产细菌素屎肠球菌F11.1G的生物学特性分析

Biological characteristics of bacteriocin-producing Enterococcus faecium F11.1G

摘要:

【背景】随着滥用抗生素导致耐药问题日益严重，逐渐破坏动物健康与生态环境。为应对这一全球性公共威胁，多国禁止把抗生素作为食品和饲料添加剂，益生菌的替代使用应运而生。【目的】对产细菌素屎肠球菌(Enterococcus faecium) F11.1G的生物学特性作全面分析，以保证该菌株在生产中的合理及安全应用。【方法】通过热稳定性试验、耐酸试验和耐胆盐试验测定其耐热、耐酸和耐胆盐性能；通过药敏试验探究其耐受药物能力；通过表面疏水性试验、自凝聚性试验间接探究其对细胞的黏附能力；通过蛋白酶耐受试验研究其在蛋白酶作用下活性作用的强弱。【结果】屎肠球菌F11.1G发酵上清液具有较强的热稳定性，在110 ℃条件下仍然能够发挥抑菌作用，而最佳生长温度为37 ℃。当pH 5.9时，菌株生长情况较好且存活率最高，随着pH的降低，其生长受到抑制，当pH值降低为1.8时，菌株仍可生长，但存活率仅有9%。胆盐浓度在0.10%时存活率达140.0%，胆盐浓度在0.15%时，存活率达126.6%，而胆盐浓度在0.20%时，存活率为96.6%；菌株对头孢吡肟、丁胺卡那、庆大霉素、诺氟沙星、红霉素、罗红霉素、多黏菌素B、林可霉素、链霉素、新霉素、甲硝唑、克林霉素、制霉菌素、甲氧苄啶、阿奇霉素和卡那霉素耐受；对头孢他啶、左氧氟沙星、环丙沙星中度敏感；对青霉素、头孢唑啉、头孢呋辛、氯霉素、四环素、多西环素、替考拉宁、克拉霉素和呋喃妥因高度敏感。菌株F11.1G对正己烷、二甲苯、乙酸乙酯和二氯甲烷的疏水率分别为84.29%、25.75%、31.26%和15.08%。菌株F11.1G在3、6、9、12、15和18 h的自凝聚率分别为51%、54%、76%、80%、83%和84%。在蛋白酶K作用下，对大肠杆菌(Escherichia coli)和沙门氏菌(Salmonella)的抑菌效果明显减弱，抑菌圈直径分别为13.05 mm和11.28 mm，抑菌率下降幅度分别为52%和58%，而在胰蛋白酶、胃蛋白酶的作用下未产生抑菌圈，抑菌效果几乎完全丧失。【结论】屎肠球菌F11.1G具有良好的耐高温、耐酸、耐胆盐能力，对丁胺卡那、庆大霉素、诺氟沙星、红霉素和罗红霉素等16种药物耐受，对正己烷有较高的疏水率且自凝聚性良好，经胰蛋白酶、胃蛋白酶处理后抑菌活性丧失，证明该抑菌物质为蛋白类或肽类，为微生态制剂的安全应用提供基础数据。

Abstract:

[Background] The abuse of antibiotics leads to increasingly severe drug resistance, which poses a growing threat to animal health and the environment. In response to this global public health threat, many countries have banned the use of antibiotics as food and feed additives. In this context, probiotics emerge as alternatives. [Objective] To comprehensively characterize bacteriocin-producing Enterococcus faecium F11.1G. [Methods] Tests were carried out to examine the thermal stability, acid tolerance, bile salt tolerance, antibiotic sensitivity, surface hydrophobicity, self-aggregation, and protease resistance of this strain. [Results] The fermentation supernatant of E.faecium F11.1G had strong thermal stability and maintained antibacterial activity at 110 ℃, with an optimal growth temperature of 37 ℃. The strain grew well and had the highest survival rate at pH 5.9. As the pH decreased, the growth was inhibited. However, at pH 1.8, the strain can still grow with a survival rate of 9%. The strain showed the survival rates of 140.0%, 126.6%, and 96.6% at the bile salt concentrations of 0.10%, 0.15%, and 0.20%, respectively. The strain was tolerant to 16 antibiotics: cefepime, amikacin, gentamicin, norfloxacin, erythromycin, roxithromycin, polymyxin B, lincomycin, streptomycin, neomycin, metronidazole, clindamycin, nystatin, trimethoprim, azithromycin, and kanamycin. It was moderately sensitive to ceftazidime, levofloxacin, and ciprofloxacin and highly sensitive to penicillin, cefazolin, cefuroxime, chloramphenicol, tetracycline, doxycycline, teicoplanin, clarithromycin and nitrofurantoin. The hydrophobicity of E.faecalis F11.1G to hexane, xylene, ethyl acetate, and dichloromethane was 84.29%, 25.75%, 31.26%, and 15.08%, respectively. The self-aggregation rates of E.faecalis F11.1G at the time points of 3, 6, 9, 12, 15, and 18 h were 51%, 54%, 76%, 80%, 83%, and 84%, respectively. In the presence of protease K, the inhibitory effects of the strain against Escherichia coli and Salmonella were significantly weakened, with the inhibition zone diameters of 13.05 mm and 11.28 mm and the inhibition rate decreases of 52% and 58%, respectively. In the presence of trypsin and pepsin, no inhibition zone was formed, which indicated that the strain almost completely lost the antibacterial effects. [Conclusion] E.faecium F11.1G demonstrates strong tolerance to high temperatures, acids, and bile salt, resistance to 16 antibiotics, high hydrophobicity to hexane, and high self-aggregation. However, it loses its antibacterial activity after treatment with trypsin and pepsin, which indicates that the antibacterial substance is a protein or peptide. The results provide basic data for the safe application of probiotics.