猪源产细菌素芽孢杆菌的筛选及抑菌特性

【背景】抗生素作为生长促进剂在畜牧业中的滥用,出现了严重的耐药基因富集和扩散问题,发掘新兴的生长促进剂作为饲料添加剂市场潜力巨大,目前益生菌制剂的开发最具潜力。【目的】通过对散养健康育肥猪粪便中芽孢杆菌的分离筛选,获得对典型肠道病原菌具有显著抑菌活性的芽孢杆菌,确定其产生的细菌素特性,以此对芽孢杆菌作为猪养殖业生长促进剂的潜力进行评价。【方法】采用梯度稀释涂平板法分离可培养细菌,利用牛津杯法检测菌株的抑菌活性。通过微生物形态及16S r RNA基因序列分析,确认6株产细菌素菌株的分类地位,并对其抗生素耐药性、细菌素稳定性及生理生化特征进行比较分析。【结果】从116株纯培养物中筛选得到6株对指示病原细菌具有显著抑菌效应的产细菌素芽孢杆菌,其中2株为贝莱斯芽孢杆菌(Bacillus velezensis),3株为枯草芽孢杆菌(Bacillus subtilis),1株为地衣芽孢杆菌(Bacillus licheniformis)。菌株B.licheniformis DY7和B.subtilis FX4对致泻、产肠毒素、出血性Escherichia coli均有显著的抑制效果,对头孢噻肟和红霉素高度敏感,其细菌素在p H 3.0-9.0、50-100°C水浴处理后仍具有明显的抑菌活性。【结论】猪源产细菌素芽孢杆菌DY7和FX4具有高效的病原细菌抑菌能力,所产细菌素稳定性较好,具有作为动物生长促进剂的应用潜力。

Selection and antibacterial activity of bacteriocin-producing Bacilli from swine

Background] Using traditional antibiotics as growth promoters in the pig industry leads to the richness and transmission of bacterial resistant genes. Thus, the identification and development of the emerging growth promoters as alternatives to antibiotics represents tremendous economic potential in feed additive industry. Probiotic cultures are interesting candidates as feed additives for food-producing animals. Objective] This work had as main objectives to isolate Bacilli from the feces of free-range swine and then to characterize bacteriocins produced by Bacilli showing inhibition of typical gastrointestinal bacterial pathogens, in order to evaluate their potential as new growth promoters. Methods] Using the dilution plate separation method and coated plate method, we screened culturable Bacilli. Strains presenting inhibitory properties for gastrointestinal bacterial pathogens were determined by Oxford cup method. The bacteriocin-producing Bacillus isolates were identified by 16S rRNA gene sequencing. Results] Screening of 116 candidate strains isolated from feces samples resulted in the selection of six isolates presenting markedly inhibitory properties for bacterial pathogens, of which two strains were identified as Bacillus velezensis, three as Bacillus subtilis and one as Bacillus licheniformis. Among of them, B. licheniformis strain DY7 and B. subtilis strain FX4 exhibited the most effective inhibitory activity against the indicator pathogens, except for Salmonella enterica CICC10420, and were susceptible to Cefotaxime and Erythromycin. Additionally, both bacteriocins were stable over a wide range of pH 3.0?9.0 and temperatures 50?100 °C. Conclusion] The effective inhibitory activity against the indicator pathogens and the stability may lead to the assumption that the strains DY7 and FX4 may be applied as pig growth promoters to control pathogens.