降胆固醇益生菌的研究
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摘要
近年来心脑血管疾病的发病率呈逐年上升的趋势,在我国心脑血管疾病死亡率已位居第一位,严重威胁人们的身体健康。心脑血管疾病主要是由高血脂症引发的动脉硬化引起的。化学药物治疗心脑血管疾病费用高、毒副作用大,且主要以治疗为主,不能起到良好的预防作用。为找到一种更安全、毒副作用小且经济的降胆固醇制品,许多科学家对益生菌的降胆固醇作用进行了研究。在我国益生菌降胆固醇的研究还处于起步阶段,筛选出具有高效降胆固醇功能的益生菌并对其功能性、安全性、作用机理和动物体内作用效果进行验证使其能够作为具有降胆固醇功能的益生菌制品的候选菌株具有重要的意义。本课题研究的结果：
1从长寿地区采集的多种发酵食品中例如辣白菜、泡菜、酸奶、奶酪、腐乳等,利用碳酸钙透明圈法和指示剂法初步筛选出19株具有较强降胆固醇功能的菌株作为筛选菌株。用硫酸铁铵法将这19株筛选菌株进行体外降胆固醇功能的测定,得到8株降解率在55%以上的菌株作为候选菌株。
2测定候选菌株在模拟胃肠道环境下的存活情况和对细胞的粘附性,结果表明DM9007、DM9054、DM9073三株菌在模拟胃肠道环境实验中能保持较高的活菌数,并且对细胞也有较好的粘附能力,将其作为后期实验菌株。
3研究三株实验菌株对6种常见致病菌的抑制生长能力,结果表明三株实验菌株具有一定程度的抑制有害菌的生长的能力。同时也对三株实验菌株进行抗生素类药物敏感性测定,菌株总体对抗生素类药物较稳定,仅对糖肽类和喹诺酮类药物具有耐药性。
4对三株实验菌株进行降胆固醇能力的优化,DM9007、DM9054、DM9073胆固醇降解率分别升高了2.60%、8.27%和2.78%,胆固醇的降解率最终分别达到58.24%、66.48%和60.99%。
5将各方面性状较好的菌株DM9054作为优选菌株,对其进行菌种鉴定,分别从形态学方面、生物化学方面和分子生物学方面对菌株进行鉴定,结果表明菌株为鼠李糖乳杆菌。
6对优选菌株DM9054进行安全性检验,以昆明小鼠为实验对象,检测菌株对小鼠的急性毒性和肝肠易位现象。实验过程中小鼠未出现死亡和肝肠易位现象,表明菌株安全性较好,未为对小鼠造成明显毒副作用。
7对优选菌株DM9054进行动物实验,从菌株对高血脂症的预防方面和治疗方面检测菌株的降胆固醇作用。结果表明菌株对小鼠各个血清指标和脏器指数均具有较好的恢复作用,预防方面血清胆固醇和肝脏胆固醇降解率分别达到48.01%和64.30%；治疗方面血清胆固醇和肝脏胆固醇降解率分别达到61.34%和68.82%。
8对DM9007、DM9054、DM9073三株具有较强体外将胆固醇能力的菌株进行降解机理研究,结果发现共沉淀作用、吸收作用和其他降胆固醇机理在三株菌降胆固醇作用中占据不同比例。DM9007、DM9054、DM9073共沉淀作用占的比例分别为28.59%、40.98%和42.07%；吸收作用占的比例分别为22.50%、20.33%和15.98%。
通过一系列的实验得到一株降胆固醇能力较强、性状优良、安全性高的菌株,为其后期开发出具有降胆固醇功能益生菌制品提供了潜力菌株、奠定了实验基础。
In recent years, the incidence of cardiovascular disease showed a trend of sustained growth, in China cardiovascular disease mortality has been ranked the first, which has been serious threat to people's health. Cardiovascular disease is mainly caused by atherosclerosis which is caused by the hyperlipidemia. Using drug to therapy cardiovascular and cerebrovascular diseases has some problems which are high cost、toxic side effects mainly treatment-oriented and can not play a good prevention. To find a safer、less toxic side effects and affordable cholesterol-lowering products many scientists has been studied the effect of lower cholesterol. In China the study of using probiotics to lower cholesterol still at the initial stage. It has great importance to selected probiotics which has the function of cholesterol-lowering and verify the function,safety, mechanism and the effect in vivo to make it be a candidate strains of cholesterol-lowering function.The results of the research:
1 19 stains probiotics are selected using calcium carbonate transparent circle method and indicator method form fermented foods such as spicy cabbage, pickles, yogurt, cheese, fermented bean curd, homemade wine. The ability of 19 strains of cholesterol-lowering function is determined by ammonium iron sulfate, and 8 strains which degradation rate more than55% are chosen as candidate strains.
2 3 stains, DM9007、DM9054、DM9073 are chosen as a post-experimental strains because they maintain a high number of viable cells, and also have a good function of cells adhesion in the determination of the simulated gastrointestinal tract environment survival and cell adhesion of candidate strains which have high cholesterol-lowering function in vitro.
3 Experimental strains have some degree of inhibition of the capacity growth of harmful bacteria in the research the ability of three experimental strains to inhibit the growth of 6 pathogenic bacteria. Also on research the three experimental strain sensitivity to antibiotics, the strains to the antibiotic drugs are stable in general, only resistant to individual drugs.
4 Optimization the three experiments strains of cholesterol-lowering ability, all of them have a certain level of cholesterol elevation.
5 Identify the strain DM9054 which has good functions and well cholesterol-lowering capacity.Respectively it from morphology, biochemistry and molecular biology.
6 Taking security experiments to DM9054, detect the acute toxicity and translocation of the strain to the mice. The results show that the strain was security and don't has obvious toxicity to the mice.
7 Carry out animal experiments to DM9054, study the treatment and prevention to hyperlipemia of the strain. The results show that the strain has a effect of recovery the each hyclones and viscera index of the mice, and the effect of treatment is better than prevention.
8 The total deposits, the absorption and other lower cholesterol mechanism with different ratio in cholesterol-lowering ability in the research of the mechanism of cholesterol degradation of the 3 experiments strains which have the function of lowing cholesterol. The features of cholesterol-lowering of each strain are confirmed.
A strain which has strong ability of lowing cholesterol, good properties and security is obtained through a series of experiments. It provide potential strain and basis for future study of a cholesterol lowing product made by probiotics.
1从长寿地区采集的多种发酵食品中例如辣白菜、泡菜、酸奶、奶酪、腐乳等,利用碳酸钙透明圈法和指示剂法初步筛选出19株具有较强降胆固醇功能的菌株作为筛选菌株。用硫酸铁铵法将这19株筛选菌株进行体外降胆固醇功能的测定,得到8株降解率在55%以上的菌株作为候选菌株。
2测定候选菌株在模拟胃肠道环境下的存活情况和对细胞的粘附性,结果表明DM9007、DM9054、DM9073三株菌在模拟胃肠道环境实验中能保持较高的活菌数,并且对细胞也有较好的粘附能力,将其作为后期实验菌株。
3研究三株实验菌株对6种常见致病菌的抑制生长能力,结果表明三株实验菌株具有一定程度的抑制有害菌的生长的能力。同时也对三株实验菌株进行抗生素类药物敏感性测定,菌株总体对抗生素类药物较稳定,仅对糖肽类和喹诺酮类药物具有耐药性。
4对三株实验菌株进行降胆固醇能力的优化,DM9007、DM9054、DM9073胆固醇降解率分别升高了2.60%、8.27%和2.78%,胆固醇的降解率最终分别达到58.24%、66.48%和60.99%。
5将各方面性状较好的菌株DM9054作为优选菌株,对其进行菌种鉴定,分别从形态学方面、生物化学方面和分子生物学方面对菌株进行鉴定,结果表明菌株为鼠李糖乳杆菌。
6对优选菌株DM9054进行安全性检验,以昆明小鼠为实验对象,检测菌株对小鼠的急性毒性和肝肠易位现象。实验过程中小鼠未出现死亡和肝肠易位现象,表明菌株安全性较好,未为对小鼠造成明显毒副作用。
7对优选菌株DM9054进行动物实验,从菌株对高血脂症的预防方面和治疗方面检测菌株的降胆固醇作用。结果表明菌株对小鼠各个血清指标和脏器指数均具有较好的恢复作用,预防方面血清胆固醇和肝脏胆固醇降解率分别达到48.01%和64.30%；治疗方面血清胆固醇和肝脏胆固醇降解率分别达到61.34%和68.82%。
8对DM9007、DM9054、DM9073三株具有较强体外将胆固醇能力的菌株进行降解机理研究,结果发现共沉淀作用、吸收作用和其他降胆固醇机理在三株菌降胆固醇作用中占据不同比例。DM9007、DM9054、DM9073共沉淀作用占的比例分别为28.59%、40.98%和42.07%；吸收作用占的比例分别为22.50%、20.33%和15.98%。
通过一系列的实验得到一株降胆固醇能力较强、性状优良、安全性高的菌株,为其后期开发出具有降胆固醇功能益生菌制品提供了潜力菌株、奠定了实验基础。
In recent years, the incidence of cardiovascular disease showed a trend of sustained growth, in China cardiovascular disease mortality has been ranked the first, which has been serious threat to people's health. Cardiovascular disease is mainly caused by atherosclerosis which is caused by the hyperlipidemia. Using drug to therapy cardiovascular and cerebrovascular diseases has some problems which are high cost、toxic side effects mainly treatment-oriented and can not play a good prevention. To find a safer、less toxic side effects and affordable cholesterol-lowering products many scientists has been studied the effect of lower cholesterol. In China the study of using probiotics to lower cholesterol still at the initial stage. It has great importance to selected probiotics which has the function of cholesterol-lowering and verify the function,safety, mechanism and the effect in vivo to make it be a candidate strains of cholesterol-lowering function.The results of the research:
1 19 stains probiotics are selected using calcium carbonate transparent circle method and indicator method form fermented foods such as spicy cabbage, pickles, yogurt, cheese, fermented bean curd, homemade wine. The ability of 19 strains of cholesterol-lowering function is determined by ammonium iron sulfate, and 8 strains which degradation rate more than55% are chosen as candidate strains.
2 3 stains, DM9007、DM9054、DM9073 are chosen as a post-experimental strains because they maintain a high number of viable cells, and also have a good function of cells adhesion in the determination of the simulated gastrointestinal tract environment survival and cell adhesion of candidate strains which have high cholesterol-lowering function in vitro.
3 Experimental strains have some degree of inhibition of the capacity growth of harmful bacteria in the research the ability of three experimental strains to inhibit the growth of 6 pathogenic bacteria. Also on research the three experimental strain sensitivity to antibiotics, the strains to the antibiotic drugs are stable in general, only resistant to individual drugs.
4 Optimization the three experiments strains of cholesterol-lowering ability, all of them have a certain level of cholesterol elevation.
5 Identify the strain DM9054 which has good functions and well cholesterol-lowering capacity.Respectively it from morphology, biochemistry and molecular biology.
6 Taking security experiments to DM9054, detect the acute toxicity and translocation of the strain to the mice. The results show that the strain was security and don't has obvious toxicity to the mice.
7 Carry out animal experiments to DM9054, study the treatment and prevention to hyperlipemia of the strain. The results show that the strain has a effect of recovery the each hyclones and viscera index of the mice, and the effect of treatment is better than prevention.
8 The total deposits, the absorption and other lower cholesterol mechanism with different ratio in cholesterol-lowering ability in the research of the mechanism of cholesterol degradation of the 3 experiments strains which have the function of lowing cholesterol. The features of cholesterol-lowering of each strain are confirmed.
A strain which has strong ability of lowing cholesterol, good properties and security is obtained through a series of experiments. It provide potential strain and basis for future study of a cholesterol lowing product made by probiotics.
引文
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