酸马奶提取Kluyveromyces marxianus代谢抗菌复合物对致病性大肠杆菌的抑菌和细胞表面特性的影响

Effects of antimicrobial compounds of Kluyveromyces marxianus in Koumiss on pathogenic Escherichia coli and its cell surface characteristics

【目的】探究酸马奶提取马克斯克鲁维酵母(Kluyveromyces marxianus)代谢产生的抗菌复合物K.marxianus p H 2.0和K.marxianus p H 8.0(简称为K2和K8)对致病性大肠杆菌Escherichia coli O8的抑菌效果和细胞表面特性的影响。【方法】乙酸乙酯萃取法制备K2和K8,牛津杯法测定其对E.coli O8的抑菌圈,高效液相色谱法测定其有机酸的组成,试剂盒测定其毒素蛋白浓度,肉汤稀释法测定其对E.coli O8的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),酶标比浊法测定其对E.coli O8生长曲线的影响,微生物粘附法测定其对E.coli O8细胞表面疏水性的影响,邻硝基苯β-D-半乳吡喃糖苷(ONPG)法测定其对E.coli O8细胞膜渗透性的影响。【结果】乙酸乙酯萃取法获得抗菌复合物溶液,其中p H 2.0水相与p H 8.0水相抑菌圈最大,冻干得K2和K8,主要组分为丙酸等有机酸和毒素蛋白。K2和K8对E.coli O8的MIC分别为0.025 g/m L和0.100 g/m L,MBC分别为0.100 g/m L和0.200 g/m L。K2和K8能影响E.coli O8的生长曲线,增加E.coli O8的疏水性和渗透性,且K2优于K8。【结论】酸马奶提取K.marxianus代谢抗菌复合物K2和K8能抑制致病性E.coli O8生长,影响其细胞表面特性。

[Objective] The aim of this study was to investigate the effects of antimicrobial compounds of Kluyveromyces marxianus（K2 and K8） on wild pathogenic Escherichia coli and its cell surface characteristics. [Methods] K2 and K8 were extracted by ethyl acetate, and the inhibition zones of K2 and K8 against E. coli O8 were determined by Oxford cup method. The organic acids were determined by HPLC, and the concentrations of killer toxin were determined by enhanced BCA Protein Assay Kit. The minimum inhibition concentration（MIC） and the minimum bactericidal concentration（MBC） were determined by broth dilution method, the effects of K2 and K8 on the growth curve of E. coli O8 were determined by turbidimetry. Moreover, the effects of K2 and K8 on the hydrophobicity of the E. coli O8 cell surface was determined using the microbial adhesion to solvents method, and the permeation of E. coli O8 cell membrane were determined by measuring the release of β-galactosidase activity into the culture medium using ONPG as a substrate. [Results] The aqueous phases of p H 2.0 and p H 8.0 had higher inhibition zones, they were dried for 48 h by freeze-drying, then, K2 and K8 were obtained, the main components were propanoic acid and some other organic acids, and killer toxins. The MICs of K2 and K8 were 0.025 g/m L and 0.100 g/m L, respectively. The MBCs of K2 and K8 were 0.100 g/m L and 0.200 g/m L, respectively. The growth curve of E. coli O8 was S-shape. It changed obviously after adding K2 and K8. E. coli O8 was basic character, and had a hydrophilic surface. The hydrophobicity increased after adding K2 and K8. In addition, the release of the β-galactosidase in permeation of E. coli O8 was promoted gradually by K2 and K8, and it also caused membrane lesions allowing ONPG uptake into cells. These two factors resulted in the increasing permeation. K2 was better than K8. [Conclusion] K2 and K8 could inhibit the growth of pathogenic E. coli O8 and influence its cell surface characteristics.