利用原生质体融合技术改良巴氏醋酸杆菌发酵性能  

Improvement of Acetobacter pasteurianus Fermentation Performance through Protoplast Fusion Technology

作　　者：任汐月 郑传洋 陈海银 胡勇[1] REN Xiyue;ZHENG Chuanyang;CHEN Haiyin;HU Yong(College of Life Science and Health Engineering,Hubei University of Technology,Hubei Provincial Research Center for Food Fermentation Engineering Technology,Wuhan 430070,China)

机构地区：[1]湖北工业大学生命科学与健康工程学院,湖北省食品发酵工程技术研究中心,湖北武汉430070

出　　处：《食品科技》2024年第7期1-9,共9页Food Science and Technology

基　　金：湖北省粮食局流通产业发展项目(鄂粮发[2020]14号)。

摘　　要：运用原生质体融合技术对醋酸发酵工业菌株巴氏醋酸杆菌(Acetobacter pasteurianus, A.pasteurianus)定向改造,提高醋酸菌乙醇、乙酸耐受能力及产酸量。通过探究高渗溶液成分、酶解时间、酶解液浓度及酶解温度4个因素,确定醋酸菌原生质体制备及再生的最适条件;使用原生质体融合技术对乙酸耐受较强的工业菌株A. pasteurianus 1.41及乙醇耐受较强的野生株A.pasteurianus 503进行原生质体融合,并研究融合子的乙酸乙醇耐受性、产乙酸能力和遗传稳定性。结果表明,使用10%蔗糖、0.5 mol/L乙二胺四乙酸、0.1 mol/L Tris-HCl作为高渗溶液,在酶解时间40 min、酶解温度为37℃、溶菌酶浓度为50μg/m L条件下,原生质体制备率及再生率达到最优,分别为98.50%~99.12%和40.51%~42.70%。融合菌株RH-7耐受13%乙醇和1.5%乙酸,优于A.pasteurianus 1.41和A.pasteurianus 503的耐受性。在含10%乙醇的发酵培养基中,产酸量达45.78g/L;遗传稳定实验表明,菌株RH-7生长及产酸量稳定性较好。该研究提供A. pasteurianus原生质体制备的最适条件,使用原生质体融合技术筛选出一株具有良好发酵性能的菌株RH-7,为优化醋酸菌发酵工业应用提供参考。Native protoplast fusion technology was applied to obtain strains with high ethanol and acetic acid tolerance and acid production capability.The influence of compositions,enzyme hydrolysis time,enzyme hydrolysis concentration,and enzyme hydrolysis temperature on the preparation and regeneration of protoplasts were determined.Using protoplast fusion technology,the A.pasteurianus 1.41 with the high acetic acid-tolerance were fused with A.pasteurianus 503 with the high ethanol-tolerance.Then,the fusant were screened out,and ethanol tolerance,acetic acid production capability,and genetic stability were studied.The results indicate that the optimal lysozyme concentration,hydrolysis time and temperature were 50μg/mL,40 minutes and 37℃,respectively.Using a high-osmotic solution composed of 10%sucrose,0.5 mol/L EDTA and 0.1 mol/L Tris-HCl,the protoplast preparation and regeneration rates were from 98.50%to 99.12%and from 40.51%to 42.70%,respectively.The fused strain RH-7 was demonstrated to grow at 13%ethanol or 1.5%acetic acid.In a fermentation containing 10%ethanol,RH-7exhibited the highest acid production reaching 45.78 g/L.Genetic stability experiments revealed that strain RH-7 exhibited good stability in both growth and acid production.This study provides the optimal conditions for the preparation of A.pasteurianus protoplasts and identifies a strain RH-7 with excellent fermentation performance.The study contributed to an improved application of acetic acid bacteria in industrial.

关 键 词：巴氏醋酸杆菌 原生质体融合 乙酸耐受 乙醇耐受 

分 类 号：TS201.3[轻工技术与工程—食品科学]