5 4 Vol. 5 No. 4 2014 4 Journal of Food Safety and Quality Apr., 2014 1, 李梓铭 2 1,, 谢靓 2 2,, 蒋立文 3*, 李云倩 (1., 410128; 2., 410128; 3., 410128) 1 摘要 : 目的 MRS, 方法 18% NaCl, PCR 16S rdna, 结果,, 结论 16S rdna 关键词 : ; ; Isolation and identification of salt-tolerance lactic acid bacterium from high-salt chili LI Zi-Ming 1, 2, XIE Jing 1, 2, JIANG Li-Wen 2, 3*, LI Yun-Qian 1 (1. College of Food Science and Technology, Hunan Agricultural University, Changsha 410128, China; 2. Hunan Provincial Key Laboratory of Food Science and Biotechnology, Changsha 410128, China; 3. Hunan Provincial Research Center of Engineering and Technology for Fermented Food, Changsha 410128, China) ABSTRACT: Objective To isolate some salt-tolerance strains with different colony morphologies from Hunan characteristic fermented chilli samples using selected MRS medium. Methods One isolated strain which could grow on an 18% NaCl salt tolerant bacteria medium was identified base on the physiological and biochemical experiments with 16S rdna sequencing and phylogenetic tree analyses. Results This strain had a good ability of producing lactic acid and high tolerance to salt. Conclusion This strain was identified as Lactobacillus plantarum through 16S rdna sequence analysis. KEY WORDS: fermented chili; lactic acid bacteria; salt-tolerance 1 引言,,,,, NaCl, NaCl, NaCl 10%~15%,,, 基金项目 : (SCX1220) Fund: Supported by National Undergraduate Innovation Project (SCX1220) * 通讯作者 :,,, E-mail: hnndjlw@163.com *Corresponding author: JIANG Li-Wen, Ph.D, Professor, Hunan Provincial Key Laboratory of Food Science and Biotechnology, Changsha 410128, China. E-mail: hnndjlw@163.com
4, : 1017 (Lactobacillus reuteri) / (Lactobacillus casei/paracasei) / (Lactobacillus paraplantarum/ pentosus) (Lactobacillus acidophilus) (Lactobacillus plantarum) (Lactobacillus rhamnosus) (Bifidobacterium ) [1], [2] ( ) [3,4],,,,,, [5,6],,, 2 材料与方法 2.1 实验材料 2.1.1 材料 20% 2.1.2 试剂与仪器 : (95%),, ; : GZ-250-S ( ); PHS-3C ph ( ); 5417R ( Eppendorf); ( ); WS2-84-64 ( ) 2.1.3 培养基 MRS [7,8] ( ): 10 g 5 g 4 g 20 g -801 ml 2 g 5 g 2 g 0.2 g 0.05 g 15 g 2% 1000 ml ph 6; 10 g 5 g 4 g 20 g -801 ml 2 g 5 g 2 g 0.2 g 0.05 g 25 g 2% 10% 1000 ml ph 6; MC [9, 10] 2.2 试验方法 2.2.1 耐盐菌株的分离 25 g, 225 ml, 1 10-5 1 10-6 1 10-7, 0.1 ml, 37 48~72 h,,, G(+), NaCl (5 % 10 % 12 % 15 % 18 % 20 %),, NaCl,, MRS, 37 24 h, [11] 2.2.2 耐盐菌株的生理生化特性 (1) 0.1 ml, 37 24 h,,,, (2), 22 37 7~14 d,,, 10~20 min,, (3), 37 7~14 d, (4), 37
1018 5 48 h, 0.2 ml,, 10~15 min (5),, (, B.C.P, ph 5.2, ph 6.8 ), (6) 5 ml, 10 % H2SO4 0.5 ml, 2% KMNO4 0.5 ml,,,,, (7), 2.2.3 16S rdna 的扩增与序列分析 DNA [12,13], 16S rdna, primer1 : AGAGTTTGATCCTGGCTCAG; primer2 : AAGGAGGTGATCCAGCCGCA, PCR : 95 5 min; 95 45 s, 52 30 s, 72 60 s, 35 72 10 min,, NCBI BLAST 3 结果与分析 3.1 菌株的分离筛选,, G(+) ly-1 Y-2 Y-3 ly-4 NaCl 5% 10 % 12 % 15 % 18 % 20 %,, ly-4, NaCl 18% ly-4,,,,,,, 3.2 菌株生理生化性质试验结果,,,,, 1 Fig. 1 Table 1 1 ly-4 ( 1000) Colonial morphology and thallus appearance of ly-4 ( 1000) 表 1 耐盐菌株生理生化特性 The salt-tolerance strains of physiological and biochemical characteristics : + ( ), ly-4 ( Lactobacillus ), (Lactobacillus ) [14]
4, : 1019 3.3 16S rdna 序列鉴定 ly-4 16S rdna PCR 3, 1000 2000 bp, 1500 bp 2 ly-4 PCR Fig. 2 Electrophoresis patterns of purified PCR products 2, 1%, 1500 bp,,, GenBank( KJ636841), NCBI BLAST, ly-4 16S rdna NCBI BLAST Lactobacillus plantarum WCFS1 strain WCFS1 99%, GenBank 16S rdna, MEGA5.02 Neighboringjoining 3 3, ly-4 Lactobacillus plantarum, ly-4 Lactobacillus planttarum 16S rdna 99% [14] 16S rdna, (Lactobacillus) (Lactobacillus plantarum) 4 讨论 ly-4, 18%NaCl, ly-4,,,, Fig. 3 3 ly-4 16S rdna Phylogenetic tree based on16s rdna gene sequences for ly-4 strain
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