明有很强的降胆固醇活性(脱除率60.8％)。 3．对筛选出的两株高效降胆固醇乳杆菌FML-16、FMLA-1用VITEK-32全自动微生物分析系统结合形态和生理生化特征进行鉴定，结果表明FML-16、FMLA-1均为干酪乳杆菌(Lactobacillus casei)。 4．采用喂养法建立高脂血症动物模型，评价干酪乳杆菌FMLA-1、FML-16，长双歧杆菌BL-1192的降血脂功效。用含猪油、胆固醇、胆盐、甲基硫氧嘧啶的高脂饲料饲喂小鼠，10天后成功地复制了小鼠高脂血症模型。与饲喂基础日食的对照组比较，受试小鼠血清总胆固醇(TC)水平约上升了1.00mmol／L(P＜0.01)，甘油三酯(TG)值约升高了0.3mmol／L(P＜0.05)，但高密度脂蛋白胆固醇(HDL-C)含量在此期间变化 乳酸菌降胆固醇作用及机理研究不大。同时，高脂小鼠动脉硬化指数（AI）从 0．32增加到 1．05（P＜0．of），HDL－C／TC值自0．76下降至0．48（P＜0．05）。 5．与灌胃生理盐水的高脂小鼠比较，灌胃干酪乳杆菌FMLA－1、FML－16，长双歧杆菌队一119 2菌体后，高血脂＊、鼠血清n、TG水平明显下降。灌胃14天后，。］、鼠血清n、比水平恢复到高脂饲料饲喂前的水平，比灌胃生理盐水的对照组约低1．5mmo儿和0．6mmo几（P＜O．01）；而m卜C含量有上升的趋势。同时，灌胃菌体后AI值很快下降到 0．25－0．38（P＜0．01）之间，HDL－C／TC值回升到 0．75左右（P＜0．05）。但体外不降胆固醇的菌株刚二人一3灌胃后，小鼠的血脂水平和高脂组间的差异不明显。这些结果证明，具有体外降胆固醇活性的FMLA－1、FML－16、BL－11 92对小鼠高脂血症表现出较好的治疗效果。 6．胆固醇同化和沉淀分析表明，在含胆盐、蛋黄的血卜TmO中，刚卜16、刚M－1、BL－1192均表现出胆固醇同化作用和胆盐共沉淀作用，但强度有所不同。对刚L－16、FMLA－1而言，同4匕作用（25％左右）略强于共沉淀作用（20％左右），而 BL－1192的胆盐共沉淀能力（42％）明显高于同化能力（18％）。高胆固醇环境下生长的 FML－16、FMLA－1菌体对超声波的抗性有所增加，同样强度超声波处理，细胞存活率高出25％左右，间接说明有部分胆固醇同化到细胞中。胆盐对菌株降胆固醇能力的影响很大，没有胆盐存在时，三菌不表现降胆固醇活性，添加胆盐后菌株立即表现出胆固醇脱除能力，且随着胆盐浓度增加，胆固醇脱除率明显上升，胆盐浓度在0．3－0．4％时菌株降胆固醇活性最强。综上所述，初步判断刚L－16、FMLA－1、BL－119 2降胆固醇作用是由于j旦盐存在条件下菌体同化和胆盐共沉淀共同作用所致。更多还原

【Abstract】 According to the latest census, heart disease is the number one cause of death in many countries. Reduction of total plasma cholesterol can lower the incidence of coronary heart diseases in that people suffering from primary hypercholesterolemia. Because of the associated risk of heart disease, there has been an upsurge in finding ways to lower plasma cholesterol levels in such people. Several studies have indicated that consumption of certain lactic acid bacteria and its cultured dairy products resulted in reduction of serum cholesterol levels, and some Lactobacillus spp. can also reduce cholesterol from medium during growth in vitro.ZangLingGu (ZLG), a Tibet traditional yoghurt-like dairy fermented by natural formed ZLG grains, has been handed down to us from ancient times. A variety of potential beneficial effects about ZLG have already been described, such as improvement of host resistance by immunomodulation, depress of serum cholesterol levels and reduction of tumor formation. However, these are not systematic studies on hypocholesterolaemic effects of ZLG grains and its fermented milk. This paper research on the cholesterol-reducing ability and mechanisms of strains isolated from ZLG grains. The main results include:1. Determined cholesterol-reducing ability of ZLG grains, and found it could reduce 13.3% cholesterol from egg yolk. Tamed it with high-cholesterol skim milk supplemented with egg yolk and improved its cholesterol-reducing ability . The cholesterol removed form medium after 24h incubation with ZLG grains varied from 3.24mg/mL to 2.81mg/mL when it was been tamed to 40 generations.2. 11 Lactobacillus spp. strains, 4 Lactococcus spp.strains and 4 yeast strains were isolated from tamed ZLG grains. Cholesterol-reducing ability of those strains differed significantly and most Lactobacillus spp. could reduce cholesterol from medium. Two strains, FML-16, FMLA-1 were selected for next research, which could reduce 51.8%, 47.5% cholesterol form egg yolk . And Bifidobacterium longum BL-1192 was selected at the same time for its ability reducing 60.8% cholesterol from medium.3. According to their morphological and physiological characters, FML-16, FMLA-1 were identified as Lactobacillus casei. Identification results of these two isolates bybacteria auto-analytical system showed the same conclusion.4. The health effects of FML-16, FMLA-1, BL-1192 on serum cholesterol levels were demonstrated in vivo by hyperlipemial mice. Feeding mice with high-cholesterol food supplemented with bile salt, fat, etc, we succeeded in inducing hyperlipemial mice model. Compared with normal mice, the total serum cholesterol (TC) concentration, triglyceride (TG) concentration of those increased significantly after 10 days running ,but high density cholesterol (HDL-C) had very little change. At the same time, an increase in the atherogenic index (AI) were observed, and HDL-C/TC value decreased significantly.5. The effects of L.casei FML-16, FMLA-1, B.longum BL-1192 and Lactobacillus sp. FMLA-3 on serum TC, TG , HDL-C concentration were investigated in artificially-induced hyperlipemial mice. The results showed that the mean values of TC, TG concentrations and atherogenic index were decreased significantly when FML-16, FMLA-1, BL1192 strains were administrated to mice after 14 days running , and HDL-C concentration, HDL-C/TC value increased obviously at the same time. However, Lactobacillus sp.FMLA-3, which could not remove cholesterol in vitro had no effects on serum TC , TG and HDL-C levels of hyperlipemial mice.6. Studied on the mechanism of the cholesterol-reducing ability of L.casei FML-16, FMLA-1 , B.longum BL-1192 , includes: analysis of cholesterol assimilation and precipitation; effects of bile salts concentrations on cholesterol-reducing; cells resistance to lysis by ultrasonic when cultured in the presence of bile salts and cholesterol or not. The result proved that those three strains differed significantly in the amount of cholesterol更多还原