贝莱斯芽孢杆菌BR-01菌株高产抗菌肽培养基的优化及其抗菌肽的鉴定

Optimization of High-yield Antimicrobial Peptide Culture Medium of Bacillus velezensis BR-01 and Identification of Its Antimicrobial Peptide

关键词:  贝莱斯芽孢杆菌  水稻细菌性条斑病菌  抗菌肽  发酵条件  响应面法

Key words:  Bacillus velezensis  Xanthomonas oryzae pv.oryzicola  antimicrobial peptide  fermentation conditions  response surface methodology

摘要:

贝莱斯芽孢杆菌(Bacillus velezensis) BR-01菌株对水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)具有较好的拮抗作用。为了提高该菌株的抗菌肽发酵产量和生防价值，对其产抗菌肽培养基进行了优化，并初步鉴定了其抗菌肽的成分。以B.velezensis BR-01菌株无菌发酵滤液对Xoc的抑菌圈直径为评价指标，从7种常见培养基中筛选出最适培养基，通过单因素试验优化初始培养基各组分含量和发酵条件，再利用Plackett-Burman (PB)试验筛选出影响发酵滤液抑菌活性的显著因素，运用响应面分析法获得最优发酵条件。利用PCR分析B.velezensis BR-01基因组中抗菌肽合成相关基因；使用液相色谱-质谱联用(Liquid Chromatography-Mass Spectrometer,LC-MS)技术鉴定其产生的抗菌肽。结果表明，B.velezensis BR-01菌株最佳抗菌肽培养基配方与发酵条件为麦芽糖11.89 g/L、酵母提取物13.54 g/L、NaCl 5 g/L、KH₂PO₄ 1 g/L、初始pH值为7、发酵时间48 h、发酵温度32.3 ℃。该优化方案下，B.velezensis BR-01菌株的无菌发酵滤液对Xoc的抑菌圈直径为(42.5±0.2) mm，显著高于优化前的(28.0±0.9) mm，比优化前增大51.79%。抗菌肽主要成分初步鉴定为溶杆菌素(Bacilysin)、表面活性素(Surfactin)、伊枯草菌素(Iturin)和丰原素(Fengycin)。本研究为B.velezensis BR-01菌株用于快速、批量发酵制备生防菌液，以及其抗菌肽的分离纯化、结构鉴定、田间生防利用等后续研究提供了理论基础。

Abstract:

Bacillus velezensis BR-01 strain has a good antagonistic effect on Xanthomonas oryzae pv.oryzicola (Xoc).In order to improve the antimicrobial peptide fermentation yield and biocontrol value of the strain,the medium for producing antimicrobial peptides was optimized,and the components of antimicrobial peptides were preliminarily identified.The diameter of the inhibition zone of B.velezensis BR-01 sterile fermentation filtrate against Xoc was used as the evaluation index,and the optimal medium was selected from 7 common media.The content of each component of the initial medium and the fermentation conditions were optimized by single factor test.Plackett Burman (PB) test was used to screen out the significant factors affecting the antibacterial activity of the fermentation filtrate,and the optimal fermentation conditions were obtained by response surface analysis.The genes related to antimicrobial peptide synthesis in B.velezensis BR-01 genome were analyzed by PCR.Liquid Chromatography-Mass Spectrometer (LC-MS) was used to identify the antimicrobial peptides.The results showed that the optimal antimicrobial peptide medium and fermentation conditions of B.velezensis BR-01 strain were as follows:maltose 11.89 g/L,yeast extract 13.54 g/L,NaCl 5 g/L,KH₂PO₄ 1 g/L,initial pH value is 7,fermentation time 48 h,and fermentation temperature 32.3 ℃.Under this optimization scheme,the inhibition zone diameter of the sterile fermentation filtrate of B.velezensis BR-01 strain against Xoc was (42.5±0.2) mm,which was significantly higher than that before optimization (28.0±0.9) mm,which was 51.79% higher than that before optimization.The main components of the antimicrobial peptide were preliminarily identified as Bacilysin,Surfactin,Iturin and Fengycin.This study provides a theoretical basis for the rapid and batch fermentation of B.velezensis BR-01 strain for the preparation of biocontrol bacterial liquid,and provides a theoretical basis for the subsequent research on the separation and purification,structural identification and field biocontrol utilization of its antimicrobial peptides.