Combinatorial mutagenesis of high-yielding esterase bacteria and optimization of solid-state fermentation
ZHAO Zhijun1, ZHANG Yanzhu1,3, LIU Yanbo1, ZHOU Pingping1, GE Shaohua2, SUN Xiyu1*
1(Liquor College,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China) 2(Baofeng Liquor Industry Co.Ltd,Pingdingshan 467500,China) 3(College of Food and Pharmaceutical Science,Ningbo University,Ningbo 315211,China)
Abstract: In order to obtain bacteria with high esterase production,combinatorial mutagenesis using ultraviolet (UV) and diethyl sulfate (DES) technology was applied to previous isolated strain Bacillus velezensis Nz 1.201.The high-yielding mutant strain D27 was selected by the transparent circle method for primary screening and the esterase activity measurement for rescreening.After five consecutive subcultures,its genetic performance was relatively stable.The enzyme activity reached 62.45 U/100 g,which was 66% higher than that of the original strain.Mutant strain D27 was further used in solid-state fermentation.Based on single factor experiment,Box-Behnken design-response surface methodology was used to optimize culture conditions for mutant D27 with esterase activity as the response value.The results showed that the optimal culture conditions were:33℃,6 days,strain inoculation volume of 3%,and raw material moisture content of 73%.Under the optimal condition,the highest esterase activity was 89.52 U /100 g,an increase of 137% compared with the initial strain.Studies have shown that the mutant strain D27 has a higher ability to produce esterase under solid-state fermentation,and has certain application potential in liquor production.
[1] LIU H L,SUN B G.Effect of fermentation processing on the flavor of Baijiu[J].Journal of Agricultural and Food Chemistry,2018,66(22):5 425-5 432. [2] GOU M,WANG H Z,YUAN H W,et al.Characterization of the microbial community in three types of fermentation starters used for Chinese liquor production[J].Journal of the Institute of Brewing,2015,121(4):620-627. [3] ZHENG X W,HAN B Z.Baijiu (白酒),Chinese liquor:History,classification and manufacture[J].Journal of Ethnic Foods,2016,3(1):19-25. [4] WANG M Y,YANG J G,ZHAO Q S,et al.Research progress on flavor compounds and microorganisms of Maotai flavor Baijiu[J].Journal of Food Science,2019,84(1):6-18. [5] ZOU W,ZHAO C,LUO H.Diversity and function of microbial community in Chinese strong-flavor Baijiu ecosystem:A review[J].Frontiers in Microbiology,2018,9:671. [6] FAN G S,SUN B G,FU Z L,et al.Analysis of physicochemical indices,volatile flavor components,and microbial community of a light-flavor Daqu[J].Journal of the American Society of Brewing Chemists,2018,76(3):209-218. [7] 罗小叶,邱树毅,王晓丹.微生物发酵产酯化酶在浓香型白酒品质提升中研究进展[J].中国酿造,2019,38(8):6-8. LUO X Y,QIU S Y,WANG X D.Research progress in esterifying enzyme produced by microbial fermentation in the quality improvement of strong-flavor Baijiu[J].China Brewing,2019,38(8):6-8. [8] JIA W,FAN Z,DU A,et al.Recent advances in Baijiu analysis by chromatography based technology:A review[J].Food Chemistry,2020,324:126 899. [9] 郭学武,范恩帝,马冰涛,等.中国白酒中微量成分研究进展[J].食品科学,2020,41(11):267-276. GUO X W,FAN E D,MA B T,et al.Recent progress in micro components of Chinese Baijiu[J].Food Science,2020,41(11):267-276. [10] 滕巍,李国莹,刘小波,等.大曲中产酯化酶菌株的分离鉴定及固体发酵工艺优化[J].食品与生物技术学报,2016,35(9):971-977. TENG W,LI G Y,LIU X B,et al.Isolation and identification of ethyl caproate esterifying enzyme in Daqu and its fermentative technology optimization[J].Journal of food science and biotechnology,2016,35(9):971-977. [11] ZHANG W X,WU Z Y,ZHANG Q S,et al.Combination of newly developed high quality Fuqu with traditional Daqu for Luzhou-flavor liquor brewing[J].World Journal of Microbiology and Biotechnology,2009,25(10):1 721-1 726. [12] 谢军,罗惠波,边名鸿,等.酒曲中产香微生物的筛选及其生长特性研究[J].食品与机械,2016,32(1):22-25;86. XIE J,LUO H B,BIAN M H,et al.Study on screening of aroma-producing microbe and growing characters[J].Food &Machinery,2016,32(1):22-25;86. [13] 田宇敏.基于风味导向产香菌M5的分离鉴定及应用研究[D].晋中:山西农业大学,2019. TIAN Y M.Isolation,identification and application of Flavor Bacteria M5 based on flavor-iriented[D] Jinzhong:Shanxi Agricultural University,2019. [14] 单淑芳,丁咚,赵颖,等.酯化红曲霉的分离纯化及其在固态发酵中的应用研究[J].酿酒科技,2018(2):69-73;82. SHAN S F,DING D,ZHAO Y,et al.Separation &purification of esterified Monascus and its application in solid-state fermentation[J].Liquor-Making Science &Technology,2018(2):69-73;82. [15] 赵志军,位宁,刘延波,等.窖泥中酯化型细菌的筛选及产酶条件优化[J].中国酿造,2019,38(9):74-79. ZHAO Z J,WEI N,LIU Y B,et al.Screening of esterifying bacteria in pit mud and optimization of enzyme producing conditions[J].China Brewing,2019,38(9):74-79. [16] ZHOU X,JIN W B,HAN S F,et al.The mutation of Scenedesmus obliquus grown in municipal wastewater by laser combined with ultraviolet[J].Korean Journal of Chemical Engineering,2019,36(6):880-885. [17] 蒋汶,张庆庆,汤文晶,等.紫外-等离子体复合诱变红曲霉产胞外多糖[J].食品与发酵工业,2016,42(1):64-69. JIANG W,ZHANG Q Q,TANG W J,et al.Research on screening and breeding of Monascus on the production of extracellular polysaccharides by UV-ARTP composite mutagenesis[J].Food and Fermentation Industries,2016,42(1):64-69. [18] HAMID Z,SADIQ T,ABBAS A,et al.Enhanced production of cellulase by Escherichia coli engineered with UV-mutated cellulase gene from Aspergillus niger UVMT-I[J].BioResources,2019,14(4):9 054-9 063. [19] ONG H R,GANASEN P,ABUL KALAM M,et al.Effect of light irradiation on esterification of oleic acid with ethanol catalyzed by immobilized Pseudomonas cepacia lipase[J].The Canadian Journal of Chemical Engineering,2019,97(11):2 876-2 882. [20] 袁超,袁丹丹,赵建刚,等.硫酸二乙酯与紫外线复合诱变选育糖化酶高产菌株的研究[J].食品与药品,2018,20(3):219-223. YUAN C,YUAN D D,ZHAO J G,et al.Breeding of glucoamylase high-yield strains using compound Mutagenesis induced by diethyl sulfate and ultraviolet rays[J].Food and Drug,2018,20(3):219-223. [21] SONG X Q,ZHANG Y,ZHU X D,et al.Mutation breeding of high avermectin B1a-producing strain by the combination of high energy carbon heavy ion irradiation and sodium nitrite mutagenesis based on high throughput screening[J].Biotechnology and Bioprocess Engineering,2017,22(5):539-548. [22] 刘文龙,钱娟娟,王兴吉,等.ARTP与紫外线复合诱变选育高产酸性蛋白酶菌株的研究[J].食品与发酵科技,2018,54(6):5-8. LIU W L,QIAN J J,WANG X J,et al.Study on selected breeding of high-yielding strains of producing acidic protease by UV/ARTP Mutagenesis[J].Food and Fermentation Sciences &Technology,2018,54(6):5-8. [23] 刘刚,梁琪,宋雪梅,等.复合诱变选育高产胞外多糖嗜热链球菌菌株[J].食品与发酵科技,2019,55(1):11-18;70. LIU G,LIANG Q,SONG X M,et al.Compound mutation breeding of high-yield extracellular polysaccharide Streptococcus thermophilus[J].Food and Fermentation Sciences &Technology,2019,55(1):11-18;70. [24] 中华人民共和国工业和信息化部.QB/T 4257—2011酿酒大曲通用分析方法[S].北京:中国标准出版社,2011. Ministry of industry and information technology of the people′s Republic of China.QB/T 4257—2011 General methods of analysis for Daqu[S].Beijing:China Standards Press,2011. [25] 潘俊潼,寇凤雨,刘瑞艳,等.常压室温等离子体-紫外复合诱变选育β-法尼烯高产菌株[J].微生物学通报,2020,47(2):542-551. PAN J T,KOU F Y,LIU R Y,et al.Breeding of high β-farnesene producing strain by ARTP-UV combined mutation[J].Microbiology China,2020,47(2):542-551. [26] 赵云彤.侧孢芽孢杆菌BL-1的诱变选育及发酵条件优化[D].哈尔滨:黑龙江大学,2009. ZHAO Y T.Mutation breeding and fermentation conditions optimization of Bacillus subtilis BL-1[D].Harbin:Heilongjiang University,2009.
2021, Vol. 47 
