短头熊蜂肠道拮抗菌株的分离筛选鉴定及其生物特性评价

Isolation, screening, identification, and biological characterization of antagonistic strains in intestinal tract of Bombus breviceps

摘要:

【背景】近年来，由于栖息地减少、农药的大量使用及病原菌侵染等综合因素，导致全世界的熊蜂种类与数量逐年减少，病原菌的侵染可通过微生物在自身生长过程中会产生的抑菌物质进行有效抑制或杀灭。【目的】短头熊蜂(Bombus breviceps)长期生存在野外环境中，其肠道内存在着大量微生物资源。从短头熊蜂肠道内筛选拮抗菌株，并对其抑菌特性进行研究。【方法】采用牛津杯双层法筛选拮抗菌株，测定抑菌活性最佳菌株发酵液的抑菌物质稳定性与抑菌广谱性等抑菌特性，并借助细胞膜通透性、流式细胞仪检测等试验探究其抑菌机制。【结果】得到了5株具有明显抑菌作用的拮抗菌株，其中果杆菌(Fructobacillus tropaeoli)CZ01对金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌(Salmonella choleraesuis)、大肠杆菌(Escherichia coli)、福氏志贺氏菌(Shigella flexneri)和无乳链球菌(Streptococcus agalactiae)这5种病原指示菌都具有高度抑菌效果。菌株CZ01对金黄色葡萄球菌的抑菌效果最佳，抑菌圈直径可达到(21.21±0.25) mm，在121 ℃处理后仍具有67.36%以上的抑菌活性，调整pH值为10.0时仍具有78.16%的抑菌活性。【结论】短头熊蜂肠道微生物资源较丰富，尤其是果杆菌(F.tropaeoli)CZ01具有抑菌活性高、稳定性好、抑菌谱广等特性，对金黄色葡萄球菌具有良好的杀灭效果，显示出良好的应用潜能。

关键词:短头熊蜂;拮抗菌株;抑菌作用;肠道微生物

Abstract:

[Background] In recent years, bumblebee species and number have been decreasing worldwide due to habitat reduction, abuse of pesticides, and infection of pathogens. The pathogens can be effectively killed by the antimicrobial substances produced by microorganisms during their growth. [Objective] Bombus breviceps lives in the wild for a long time and has rich microbial resources in the intestinal tract. We screened antagonistic strains from the intestinal tract of B. breviceps and studied their antibacterial properties. [Methods] The Oxford cup double-layer method was used to screen out the antagonistic strains and determine the stability of antibacterial substances and the inhibition spectrum of the fermentation broth of the strains with strong inhibitory activity. Further, cell membrane permeability and flow cytometry were employed to investigate the inhibition mechanism. [Results] Five antagonistic strains with significant antibacterial effect were obtained, among which Fructobacillus tropaeoli CZ01 demonstrated strong inhibitory effect on all the five indicator bacteria: Staphylococcus aureus, Salmonella choleraesuis, Escherichia coli, Shigella flexneri, and Streptococcus agalactiae. It showed the strongest inhibitory effect on Staphylococcus aureus, with an inhibition zone diameter of (21.21±0.25) mm. Moreover, the inhibitory activity was still 67.36% after treatment at 121 ℃ and 78.16% after the medium was adjusted to pH 10.0. [Conclusion] B. breviceps carries rich microbial resources in intestinal tract. In particular, F. tropaeoli CZ01 with high antibacterial activity, good stability, and wide intestinal spectrum, has good killing effect on S. aureus and demonstrates good application potential.

Key words:Bombus breviceps;antagonistic strain;antibacterial effect;intestinal microbiota