Nisin高产菌株的高通量筛选

High-throughput screening method for high-yield nisin strain

【目的】乳酸链球菌素(nisin)是一种天然生物活性抗菌肽,对包括食品腐败菌和致病菌在内的许多革兰氏阳性菌具有强烈的抑制作用,而用作食品的防腐剂。本研究通过建立高通量筛选方法,实现高效快速省力的高产菌株筛选,为工业上筛选高产菌株提供研究方案。【方法】通过对Lactococcus lactis ATCC11454菌株进行紫外诱变,获得2511株突变株。利用Biomek FXP自动工作站建立96微孔板的高通量筛选方法,突变株经高通量挑选、菌种培养及菌液稀释后,加入到生长至对数中期的藤黄微球菌中,采用改进后的比浊法快速检测nisin生物活性。用此方法对突变株进行初筛、复筛后可得到nisin高产菌株,并通过摇瓶发酵评估高通量筛选方法。【结果】确定比浊法检测的条件为:nisin活性稀释在10–25 IU/m L范围内,与藤黄微球菌反应2 h后检测藤黄微球菌的菌体量(OD600)。2511株突变株经过2轮高通量筛选,最终获得约50株产量提升的菌株,对其中8株进行摇瓶精确测量,显示产量均有提高,并且其中一株产量提升了30%,成功建立了高通量筛选nisin高产菌株的方法。【结论】利用比浊检测法,在其基础上成功建立高通量筛选高产nisin菌的方法,经过初筛复筛,整个周期由1人耗时5 d即可完成2511株突变株的筛选工作。相较于传统的选育方法,高通量筛选具有快速、稳定、高效的特点,提高了筛选效率,缩短了选育周期,是工业上筛选高产nisin菌的有效手段。

[Objective] Nisin,a bioactive antimicrobial peptide,inhibits many Gram-positive bacteria causing food spoilage and infection.In this study,cost-effective screening method for high-yield nisin strains was developed using high-throughput screening technology to provide a practical method for screening during the industrial production process.[Methods] Mutants（2511） were obtained by applying ultraviolet mutagenesis treatments to Lactococcus lactis ATCC 11454,followed by development of a 96-well plate high-throughput screening method using a Biomek FXP automatic workstation to select mutant strains for culture dilution.Diluted bacterial solutions were transferred to another 96-well plate with Micrococcus luteus grown to logarithmic phase,and an improved turbidimetric method was used to rapidly detect the biological activity of nisin and screen the mutants associated with the high-yield strains.High-yield strains were fermented by shake-flask fermentation to evaluate the screening method.[Results] Turbidimetric detection revealed nisin activity in the range between 10 and 25 IU/m L,with M.luteus biomass detected after a 2 h reaction with nisin.After two rounds of high-throughput screening,about 50 high-yield strains were obtained from the 2511 mutants,with 8 accurately detected by shake-flask fermentation.Results indicated that the nisin yield of the tested strains all increased,with one strain showing a 30% increase in production,indicating the efficacy of the high-throughput screening method.[Conclusion] Our high-throughput screening method was capable of screening high nisin-producing bacteria.