两株茶叶籽水浆发酵杆菌的分离鉴定及其数量动态分析  

Isolation and identification of two strains from Camellia sinensis water milk and their dynamic amount

作　　者：任翠娟[1,2] 姜金仲 王自布[1,2] 穆安德 王兴春[1,2] 黄燕芬 REN Cui-juan;JIANG Jin-zhong;WANG Zi-bu;MUAn-de;WANG Xing-chun;HUANG Yan-fen(Guizhou Education University,Guiyang 550018,China;Guizhou Yuanheng Mountain Tea Seed Biological Technology Co.,Ltd.,Guiyang 550018,China)

机构地区：[1]贵州师范学院,贵阳550018 [2]贵州元亨山茶叶籽生物科技有限公司,贵阳550018

出　　处：《南方农业学报》2018年第8期1603-1611,共9页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31460405);贵州省科技计划项目(20174127);贵州省茶籽资源综合开发利用工程研究中心建设项目(2017020);贵州省一流师资团队建设项目(2017158)

摘　　要：【目的】鉴定茶叶籽水浆发酵分层过程中后期的微生物种类,并研究其数量动态,为优化茶叶籽油生物发酵生产工艺及揭示茶叶籽水浆发酵分层机理提供参考依据。【方法】利用添加放线菌酮的MRS固体培养基对发酵5 h的茶叶籽水浆发酵液划线培养,并进行微生物分离与提纯;通过形态学观察、酶学反应、碳源反应、16S rDNA及pheS基因测序分析、系统发育树分析,对分离的微生物进行鉴定;再利用涂布培养法观察菌体的数量动态变化情况。【结果】从茶叶籽水浆发酵液中分离纯化获得两株菌株,分别编号为JJZ12和JJZ21。两株菌株的菌落均呈圆形,白色,凸起,表面光滑湿润,边缘整齐。其中,JJZ12菌株的菌体呈杆状,大小0.6~1.2μm×1.0~3.0μm,成对或短链排列,革兰氏阳性;JJZ21菌株的菌体大小为0.5~0.7μm×0.8~1.7μm,单个或成对排列,革兰氏阳性。经鉴定,JJZ12和JJZ21分别为Gluconacetobacter liquefaciens(产液葡糖酸醋杆菌)和Lactobacillus plantarum subsp. plantarum(植物乳杆菌)的一个菌株;结合本研究编号,两种杆菌分别命名为G. liquefaciens JJZ12和L. plantarum subsp. plantarum JJZ21。在茶叶籽水浆发酵过程中,发酵液中JJZ12和JJZ21菌体的总数量在发酵5 h后逐渐快速增加,至15 h时达最大值,之后又逐渐回落进入稳定状态。【结论】JJZ12和JJZ21两株杆菌是茶叶籽水浆发酵中后期起主要作用的发酵微生物,该结论为解释茶叶籽水浆发酵分层现象的发生机理提供了理论依据。【Objective】The species and count dynamic of microorganism in fermentation layering of Camellia sinensis water milk were clarified to provide reference for optimization of fermentation technique of C.sinensis and revealing the fermentation layering mechanism of C.sinensis water milk.【Method】C.sinensis water milk that fermented for 5 h was cultured on MRS solid culture medium with cycloheximide.The microorganism was isolated and purified.Through morphological observation,enzymatic reaction,carbon source reaction gene sequencing and comparison of 16S rDNA and pheS,establishment and analysis of phylogenetic tree,the isolated microorganism were identified.Spread plate method was used to observe the dynamic amount of bacteria.【Result】Two strains were isolated and purified from fermented C.sinensis water milk,their serial numbers were JJZ12 and JJZ21 respectively.The colony of JJZ12 was round,white and convex,with smooth and moist surface and neat edges;and its thallus were rod-shaped,with a size of 0.6-1.2μm?á1.0-3.0μm,and in pairs or short chains,gram-positive.The morphology of JJZ21 colony was the same as JJZ12.However,its size of thallus was 0.5-0.7μm?á0.8-1.7μm,and single or paired,gram positive.JJZ12 and JJZ21 were authenticated as a strain of Gluconacetobacter liquefaciens and Lactobacillus plantarum subsp.plantarum respectively.Combined with the research number,the full name of these two zymobacter strains were G.liquefaciens JJZ12 and L.plantarum subsp.plantarum JJZ21.In the fermentation process of C.sinensis water milk,the total number of JJZ12 strains and JJZ21 strains in fermented milk increased rapidly after 5 h,and peaked at 15 h,and after that gradually reduced to a stable status.【Conclusion】JJZ12 and JJZ21 are two strains that play a major role in the middle and later periods of C.sinensis water milk fermentation as the fermentation microorganism.The conclusion afford preliminary evidences for explaining occurrence mechanism of fermentation layering of C.sinensis water milk.

关 键 词：茶叶籽水浆 杆菌 发酵工艺 菌体数量动态 

分 类 号：S794.4[农业科学—林木遗传育种] TS222.1[农业科学—林学]