产抗真菌肽的爱媛类芽孢杆菌HD的筛选、鉴定及发酵条件优化  

Screening,Identification and Optimization the Fermentation Conditions of Paenibacillus ehimensis HD Producing Antifungal Peptides

作　　者：鲁雷震 宏丹 封成玲 贾紫伟 周景波 宁亚维[1] 贾英民[2] 王志新[1] Lu Leizhen;Hong Dan;Feng Chengling;Jia Ziwei;Zhou Jingbo;Ning Yawei;Jia Yingmin;Wang Zhixin(College of Food Science and Biology,Hebei University of Science and Technology,Shijiazhuang 050018;School of Food and Health Engineering,Beijing Technology and Business University,Beijing 100048)

机构地区：[1]河北科技大学食品与生物学院,石家庄050018 [2]北京工商大学食品与健康学院,北京100048

出　　处：《中国食品学报》2022年第3期212-224,共13页Journal of Chinese Institute Of Food Science and Technology

基　　金：河北省自然科学基金项目(C2020208014);河北省重点研发计划项目(20327124D)。

摘　　要：目的:从土壤中筛选产抗真菌肽的菌株,并通过优化其发酵条件提高产量。方法:用琼脂扩散法筛选菌株;通过形态观察、理化测定和16S rDNA序列分析鉴定菌株;采用蛋白酶处理、硫酸铵沉淀、膜透析、热稳定性研究确定抑菌物质;采用单因素和正交试验优化其发酵条件,并在发酵罐中放大验证。结果:获得1株产抗真菌物质的菌株,鉴定为爱媛类芽孢杆菌,命名为爱媛类芽孢杆菌(Paenibacillus ehimensis)HD。所产抑菌物质为小分子多肽,分子质量约1 ku,其抑菌谱广,能抑制多株腐败真菌和致病细菌。爱媛类芽孢杆菌HD发酵产抗真菌肽最佳条件为:甘露醇2.5 g/L,MgSO_(4)3.0 g/L,鱼蛋白胨10 g/L,初始pH 7.5,装液量40 mL/250 mL,接种量1×10_(8) CFU/mL,摇床转速220 r/min,发酵温度32℃,发酵时间32 h。在该条件下,抗真菌肽效价为324.92 AU/mL,比优化前提高了3.63倍。在5 L和30 L发酵罐中进行发酵验证,抗真菌肽效价分别为371.01 AU/mL和364.05 AU/mL,产量达到最高时的时间提前至20 h。结论:爱媛类芽孢杆菌HD所产抗菌肽具有较好抑菌作用,且经发酵优化与放大后产量稳定。Objective:Strains for antifungal peptides production were screened and isolation from soil,and then the antifungal peptides yield was improved by optimizing its fermentation conditions.Methods:Agar plate dilution and agar diffusion method were used for the strain isolation.Morphological,physiological and biochemical characteristics and 16S rDNA sequence of the strain were used for the strain analysis and identification.The antifungal properties of antifungal substances were determined by protease treatment,ammonium sulfate precipitation,membrane dialysis and thermal stability study.The fermentation medium and conditions for antifungal peptides production were optimized by single factor and orthogonal tests,and then the fermentation amplification was carried out in the fermenter.Results:An antifungal peptides-producing strain was screened and identified as Paenibacillus ehimensis.The strain was named as Paenibacillus ehimensis HD.The antifungal substances produced by P.ehimensis HD was verified as small molecular polypeptides with the molecular weight of about 1 ku.The antifungal peptides had a broad spectrum of antimicrobial activity,which could not only inhibit the spoilage fungi,but also inhibit the pathogenic bacteria.The optimum conditions for antifungal peptides production by P.ehimensis HD were as follows:mannitol 2.5 g/L,MgSO_(4)3.0 g/L,fish peptone 10 g/L,initial pH 7.5,liquid volume 40 mL/250 mL,inoculation amount 1×10^(8) CFU/mL,shaker speed 220 r/min,fermentation temperature 32℃,fermentation time 32 h.Under the optimal conditions,the titer of antifungal peptides was 324.92 AU/mL,which was 3.63-fold compared to the initial medium.Scale-up of antifungal peptides fermentation from shaking flask to 5 L and 30 L fermenters,the titers of antifungal peptides were 371.01 AU/mL and 364.05 AU/mL,respectively,moreover,the fermentation time was forward to 20 h.Conclusion:The antifungal peptides produced by P.ehimensis HD had good antifungal activity,and the yield of antifungal peptides was stable after fermenta

关 键 词：爱媛类芽孢杆菌 抗真菌肽 筛选 鉴定 发酵优化 

分 类 号：TS201.3[轻工技术与工程—食品科学]