Microsoft Word tb 呼斯楞-研究报告

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1 微生物学通报 Microbiology China May 20, 2016, 43(5): DOI: /j.microbiol.china 研究报告 内蒙古呼伦贝尔地区传统发酵乳中乳酸菌的多样性分析 * 呼斯楞刘红新于洁刘文俊孙天松 ( ) 摘要 : 目的 对内蒙古呼伦贝尔地区传统发酵乳制品中乳酸菌资源的生物多样性进行研究 方法 采用纯培养和 16S rrna 基因序列分析法对内蒙古呼伦贝尔地区传统发酵乳中的乳酸菌进行多样性分析 结果 从 8 份传统发酵乳制品 (6 份酸牛奶和 2 份酸马奶 ) 样品中分离到 24 株乳酸菌, 通过 16S rrna 基因序列分析和系统进化关系分析将 24 株乳酸菌鉴定为 2 株 Lactobacillus kefiranofaciens 2 株 Lactobacillus kefiri 5 株 Lactobacillus paracasei 3 株 Lactobacillus plantarum 1 株 Lactobacillus rhamnosus 6 株 Lactococcus lactis subsp. lactis 2 株 Leuconostoc mesenteroides subsp. dextranicum 2 株 Streptococcus thermophilus 和 1 株 Enterococcus faecium 结论 Lactococcus lactis subsp. lactis 为内蒙古呼伦贝尔地区传统发酵乳制品的优势菌种, 占总分离株的 25%, 其次为 Lactobacillus paracasei, 占总分离株的 20.83% 关键词 : 乳酸菌, 多样性分析,16S rrna 基因序列分析, 系统发育树 Diversity of lactic acid bacteria associated with traditional fermented milk from Hulun Buir in Inner Mongolia HU Si-Leng LIU Hong-Xin YU Jie LIU Wen-Jun SUN Tian-Song * (Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia , China) Abstract: [Objective] The diversity of lactic acid bacteria in traditional fermented milk from Hulun Buir in Inner Mongolia was studied. [Methods] The samples were analyzed using pure culture and 16S rrna gene sequence analysis. [Results] A total of 24 strains of lactic acid bacteria were obtained from eight fermented milk samples (6 fermented cow milk and 2 fermented mare milk). All the isolates were classified to 5 genera and 9 species: Lactobacillus kefiranofaciens (2 strains), Lactobacillus kefiri (2 strains), Lactobacillus paracasei (5 strains), Lactobacillus plantarum (3 strains), Lactobacillus rhamnosus (1 strain), Lactococcus lactis subsp. lactis (6 strains), Leuconostoc mesenteroides subsp. dextranicum (2 strains), Streptococcus thermophilus (2 strains), and Enterococcus faecium (1 strain). [Conclusion] Lactococcus lactis subsp. lactis is the dominant species, held 25% of the total isolated lactic acid bacteria, and Lactobacillus paracasei occupied Foundation item: Key Technologies R & D Program of China (No. 2013BAD18B01); National Natural Science Foundation of China (No ) *Corresponding author: sts9940@sina.com Received: November 15, 2015; Accepted: March 14, 2016; Published online ( March 14, 2016 基金项目 : (No. 2013BAD18B01) (No ) * 通讯作者 : sts9940@sina.com 收稿日期 : 接受日期 : 优先数字出版日期 (

2 : % of the total lactic acid bacteria isolates as the predominant species of traditional fermented milk from Hulun Buir in Inner Mongolia. Keywords: Lactic acid bacteria, Diversity, 16S rrna gene sequence analysis, Phylogenetic tree [1] (Lactobacillus) (Streptococcus) (Lactococcus) (Leuconostoc) (Pediococcus) (Bifidbacterium) (Enterococcus) (Probiotics) [2] [3-4] [5] [6] 1 材料与方法 1.1 样品来源 C 1.2 培养基及主要试剂 MRS ( BD) MRS (CM0359 OXOID ) M17 (CM0785B OXOID ) M17 (CM0817B OXOID ) K DNA 10 EasyTaq Buffer DNA Marker (TaKaRa) PCR 表 1 内蒙古呼伦贝尔地区传统发酵乳制品种类及来源 Table 1 Information of traditional fermented dairy products from Hulun Buir of Inner Mongolia Sample number Sample type Site HM2 HM7 HM18 HM19 HM20 HM22 HM23 HM24

3 986 微生物学通报 Microbiol. China 2016, Vol.43, No 样品采集 1.5 ml ( 0.5 g : =50:1) MRS (4 C) 1.4 样品中乳酸菌的计数 0.5 ml 4.5 ml ( 0.85%) 10 1 ml ( 2 ) 20 ml ( CO 2 :H 2 :N 2 =10:10:80) 30 C h [7] 1.5 乳酸菌的组成分析 分离纯化与保存 0.85% ml MRS M17 30 C 48 h [8] % ( ) MRS M17 30 C 48 h 30 C 24 h 0.1% 80 C [9] 1.6 乳酸菌基因组 DNA 的提取以及纯度检测 CTAB DNA [8-9] ND-1000 DNA 1.7 乳酸菌 16S rrna 基因的 PCR 扩增 PCR 16S rrna FA-27F (5 -GCAGAGTTCTCGGA GTCACGAAGAGTTTGATCCTGGCTCAG-3 ) RA-1495R (5 -AGCGGATCACTTCACA CAGGACTACGGCTACCTTGTTACGA-3 ) [10] 16S rrna PCR 5 μl 10 EasyTaq Buffer ( Mg 2+ ) 4 μl High Pure dntps (2.5 mmol/l) 1.5 μl FA-27F (10 mmol/l) 1.5 μl RA-1495R (10 mmol/l) 0.5 μl EasyTaq DNA polymerase (5 U/μL) 2 μl DNA (100 mg/l) 35.5 μl ddh 2 O 16S rrna PCR 94 C 5 min 94 C 1 min 58 C 1 min 72 C 2 min C 10 min [11] 2 μl PCR 1.0% bp PCR S rrna 基因序列测定和构建系统发育树 bp DNAStar 5.01 SeqMan 16S rrna bp NCBI BLAST ( nih.gov/blast.cgi) GenBank 16S rrna [12] MEGA 6.0 Neighbor-Joining 24 [7,13]

4 : 结果与分析 2.1 乳酸菌计数 lgcfu/ml CFU/mL CFU/mL CFU/mL 10 6 CFU/mL [14] [15] 2.2 乳酸菌的鉴定结果 乳酸菌的表型特征 : 8 24 MRS 表 2 内蒙古呼伦贝尔地区传统发酵乳制品中乳酸菌活菌计数结果 Table 2 Lactic acid bacteria viable counts in traditional fermented dairy products from Hulun Buir of Inner Mongolia Lactic acid bacteria viable counts Sample number (lgcfu/ml) HM2 8.54±0.02 HM7 7.50±0.08 HM ±0.08 HM ±0.04 HM ±0.07 HM ±0.01 HM ±0.06 HM ± mm 菌种 DNA 提取和 16S rrna 基因扩增 :DNA OD 260/280 OD 260/ DNA 100 mg/l 16S rrna 16S rrna 1.0% bp 16S rrna 16S rrna 16S rrna GenBank 99% 系统发育树的构建和乳酸菌鉴定 : NCBI BLAST 16S rrna GenBank MEGA 6.0 ( MEGA IMAU11180 IMAU11181 IMAU11184 IMAU11204 IMAU11201 Lactobacillus paracasei ATCC T 100% 99% Lactobacillus paracasei IMAU11190 Lactobacillus rhamnosus ATCC 7469 T 100% Lactobacillus rhamnosus IMAU11191 IMAU11209 IMAU11205

5 988 微生物学通报 Microbiol. China 2016, Vol.43, No.5 图 1 内蒙古呼伦贝尔地区分离株的 16S rrna 基因序列的系统发育树 Figure 1 Phylogenetic analysis of 16S rrna gene sequences of the isolates in Hulun Buir of Inner Mongolia Lactobacillus plantarum ATCC T 100% Lactobacillus plantarum IMAU11197 IMAU11200 Lactobacillus kefiri LMG 9480 T 100% 99% Lactobacillus kefiri IMAU11193 Enterococcus faecium LMG T 100% Enterococcus faecium IMAU11195 IMAU11199 Lactobacillus kefiranofaciens DSM 5016 T

6 : % 100% Lactobacillus kefiranofaciens IMAU11185 IMAU11186 IMAU11192 IMAU11202 IMAU11207 IMAU11183 Lactococcus lactis subsp. lactis ATCC T 100% 99% Lactococcus lactis subsp. lactis IMAU11203 IMAU11194 Streptococcus thermophilus ATCC T 100% Streptococcus thermophilus IMAU11208 IMAU11187 Leuconostoc mesenteroides subsp. dextranicum ATCC T 100% Leuconostoc mesenteroides subsp. dextranicum 2.3 内蒙古呼伦贝尔地区传统发酵乳制品中优势菌种分析 Lactococcus lactis subsp. lactis 6 25% Lactococcus lactis subsp. lactis 3 Lactobacillus paracasei (20.83%) 3 Lactobacillus plantarum (12.5%) Enterococcus faecium Lactobacillus kefiranofaciens Lactobacillus kefiri Lactobacillus rhamnosus Leuconostoc mesenteroides subsp. dextranicum Streptococcus thermophilus Lactobacillus rhamnosus Lactobacillus paracasei 22% 4 6 Lactococcus lactis subsp. lactis 50% Lactococcus lactis subsp. lactis Lactobacillus paracasei Lactobacillus helveticus Lactococcus lactis subsp. lactis Lactobacillus casei Leuconostoc mesenteroides subsp. mesenteroides 表 3 内蒙古呼伦贝尔地区传统发酵乳制品样品中菌株分离鉴定结果 Table 3 The distribution of lactic acid bacteria from traditional fermented milk from Hulun Buir in Inner Mongolia Identification result Sample number HM2 HM7 HM18 HM22 HM23 HM24 HM19 HM20 E. faecium IMAU L. kefiranofaciens IMAU11195 IMAU L. kefiri IMAU11197 IMAU L. paracasei IMAU11201 IMAU11204 IMAU11180 IMAU11181 IMAU L. plantarum IMAU11205 IMAU11209 IMAU L. rhamnosus IMAU Lac. lactis subsp. lactis IMAU11202 IMAU11207 IMAU11192 IMAU11183 IMAU11185 IMAU IMAU11208 IMAU Leu. mesenteroides subsp. dextranicum S. thermophilus IMAU11203 IMAU Total Total

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