酸马奶提取Kluyveromyces marxianus代谢抗菌复合物对感染致病性Escherichia coli O_8小鼠的免疫机能及其盲肠菌群的影响

Effect of antimicrobial compounds of Kluyveromyces marxianus in Koumiss on immune function and caecal microflora in mice challenged with pathogenic Escherichia coli O_8

【目的】酸马奶可防治心血管、消化系统、肺结核、糖尿病和腹泻等疾病,尤其马克斯克鲁维酵母Kluyveromyces marxianus对单核细胞增生李斯特菌Listeria monocytogenes有抑菌作用,但对酸马奶提取K.marxianus代谢抗菌复合物抑菌作用的研究报道较少。为此,研究酸马奶提取K.marxianus代谢抗菌复合物对感染致病性大肠杆菌Escherichia coli O_8小鼠免疫机能及其盲肠菌群的影响。【方法】将128只小鼠随机分为4组,空白组、致病对照组、K2组和K8组,致病对照组小鼠连续7 d灌胃无菌PBS,并于第4天注射E.coli O_8;K2组灌胃抗菌复合物K.marxianus p H 2.0,并注射E.coli O_8;K8组灌胃抗菌复合物K.marxianus p H 8.0,并注射E.coli O_8。采用常规HE染色法观察0、4和7 d小鼠小肠病理切片,称重法测定小鼠免疫器官指数,ELISA法测定血清中免疫球蛋白,流式细胞术测定T细胞亚群,平板涂布法测定盲肠菌群。【结果】致病对照组小鼠在实验第4天注菌后出现一系列患病临床症状和小肠组织病理变化。K2组和K8组小鼠整体精神状态好于致病对照组,死亡小鼠数量较少,可一定程度上缓解和改善感染致病性E.coli O_8小鼠的小肠组织病理变化。致病对照组在第7天胸腺指数显著低于空白组(P<0.05)。K2组和K8组在第4天和第7天脾脏指数显著高于空白组(P<0.05)。致病对照组在第7天Ig A显著低于空白组(P<0.05)。K2组在第4天Ig A、Ig G显著高于空白组(P<0.05)。K2组在第7天Ig G和Ig M显著高于空白组(P<0.05)。K8组在第7天Ig M显著高于空白组(P<0.05)。K2组在第4天和第7天CD8+显著低于空白组,CD4+/CD8+显著高于空白组(P<0.05)。K8组在第4天CD8+显著低于空白组(P<0.05)。K8组在第7天CD8+显著低于空白组,CD4+/CD8+显著高于空白组(P<0.05)。致病对照组在第7天盲肠E.coli数量显著高于空白组,双歧杆菌数量显著低于空白组(P<0.05)。K2组在第7天盲肠E.coli数量显著低于空白组,肠球菌数量显著低于空白组,乳酸杆菌数量显著高于空白组(P<0.05)。K8组在第7天盲肠肠球菌数量显著低于空白组,乳酸杆菌数量显著高于空白组(P<0.05)。【结论】酸马奶提取K.marxianus代谢抗菌复合物K2和K8能缓解感染致病性E.coli O_8小鼠临床症状,提高其免疫机能,并影响其盲肠菌群,提高双歧杆菌和乳酸杆菌,降低E.coli和肠球菌的数量。

[Objective] Koumiss had beneficial therapeutic effects on cardiovascular disease, digestion disease, tuberculosis, diabetes and diarrhea, especially Kluyveromyces marxianus had antibacterial effect on Listeria monocytogenes. There was limited knowledge about antimicrobial compounds of K. marxianus in Koumiss, so, we evaluated the effect of antimicrobial compounds of K. marxianus in Koumiss on immune function and caecal microflora in mice challenged with pathogenic Escherichia coli O8. [Methods] Kunming strain mice （128 heads, 20±2 g） were divided into 4 groups, the control group, the challenged control group, K2 group, and K8 group. Mice in the challenged control group were oral administrated sterile PBS by gavage for 7 d, then were administered intraperitoneally E. coliO8 at 4 d. Mice in K2 group were oral administrated K. marxianus pH 2.0 and K8 group were oral administrated K. marxianus pH 8.0, then were administered intraperitoneally E. cob O8. The pathological section of small intestine of mice were observed by HE staining at 0, 4, 7 d. The thymus index and spleen index were weighed and calculated. Immunoglobulins in serum were monitored by enzyme-linked immunosorbent assay （ELISA）. T subsets were analyzed by flow cytometry. The eaeeal mieroflora was calculated by the spread plate method. [Results] Mice in the challenged control group appeared clinical symptoms and pathological changes of small intestine after they were challenged with E. coli O8 at 4 d. Mice in K2 and K8 groups had better mental state, and less died mice than the challenged control group, K2 and K8 could improve pathological changes of small intestine after mice were challenged with E. coli O8. Compared with the control group, the thymus index was decreased at 7 d by the challenged control group, while the spleen index was increased at 4 d and 7 d by K2 and K8 groups （P〈0.05）. Compared with the control group, IgA was decreased at 7 d by the challenged control group, while IgA and IgG were increased at 4 d, IgG and IgM were increased at 7 d by K2 group, IgM was increased at 7 d by K8 group （P〈0.05）. Compared with the control group, CD8＋ was decreased and CD4＋/CD8＋ was increased at 4 d and 7 d by K2 group, CD8＋ was decreased at 4 d by K8 group, CD8＋ was decreased and CD4＋/CD8＋ was increased at 7 d by K8 group （P〈0.05）. Compared with the control group, E. coli was increased and Bifidobacterium was decreased at 7 d by the challenged control group （P〈0.05）, while E. coli was decreased, Enterococcus was decreased, and Lactobacillus was increased at 7 d by K2 group （P〈0.05）, Enterococcus was decreased, Lactobacillus was increased at 7 d by K8 group （P〈0.05）. [Conclusion] These results suggested that antimicrobial compounds of K. marxianus in Koumiss named K2 and K8 could ease clinical symptoms after mice were challenged with pathogenic E. coli O8, enhance their immune function, influence their caecal microflora as Bifidobacterium and Lactobacillus increased, E. coli and Enterococcus decreased.