紫外诱变选育高产丁二酮乳酸菌株及其低成本发酵优化  

Ultraviolet mutagenic selection of a lactobacillus with high productivity of butanedione and low cost optimization of fermentation

作　　者：张白梅 徐辉[1] 曹瑜[1] 刘博[1] 胡超[1] 李宪玲[1] 史红霞[1] 曹毅[1] 乔代蓉[1] 

机构地区：[1]四川大学生命科学学院,微生物与代谢工程四川省重点实验室,成都610065

出　　处：《应用与环境生物学报》2017年第3期437-442,共6页Chinese Journal of Applied and Environmental Biology

基　　金：“十二五”国家科技支撑计划项目(2014BAD02B02);四川省科技厅项目(2012GZ0008,2014GZX0005,2017TJPT0001)资助

摘　　要：2,3-丁二酮是一种常用的安全食品添加剂,为了提高微生物发酵中菌株的丁二酮产量,从泡菜水样品中分离筛选出一株野生型高产丁二酮菌株,并进行紫外诱变选育以及发酵条件优化.筛选获得高产丁二酮野生型乳酸菌株(1)-2,产量为67.02 mg/L,经16S r DNA分子鉴定为植物乳杆菌(Lactobacillus plantarum).紫外诱变获得高产丁二酮突变株U13,产量为127.88 mg/L.对突变前后菌株几种丁二酮代谢相关酶酶活变化的比较结果显示,突变株丁二酮产量的提高是因为乳酸脱氢酶减少及乙酰乳酸合成酶增加.正交实验优化突变株U13的最佳丁二酮发酵条件为接种量3%,初始pH 6.6,葡萄糖30 g/L,组合氮源(蛋白胨:酵母粉:牛肉膏=2:1:2)20 g/L,柠檬酸氢二铵3 g/L,乙酸钠2 g/L,吐温-80 1 m L/L,K_2HPO_4 2 g/L,Mg^(2+)2 mmol/L,Mn^(2+)0.7 mmol/L,Cu^(2+)2 mmol/L,温度为37℃.利用廉价碳氮源淀粉及小麦麸皮替代原有碳氮源,淀粉替代率不超过20%、小麦麸皮替代率不超过40%时,丁二酮产量降幅比较低.本研究通过诱变选育和发酵条件优化,提高了菌株丁二酮产量,并通过廉价碳氮源替换降低了成本,可为丁二酮的微生物工业发酵提供参考.Because 2,3-butanedione is a safe food additive, it is generating increasing interest among scientists. Therefore, the aim of this study was to screen bacteria for a strain with high production of 2,3-butanedione. First, we found （by screening） several wild-type strains yielding high concentration of 2,3-butanedione in pickle samples; then ultraviolet （UV） mutagenesis and fermentation optimization were used to improve the yield; additionally, the strains were identified by 16S rDNA molecular subtyping. Finally, to reduce the production cost, cheaper carbon and nitrogen sources were used to partially replace the original sources, thereby we investigated the relation between the nutrient replacement and 2,3-butanedione production. The results were as follows： （1） Using the screening, we obtained a wild-type strain （named 1-2） with 2,3-butanedione production of 67.02 mg/L; （2） the strain was identified as Lactobacillus plantarum; （3） we created an improved mutant strain （named U13） with 2,3-butanedione production of 127.88 mg/L; （4） using the orthogonal optimization test, we obtained an optimized strain with 2,3-butanedione production of 169.23 mg/L, and the conditions were as follows： inoculum concentration of 3%, initial pH of 6.6, 30 g/L glucose, 20 g/L nitrogen source （peptone：yeast powder：beef extract at 2：1：2）, 3 g/L ammonium citrate dibasic, 2 g/L sodium acetate, 1 ml/L Twain-80, 2 g/L K2HPO4, 2 mmol/L Mg2＋, 0.7 mmol/L Mn2＋, 2 mmol/L Cu2＋, temperature of 37 ℃. （5） The reduction in 2,3-butanedione production was relatively small when the replacement rate of a cheaper carbon source （i.e., starch） was not more than 20% and replacement rate of a cheaper nitrogen source （i.e., wheat bran） was not more than 40%. Thus, we successfully obtained a strain with high production （169.23 mg/L） of 2,3-butanedione, based on UV mutagenesis and fermentation optimization and successfully reduced the production cost by replacing the original carbon and nitro

关 键 词：2 3-丁二酮 植物乳杆菌 紫外诱变 发酵优化 小麦麸皮 

分 类 号：Q93[生物学—微生物学] TQ920.6[轻工技术与工程—发酵工程] TS202.3