Microsoft Word _二校

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1 7 9 Vol. 7 No Journal of Food Safety and Quality Sep., 2016 CGMCC 马凯 1, 安云鹤 1, 武会娟 1, 刘悦 1, 陈尔凝 1, 王衍生 2, 李太华 2, 高丽娟 1* (1.,, ; 2., ) 摘要 : 目的 CGMCC 方法 CGMCC 10624, 16S rrna, 结果 CGMCC (Weissella cibaria) 结论, 关键词 : ; ; ; Isolation and identification of a dominant lactic acid bacteria strain CGMCC from a traditional steamed buns Jiaozi MA Kai 1, AN Yun-He 1, WU Hui-Juan 1, LIU Yue 1, CHEN Er-Ning 1, WANG Yan-Sheng 2, LI Tai-Hua 2, GAO Li-Juan 1* (1. Beijing Centre for Physical and Chemical Analysis, Beijing Engineering Technology Research Centre of Gene Sequencing and Gene Function Analysis, Beijing Engineering Research Center of Food Safety Analysis, Beijing , China; 2. Beijing Qijian Food Group Co. Ltd., Beijing , China) ABSTRACT: Objective To identify and analyze a dominant lactic acid bacteria strain CGMCC isolated from the traditional steamed buns Jiaozi by polyphasic taxonomy. Methods The lactic acid bacteria strain CGMCC was isolated and purified from the traditional steamed buns Jiaozi. Taxonomic status of the CGMCC was conducted by bacterial morphology observation, phylogenetic analysis based on the 16S rrna gene sequence and physiological and biochemical characteristics identification method based on carbon source metabolic utilization. Results The lactic acid bacteria strain CGMCC was identified as Weissella cibaria. Conclusion A dominant lactic acid bacteria strain was isolated and purified from the traditional steamed buns Jiaozi in this study which would lay the foundation of resources for its study on the biologic function and production of composite microbial agent containing lactic acid bacteria and yeasts. KEY WORDS: Jiaozi; isolation; identification; Weissella cibaria 基金项目 : ( ) ( ) Fund: Supported by Special Fund for Agro-scientific Research in the Public Interest ( ) * 通讯作者 :,, aglj889@163.com *Corresponding author: GAO Li-Juan, Associate Professor, Beijing Centre for Physical and Chemical Analysis, Beijing , China. aglj889@163.com

2 9, : CGMCC 引言,, [1,2],,,, [3,4],,,,,,,,,,,,, [3], CGMCC 10624, 2 材料与方法 2.1 材料供试菌株, (CGMCC), CGMCC 培养基及主要试剂 MRS ( ); Taq DNA D2000 Marker( ); DNA ( ); AN-IF AN BUA ( Biolog ); 仪器 C1000TM Thermal Cycler PCR ( Bio-Rad ); ZSD-A1090 ( ); ( ); Gen III Microstation ( Biolog ) 2.2 实验方法菌株的分离纯化 5 g, 90 ml (PBS, ph 7.4),, 150 r/min 20 min,, PBS 10 3 ~10 6,, MRS, h, 菌株形态学观察 MRS, h, MRS ; 菌株 16S rdna 序列分析 DNA CGMCC DNA, 1%, DNA, 27F(5 -AGAGTTTGAT CMTGGCTCAG-3 ) 1492R(5 -TACGGHTACCTTGTTAC GACTT-3 ) 16S rdna PCR 50 μl: 10 Buffer 5 μl dntp 4 μl MgCl 2 4 μl (10 μmol/l) 1 μl DNA 40~50 ng Ex Taq 1.5 U 50 μl : 94 5 min, 94 1 min, 48 1 min, 72 1 min, 30, min PCR 1% PCR ABI 3730 Lasergene SeqMan NCBI BLAST, MEGA 5 CGMCC S rrna, (Neighbor-Joining) 菌株的生理生化特性分析 MRS CGMCC BUA, h AN-IF,,,, 65% ( 2%) 100 µl AN, 20 min 20~24 h, 590 nm AN, 0.25, Biolog, 95 3 结果与讨论 3.1 菌株形态学观察 CGMCC MRS

3722 第7卷食品安全质量检测学报小菌落, 有微隆起圆状轮廓, 表面光滑(见图 1 a) 光学显 3.

)下观察显示其为革兰氏阳菌株 CGMCC 10624 的基因组 DNA 提取产物的 1%琼性菌,

片段大小约为 23 kb 以描电镜的观察结果见图 1 c 此基因组 DNA 为模板扩增 16S rdna

扩增序列经测序及拼接处理后, 提交至 GenBank, 登录号为 KU550241 以

CGMCC 10624 与相关近缘模式菌株的系统发育树(图 3) 由系统发育分析判断, 菌株 CGMCC

Fig. 2 Electrophoresis of genomic DNA and 16S rdna

λdna/hindⅢ Marker; A1. CGMCC 10624 基因组 DNA ; M2.

CGMCC 10624 16S rdna 序列 PCR 产物图 3 CGMCC 10624 16S

3 3722 第7卷食品安全质量检测学报小菌落, 有微隆起圆状轮廓, 表面光滑(见图 1 a) 光学显 3.2 菌株 16S rdna 序列分析微镜 1000 (目镜 10, 物镜 100 )下观察显示其为革兰氏阳菌株 CGMCC 的基因组 DNA 提取产物的 1%琼性菌, 菌体细胞呈短杆状, 两端呈圆形(见图 1 b), 使用扫脂糖电泳显示(图 2 a), 所提 DNA 片段大小约为 23 kb 以描电镜的观察结果见图 1 c 此基因组 DNA 为模板扩增 16S rdna 序列, 得到的 PCR 产物的 1%琼脂糖电泳显示(图 2 b), 条带单一, 大小约为 1500 bp 扩增序列经测序及拼接处理后, 提交至 GenBank, 登录号为 KU 以 Bifidobacterium longum ATCC 15707(NR044691)为外群, 构建 CGMCC 与相关近缘模式菌株的系统发育树(图 3) 由系统发育分析判断, 菌株 CGMCC 的归属为魏斯氏菌属(Weissella) (a) (b) (c) 图 1 菌株 CGMCC 的形态学观察结果 Fig. 1 Morphological observation of strain CGMCC 注: a. 宏观形态学观察; b. 光学显微镜观察; c. 扫描电镜观察图 2 CGMCC 基因组 DNA 及 16S rdna 扩增产物电泳结果 Fig. 2 Electrophoresis of genomic DNA and 16S rdna PCR product from CGMCC 注: M1. λdna/hindⅢ Marker; A1. CGMCC 基因组 DNA ; M2. D2000 Marker; B. 阴性对照; A2. CGMCC S rdna 序列 PCR 产物图 3 CGMCC S rdna 序列的系统发育分析 Fig. 3 Phylogenetic analysis of 16S rdna sequence from CGMCC 注: 采用邻接(Neighbor-Joining)法和 p 距离公式进行系统发育分析, 括号内为 GenBank 收录号, 每个分支点处的数字为 Bootstrap(1000 次抽样)的支持百分率, 标尺代表 0.2%的序列差异度

4 9, : CGMCC

5 菌株的生理生化特性分析 Biolog CGMCC ( ), CGMCC D- N- -D- D- L- D- 6-O-D- -D- α- L- D- D- D- D- α-d- D,L- 3- -D- L-, (OD ), CGMCC 10624,, 菌株的分类鉴定结果 16S rrna, [5], [6], CGMCC (Weissella cibaria),, 4 讨论 (polyphasic taxonomy) CGMCC Colwell [7],,, [8,9] (Weissella) [5], 1993 Collin [10],, D-,, [11-13], [14] [15], β- β-, (Monilia albican), ph=4.0, 78%~86% [16] Tingirikari [17], (Bifidobacterium animalis) (B. infantis) (L. acidophilus),, [18],,,, 5 结论 16S rrna, [5] [6], CGMCC (Weissella cibaria), 参考文献 [1]. [J]., 2010, 35(4): Li SG. Study of the influencing factors on steamed bread [J]. Grain Process, 2010, 35(4): [2],,,. [J]., 2014,30(9): , 148. Liu TJ, Li Y, Wu SR, et al. Isolation and identification of bacteria and yeast from traditional sour dough [J]. Mod Food Sci Technol, 2014, 30(9): , 148. [3],,,. - 4 [J]., 2014, 35(16): Ma K, Hua W, Gong P, et al. Headspace solid phase microextraction for analysis of volatile components in steamed bread made with 4 starter cultures [J]. Food Sci, 2014, 35(16): [4],. [J]., 2012, 33(2): Han DQ, Sun QS. Comparison of flavor substances from steamed bread fermented with a mixed starter culture or commercial bread yeast [J]. Food Sci, 2012, 33(2): [5],. [M]. :, Ling DW, Dong XZ. Classification and identification of lactic acid bacteria and experimental methods [M]. Beijing: China Light Industry Press, [6] Björkroth KJ, Schillinger U, Geisen R, et al. Taxonomic study of Weissella confusa and description of Weissella cibaria sp. nov., detected in food and clinical samples [J]. Int J Syst Evol Microbiol, 2002, 52(1):

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6 9, : CGMCC [7] Colwell RR. Polyphasic taxonomy of the genus vibrio: numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus, and related Vibrio species [J]. J Bacteriol, 1970, 104(1): [8],,,. [J]., 2014, 5(4): Li ZM, Xie J, Jiang LW, et al. Isolation and identification of salt-tolerance lactic acid bacterium from high-salt chili [J]. J Food Saf Qual, 2014, 5(4): [9],,,. [J]., 2015, 36(17): Li ZY, Chen Y, Shi YD, et al. Screening and identification of lactobacillus from traditional fermented dairy in inner mongolia [J]. Food Res Dev, 2015, 36(17): [10] Collins MD, Samelis J, Metaxopoulos J, et al. Taxonomic studies on some leuconostoc-like organisms from fermented sausages: description of a new genus Weissella for the Leuconostoc paramesenteroides group of species [J]. J Appl Bacteriol, 1993, 75(6): [11] Rizzello CG, Nionelli L, Coda R, et al. Use of sourdough fermented wheat germ for enhancing the nutritional, texture and sensory characteristics of the white bread [J]. Eur Food Res Technol, 2010, 230(4): [12] Nuobariene L, Cizeikiene D, Gradzeviciute E, et al. Phytase-active lactic acid bacteria from sourdoughs: isolation and identification [J]. LWT-Food Sci Technol, 2015, 63(1): [13] Moroni AV, Arendt EK, Fabio DB. Biodiversity of lactic acid bacteria and yeasts in spontaneously- fermented buckwheat and teff sourdoughs [J]. Food Microbiol, 2011, 28(3): [14],,. [J]., 2014, 9(6): Wang HJ, Dai Y, Ke PQ. Progress in research on Weissella [J]. J Chengdu Med Coll, 2014, 9(6): [15] Lee KW, Park JY, Jeong HR, et al. Probiotic properties of Weissella strains isolated from human faeces [J]. Anaerobe, 2012, 18(1): [16],,,. AT6 [J]., 2014, 35(6): Chang W, Rao Y, Hu HR, et al. Characteristic analysis and optimization of incubate condition for an antifungal Weissella cibaria AT6 [J]. Sci Technol Food Ind, 2014, 35(6): [17] Tingirikari JMR, Kothari D, Goyal A. Superior prebiotic and physicochemical properties of novel dextran from Weissella cibaria JAG8 for potential food applications [J]. Food Funct, 2014, 5(9): [18],,,. [J]., 2015, 36(17): Huang DM, Jia QS, Hu L, et al. Screening and identification of Weissella cibaria with antioxidant activity in Sichuan traditional pickles and analysis of its characteristics [J]. Sci Technol Food Ind, 2015, 36(17): 作者简介 ( 责任编辑 : 刘丹 ) 马凯, 硕士, 助理研究员, 主要研究方向为食品微生物学 dimasimk008@gmail.com 高丽娟, 副研究员, 主要研究方向为食品微生物学 aglj889@163.com

Microsoft Word 聂雪梅.doc

Microsoft Word 聂雪梅.doc 4 4 Vol. 4 No. 4 2013 8 Journal of Food Safety and Quality Aug., 2013 聂雪梅 *, 李立, 孙利, 高飞 (, 100123) 摘要 : 2011 12,, 2007~2011,,,, 关键词 : ; ; ; ; Trade and notification situation on food import and export