双歧杆菌试管内生物拮抗作用研究

Biological antagonism of Bifidobacteria in vitro

目的本实验从正常青少年体内分离出1株双歧杆菌,通过试管内研究双歧杆菌对大肠埃希菌和粪肠球菌的生物拮抗作用。为双歧杆菌临床应用及食品开发提供一定的基础理论依据和广阔的市场前景。方法本项研究以从佳木斯大学健康学生体内分离出1株双歧杆菌为试材,将大肠埃希菌、粪肠球菌接种于PYG液体培养基中,37℃厌氧培养48 h,将菌液浓度利用分光光度计法调到2麦氏。将大肠埃希菌对照组分为10支,分离的双歧杆菌+大肠埃希菌组10支,分离的双歧杆菌对照组10支,分离的双歧杆菌+粪肠球菌组10支,粪肠球菌对照组10支,将以上50个试管全部置于厌氧培养箱内37℃培养48 h,将培养好的大肠埃希菌和粪肠球菌进行梯度稀释,然后用微量移液器吸取标本于每个培养基上滴3滴,每滴滴上由高稀释度向低稀释度,液体量为10μL的菌液。晾干滴种好的培养基于适宜条件进行培养并选出适合菌落生长的稀释度,计算同一稀释度平均菌落数(x)。结果双歧杆菌12 h时对大肠埃希菌生长无显著性,在24 h时检测不到大肠埃希菌的存在。12和24 h时粪肠球菌与双歧杆菌混合组与粪肠球菌对照组相比,差异均无统计学意义(P>0.05)。结论体外对照实验研究该双歧杆菌对大肠埃希菌和粪肠球菌的生物拮抗作用,此株双歧杆菌只对大肠埃希菌有抑制作用,而对粪肠球菌无抑制作用,机制需要深入研究。

Objective To investigate Bifidobacterium＇s biological antagonism on Escherichia coli and Enterococcus faecalis in vitro, and provide theoretical basis and broad market prospects for the clinical application and foods development of Bifidobacterium. Methods A strain of Bifidobacterium isolated from healthy students of Jiamusi University was used as the test material. Escherichia coli and Enterococcus faecalis were inoculated on PYG liquid media and cultured at 37 ~C for 48 hour in an anaerobic environment; then the bacterial concentrations were regulated to McFarland 2 by spectrophotometer. 50 test tubes, including 10 test tubes of Escherichia coli controls, 10 separated Bifidobacterium ＋ Escheriehia, 10 separated Bifidobacterium controls, 10 separated Bifidobacterium ＋ Enterococcus faecalis and 10 Enterococcus faecalis controls, were cultured in an anaerobic incubator for 48 hours at 37 ℃. Then the Escherichia coli and Enterococcus faecalis were diluted in gradience and dropped onto the media, three drops on each medium from high dilution to low di- lution, 10μL each drop. After air -dried and cultured under appropriate conditions, appropriate dilutions were selected for calculation of the average colony number （x）. Results Bifidobacterium had no significant impact on the growth of Escherichia coli after 12 hours and no Escherichia coli were detected after 24 hours. Enterococcus faecalis ＋ Bifidobacterium group showed no significant difference when comparing with Enterococcus faecalis group after 12 hours and 24 hours （P 〉 0.05）. Conclusion This strain of Bifidobacterium has an inhibitory effect on Escherichia coli other than Enterococcus faecalis; the mechanism need be further studied.