【作者】 张英春；

【导师】 张兰威；

【作者基本信息】 东北农业大学 ， 食品科学， 2003， 硕士

【摘要】 益生菌在促进人体健康和防止疾病中发挥着重要的作用，是未来功能食品发展的重要领域。本课题采用体外和体内实验，测定了益生菌及联合菌株的潜在功能，筛选共生性较好的菌株。 本试验共测定9株潜在益生菌，其中PB1和A878为嗜酸乳杆菌；Inf和Pba为双歧杆菌；M1、PB2、A30、A31为链球菌；M2为枯草芽孢杆菌。分离出一株酪酸梭菌。大肠杆菌、痢疾杆菌、沙门氏菌购于黑龙江省临床检测中心。 采用比浊法测定潜在益生菌的生长曲线，结果表明，本实验所测定的9株菌中，产酸能力最强的是PB1和A878、Pba和Inf，其次为PB2、M1、A30、A31、M2。但是其中四株链球菌生长速度较快，没有明显的迟滞期。确定各菌株对数生长期末期分别为：PB1和A878：12h；Inf和Pba：24h；PB2、M1、A30、A31、M2：8-10h。 采用牛津杯法测定八种潜在益生菌对大肠杆菌、痢疾杆菌、沙门氏菌的抑制性，结果表明，嗜酸乳杆菌PB1、A878和双歧杆菌Pba、Inf具有较强的抑菌能力，对三种致病菌能够形成明显的抑菌圈，而且对三种致病菌的抑菌能力明显不同。其抑菌机理主要是分解糖类产生酸类物质发挥主要作用。 将PB1和A878分别与M1、PB2、A30、A31以1:1的比例混合发酵后，测定联合菌株对致病菌的抑制性，结果表明PB1与各球菌联合后，可以互利生长，其中复合较好的为PB1与M1和A30。而A878与各球菌混合发酵后，抑菌性降低。在PB1与各球菌混合培养的基础上，再和Inf和Pba混合培养，结果发酵液中Inf和Pba的生长受到限制，结果表明，在体外Pba和Inf很难与需氧菌共生。 将几株体外性状好的菌株Pba、PB1、M1、PB1+M1、PB1+A30，每天以0.5ml／每只发酵液的量灌胃给正常小白鼠，时间30天，测定小鼠免疫器官指数和血清中细胞因子的变化，并观察小鼠脾脏的变化。结果表明，PB1+A30、PB1、PB1+M1、Pba对脾脏的发育刺激最为明显(P＜0.01)，脾脏指数分别提高了23.4％、29％、36.6％、23.6％。Pba、PB1、A30与PB1+M1与对照组相比可以明显提高小鼠血清中IL-2的含量(P＜0.05)。Pba、PB1与对照组相比，能显著增加IL-6的含量(P＜0.01)。所选菌株均能不同程度的提高实验小鼠血清中TNF-α的含量(P＜0.01)，菌株之间差异不显著。PB1+M1、PB1+A30诱导生成TNF-α的量最多，分别为1.17ng／mL和1.14ng／mL。由此可见，所选益生菌菌株能够增强小鼠的免疫功能。 将PB1、A878分别和各球菌M1、PB2、A30、A31接种于高胆固醇培养基中，在有胆汁和厌氧条件下，37℃混合培养16-18h，测定复合菌株和单菌株降低胆固醇的效果，以未接菌培养基为对照，结果表明，PB1与M1及PB2；A878与A30及A31的协同作用较好，降培养基中胆固醇的量都高于40％以上。单菌株降胆固醇效果最好的是PB1、A878、A30、A31。更多还原

【Abstract】 Probiotic are promising field for development of functional foods in the future lies in its important function on the host for its health benefit and prophylaxis disease. This experiment determined the potential functional properties of probiotic bacteria and its symbiosis in vitro and vivo in order to screen optimal combinative probiotic strains.Ten potential probiotics stains were tested in this experiment. Including two Lactobacillus acidophilus strains FBI and A878, two Bifidobacteria strains Pba and Inf, four Enterococcus strains Ml, PB2, A30, A31.one Bacillus abticisM2, one Clostridium butyricum strain was isolated from commercial probiotics by using TPY. The enterpathogens were bought from HeilongJiang Clinical Monitor Center, Including Escherichia coll, shigellosis, SalmonellaThe growth curve of nine probiotics were tested by monitoring OD620 of fermentative fluid, The result suggested that some strains had strong ability to producing acid including L. acidophilus PB1 and A878, Bifidobacteria Pba and Inf, the following were Enterococcus Ml PB2, A30, A31. But four Enterococcus grew quickly and hadn’ t delaying period, the final period of logarithm growing of all tested strains were 12 hour for I acidophilus PB1 and A878; 24 hour for Bifidobacteria Pba and Inf; 8-10 hour for Enterococcus Ml, PB2, A30, A31 and Bacillus abticisM2The growth inhibition of Escherichia coli, shigellosis, Salmonell by the fermented liquor of eight probiotics bacteria was tested. The results suggested that the growth of three enterpathogens were strongly inhibited by L. acidophilus PB1 and A878, Bifidobacteria Pba and Inf. The diameter of inhibition zone was clear. But there were different ability of probiotics on inhibiting three enterpathogens. The inhibition mechanisms of PB1, A878, Pba, Inf were some organic acids and antibacterial substance in fermented liquor.L. acidophilus strains PB1, A878 mixed with Enterococcus strains Ml, PB2, A30, A31 respectively in the ratio of 1:1 in the MRS cultures, co-cultural and single liquor inhibition to the enterpathogens were tested in the terminal fermentation. The results suggested that L.acidophilus PBlgrew well with Enterococcus strains Ml, PB2, A30, A31.and had effective inhibition on the pathogens, the best combination were L.acidophilus PB1 with Enterococcus M1,A30. L.acidophilus A878 grew not well with Enterococcus strains Ml, PB2, A30, A31. The growth inhibition of enterpathogens was lower than L.acidophilus PB1. On the base of I.acidophilus strains PB1 mixed with Enterococcus strains M1, PB2, A30, A31, Bifidobacteria Pba and Inf were added in the culture.The results suggested Pba and Inf were limited on the co-cultures, its indicated that Pba and Inf grew badly with other probiotics in vitroEach mice were given 0.5ml fermentative liquor of probiotics strains PBl+A30,Pba, PB1,M1, PB1+M1,PB1+A30 for one month. The change of immune organs index and cytokine in the serum were tested. The results suggested that PB1+A30, PB1,PB1+Ml,Pba can improved the growth of spleen significantly with the spleen index enhanced 23.4%,29%,36.6% 23.6%(P<0.01). Amounts of IL-2 in the serum of mice of groups of Pba. PBK A30, PB1+M1were all significantly higher than control groups(P<0.05).The groups of Pba, PB1 can induced amount of IL-6 in the serum more than control groups(P< 0.01). All probiotic can improved amount of TNF-a(P<0.01). there were no difference in probiotics(P<0.01). PB1+M1, PB1+A30 Induced more amount TNF-A were 1.17ng/ml and 1.14 ng/ml respectively. The results indicated that probiotic had promoting effects to the immunity of mice.L.acidophilus strains PB1,A878 mixed with Enterococcus strains M1,PB2,A30,A31. The amounts of cholesterol removed from growth medium were tesed after anaerobical incubation at 37 CThe amount of cholesterol assimilated higher 40% by PB1+M1,PB1+PB2 A878+A30, A878+A31. The result indicated that PBlmixed with Ml, PB2 and A878 mixed with A30,A31 can assimilate cholesterol effectively. And the single strains PB1, A878, A30, A31 also can assimil更多还原