非酿酒酵母Nakazawaea ishiwadae GDMCC 60786产乙酸乙酯的诱变菌株筛选及其安全性评价

doi:10.7506/spkx1002-6630-20220919-172

食品科学 ›› 2023, Vol. 44 ›› Issue (10): 165-172.doi: 10.7506/spkx1002-6630-20220919-172

非酿酒酵母Nakazawaea ishiwadae GDMCC 60786产乙酸乙酯的诱变菌株筛选及其安全性评价

马文瑞，孙志伟，石俊，张小娜，于佳俊，裴疆森，王德良，贾士儒，薛洁，钟成

（1.天津科技大学生物工程学院，省部共建食品营养与安全国家重点实验室，天津 300457；2.中国食品发酵工业研究院，北京 100015；3.国家酒类品质与安全国际研究中心，北京 100015）

出版日期:2023-05-25 发布日期:2023-06-02
基金资助:
国家重点研发计划-政府间国际科技创新合作项目（2021YFE0192000）；国家自然科学基金地区科学基金项目（32060532）

Screening and Safety Evaluation of Ethyl Acetate-Producing Mutant of Non-Saccharomyces Yeast Nakazawaea ishiwadae GDMCC 60786

Ma Wenrui, sun Zhiwei, shi Jun, zhang Xiaona, yu Jiajun, pei Jiangsen, wang Deliang, jia Shiru, xue Jie, zhong Cheng

(1. State Key Laboratory of Food Nutrition and Safety Co-sponsored by Province and Ministry, College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China; 2. China National Research Institute of Food & Fermentation Industries Co. Ltd., Beijing 100015, China; 3. International Joint Research Center of Quality and Safety of Alcoholic Beverages, Beijing 100015, China)

Online:2023-05-25 Published:2023-06-02

摘要/Abstract

摘要： 为提升Nakazawaea ishiwadae GDMCC 60786产乙酸乙酯的能力，通过物理诱变常压室温等离子体和化学诱变甲基磺酸乙酯、亚硝基胍筛选突变菌株。利用三丁酸甘油酯初筛、摇瓶发酵二筛和外加底物发酵三筛。同时，对突变菌株进行遗传稳定性、溶血性和耐药性体外安全性评价。结果表明：对出发菌株进行诱变处理，最终获得1 株高产乙酸乙酯、遗传稳定性良好及体外安全性良好的突变菌株N5，将该诱变菌连续传代5 次，得到乙酸乙酯含量的平均值为764.52 mg/L，葡萄糖转化率达到38.22%，与出发菌株相比，乙酸乙酯提高了1.90 倍，葡萄糖转化率提高了25.03%。外加乙醇后，突变菌株N5乙酸乙酯质量浓度为1 426.81 mg/L；但外加乙酸后，该菌株不生长，并失去产酯能力，表明其对乙醇耐受性强于乙酸。同时发现24 h时，突变菌株N5的酯酶、乙酰辅酶A和醇酰基转移酶活性均达到最大值。本研究构建了一套适用于该菌株的诱变体系。

关键词: Nakazawaea?ishiwadae?GDMCC?60786；乙酸乙酯；物理化学诱变；突变菌株筛选；酶活性；体外安全性

Abstract: In order to improve the ability to produce ethyl acetate, Nakazawaea ishiwadae GDMCC 60786 was mutagenized by atmospheric and room-temperature plasma (ARTP), ethyl methanesulfonate (EMS) and nitrosoguanidine (NTG). The mutants were sequentially screened by using a plate medium containing tributyrin, shake flask fermentation and addition of substrate. The genetic stability, hemolytic activity and in vitro drug resistance of the selected mutant were evaluated. Mutant N5, which was found to be able to produce a high yield of ethyl acetate, had good genetic stability and in vitro safety. After five successive passages, the average production of ethyl acetate was 764.54 mg/L, and the glucose conversion rate was 38.22%, which were 2.90 times and 25.03% higher than those observed with the original strain, respectively. When ethanol was used as a supplementary carbon source, the yield of ethyl acetate was 1 426.81 mg/L; however, upon the addition of acetic acid, N5 did not grow and lost the ability to produce esters, indicating that the strain was more tolerant to ethanol than acetic acid. Meanwhile, the activities of esterase, acetyl-CoA and alcohol acyltransferase in N5 cells were measured to reach a maximum value after 24 h. In summary, this study successfully constructed a set of suitable mutagenesis system for this strain.

Key words: Nakazawaea ishiwadae GDMCC 60786; ethyl acetate; physicochemical mutagenesis; screening of mutants; enzymatic activity; in vitro safety

中图分类号:

TS261.1

马文瑞，孙志伟，石俊，张小娜，于佳俊，裴疆森，王德良，贾士儒，薛洁，钟成. 非酿酒酵母Nakazawaea ishiwadae GDMCC 60786产乙酸乙酯的诱变菌株筛选及其安全性评价[J]. 食品科学, 2023, 44(10): 165-172.

Ma Wenrui, sun Zhiwei, shi Jun, zhang Xiaona, yu Jiajun, pei Jiangsen, wang Deliang, jia Shiru, xue Jie, zhong Cheng. Screening and Safety Evaluation of Ethyl Acetate-Producing Mutant of Non-Saccharomyces Yeast Nakazawaea ishiwadae GDMCC 60786[J]. FOOD SCIENCE, 2023, 44(10): 165-172.

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非酿酒酵母Nakazawaea ishiwadae GDMCC 60786产乙酸乙酯的诱变菌株筛选及其安全性评价

Screening and Safety Evaluation of Ethyl Acetate-Producing Mutant of Non-Saccharomyces Yeast Nakazawaea ishiwadae GDMCC 60786

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