Microsoft Word tb 安美玲b-研究报告

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1 微生物学通报 Microbiology China Sep. 20, 2017, 44(9): DOI: /j.microbiol.china 研究报告新疆喀什地区母乳中乳酸菌的多样性分析 * 安美玲张艳刘忆冬倪永清 ( ) 摘要 : 目的对新疆喀什地区母乳中乳酸菌多样性进行分析方法采用菌落培养显微镜观察 Repetitive genomic fingerprinting (Rep-PCR) 指纹图谱和 16S rrna 基因序列分析相结合的方法研究母乳中乳酸菌的菌群分布结果从 11 份母乳中共分离出乳酸菌 193 株, 利用 16S rrna 基因序列同源分析和系统发育树对代表菌株进行了分子鉴定,193 株乳酸菌隶属于 4 个属, 分别为 Lactobacillus (22 株 ) Streptococcus (42 株 ) Lactococcus (40 株 ) Enterococcus (89 株 ) 其中,Enterococcus 所占比例最大, 达到 46% 结论新疆喀什地区母乳中乳酸菌多样性丰富, 极具开发潜力, 将为开发母乳中的益生菌以及安全可靠的微生态制剂提供理论依据关键词 : 母乳, 乳酸菌,16S rrna 基因序列 Biodiversity of lactic acid bacteria isolated from human breast milk of Kashi area in Xinjiang AN Mei-Ling ZHANG Yan LIU Yi-Dong NI Yong-Qing * (School of Food Sciences, Shihezi University, Shihezi, Xinjiang , China) Abstract: [Objective] Assessment the diversity of lactic acid bacteria in the breast milk of healthy women. [Methods] The diversity of lactic acid bacteria was analyzed by using pure culture, repetitive genomic fingerprinting (Rep-PCR) analysis patterns and 16S rrna gene sequence analysis. [Results] A total of 193 strains of lactic acid bacteria were obtained from eleven breast milk samples. The phylogenetic analysis showed that these strains belonged to four phylogenetic groups, they were Lactobacillus, Streptococcus, Lactococcus and Enterococcus respectively. The dominant genus was Enterococcus, which is up to 46%. [Conclusion] The abundant diversity of lactic acid bacteria in the human breast milk of Kashi area in Xinjiang provided rich resources for exploiting probiotic lactic acid bacteria products. Keywords: Human breast milk, Lactic acid bacteria, 16S rrna gene sequence Foundation item: Special Foundation for the Research and Commercialization of the Modern Agricultural Technologies of the Xinjiang Production and Construction Corps (No. 2015AC003) *Corresponding author: niyqlzu@sina.com Received: November 30, 2016; Accepted: February 28, 2017; Published online ( February 28, 2017 基金项目 : (No. 2015AC003) * 通讯作者 : niyqlzu@sina.com 收稿日期 : ; 接受日期 : ; 优先数字出版日期 (

2 : 2105 [1-2] [3] [4-5] Jiménez [6] Lactobacillus salivariu CECT5713 Lactobacillus gasseri CECT % Arroyo [7] 124 Lactobacillus fermentum CECT5716 PCR-DGGE/TGGE qpcr Staphylococcus Enterorococcus Streptococcus Lactobacillus Bifidobacterium Leuconostococcus [8-9] Albesharat Staphylococcus haemolyticus Streptococcus gallolyticus Streptococcus vestibularis Enterococcus durans Enterococcus hirae Enterococcus mundtii Lactobcillus brevis Lactobcillus oris Lactobcillus animalis [10] Cabrera-Rubio 700 ( 4 5 d ) Weissella Leuconostococcus Staphylococcus Lactococcus [11] [12-13] [14] ( ) 1 材料与方法 1.1 材料样品来源 : ( ) 母乳样品的采集 : 4 5 ml 4 C 12 h 20 C 培养基 : MRS MRS [15] 0.5% L- BS 参考菌株 :Lactobacillus lactis subsp. Lactis CGMCC Lactococcus lactis subsp. Cremoris CGMCC Bifidobacterium bifidum CGMCC 主要试剂和仪器 :

3 2106 微生物学通报 Microbiol. China 2017, Vol.44, No.9-80 L- BS 2 EsTaq Master Mix (Dye) Marker 5000 ( ) (LAC-5040S) LabTech (SPX) (5810R) Eppendorf (GelDoc XR) Bio-Rad PCR (TC-512 PCR) Techne 1.2 方法菌株的分离纯化 : 100 μl 1 ml MRS 37 C 24 h 2 3 h ( ) 100 μl 900 μl μl 3 37 C 48 h 镜检 : 菌株 DNA 的提取 : [16] DNA 20 C PCR 乳酸菌种属鉴定 : Lactobacilli sp. Bifidobacterium sp. PCR Enterococcus sp. Lactococcus sp. 1 3 μl PCR 1.5% S rrna 基因序列分析 : 27F/ 1492R PCR PCR (25 μl) 2 EsTaq Master Mix 12.5 μl 10 μmol/l 27F 0.5 μl 10 μmol/l 1492R 0.5 μl 100 ng/μl DNA 表 1 文中所用属间引物信息 Table 1 Oligonucleotide primers used in this study Genus Primer / R/F Sequence (5 3 ) The product size (bp) Reference Bifidobacterium sp. g-bifid R GGTGTTCTTCCCGATATCTACA [17] g-bifid F CTCCTGGAAACGGGTGG Enterococcus sp. Ent1 R TACTGACAAACCATTCATGATG 112 [18] Ent2 F AACTTCGTCACCAACGCGAAC Lactobacilli sp. LbLMA1-rev R CTCAAAACTAAACAAAGTTTC 250 [19] R16-1 F CTTGTACACACCGCCCGTCA Lactococcus sp. 1RL R TTTGAGAGTTTGATCCTGG 700 [4] 2RR F TCTACGCATTTCACCGCTA

2107 安美玲等: 新疆喀什地区母乳中乳酸菌的多样性分析 1 μl ddh2o 10.5 μl PCR 反应程序 94 C 5 min 2 94 C 30 s 55 C 30 s 72 C 1 min 35 次循环 2.1 72 C 10 min 取 3 μl PCR 扩增产物于 1.

4 2107 安美玲等: 新疆喀什地区母乳中乳酸菌的多样性分析 1 μl ddh2o 10.5 μl PCR 反应程序 94 C 5 min 2 94 C 30 s 55 C 30 s 72 C 1 min 35 次循环 C 10 min 取 3 μl PCR 扩增产物于 1.5%琼脂糖凝胶电泳检测结果与分析菌株的分离与镜检从 11 份母乳样品中共分离到 193 株革兰氏阳性过氧化氢酶阴性的纯培养物初步将其确定 Rep-PCR 指纹图谱以 GTG5 (5 -GTGGTG 为乳酸菌通过革兰氏染色显微镜观察其中 GTGGTGGTG-3 )为引物进行 Rep-PCR 扩增其体 22 株为杆状 171 株为球状它们的排列方式有单系为(25 μl) 2 EsTaq Master Mix 12.5 μl 10 μmol/l 个成对或成链菌落形态大都为白色或乳白圆 GTG5 1.5 μl 100 ng/μl DNA 模板 3 μl ddh2o 形直径为 0.5 mm 2.0 mm (图 1) 8 μl PCR 扩增程序为 95 C 5 min 95 C 1 min C 1 min 65 C 8 min 35 个循环 65 C 15 min 乳酸菌属间鉴定利用属间引物进行 PCR 扩增结果显示 PCR 扩增产物在 180 V (4.5 V/cm)电压下检测约 193 株乳酸菌中有 22 株属于 Lactobacilli 其扩 120 min 结束后在紫外凝胶成像仪中观测 DNA 增片段位于 bp 之间(图 2) 其所占比例为图谱并拍照 DNA 带型完全一致的菌株通常认为 11% 89 株属于 Enterococcus 占 46% 所占比例同一物种然后挑取不同图谱类型的 16S rrna 基最大 42 株属于 Streptococcus 有 40 株归属于因 PCR 产物送至生工生物工程(上海)股份有限公 Lactococcus 未检测到 Bifidobacterium 各属所占司进行测序分析比例如图 3 所示构建系统发育树将所测得的 16S rrna 基因序列提交到 GenBank 数据库中用 BLAST 进行 16S rrna 基因扩增及系统发育树的构建通过 Rep-PCR 指纹图谱结果(部分如图 4 所相关序列的搜索与 GenBank 数据库中现有的近示) 从相同条带中选取一株作为代表菌共选取缘菌株的序列比较并从数据库获得相关属相关 11 株代表菌将其 16S rrna 基因 PCR 产物送至种的 16S rrna 基因序列建立系统发育树序列生工生物工程(上海)股份有限公司进行测序然后用 ClustalX 软件对齐进化距离的计算应用邻接法将测序结果提交到 GenBank 数据库中与 GenBank Neighbor-Joining 采用 P-distances 法和 Kimura-2 数据库中现有的近缘菌株的序列比对构建系统发 parameter 双参数法进行进化树分支模式的稳定育树(图 5) 性用 MEGA V5.0 软件分析采用 Bootstrap 法重复次数为构建进化树属分别为 Lactobacillus Lactococcus Enterococcus 图1 Figure 1 由系统发育树可知 11 株乳酸菌归属于 4 个乳酸菌菌落形态及其细胞形态(100 ) Colonies and cell morphology of lactic acid bacteria (100 ) 注 A 菌株 SHZ1-M-6 菌落形态 B 菌株 SHZ1-R-13 细胞形态 C 菌株 YL13-M-413 细胞形态. Note: A: Colonies morphology; B: Cell morphology of SHZ1-R-13; C: Cell morphology of YL13-M-413.

2108 微生物学通报 Microbiol. China 2017, Vol.44, No.9 图 2 Lactobacilli sp. 扩增产物 Figure 2 PCR amplification products from Lactobacilli sp.

TX33-B-7; 12: TX33-B-6; 13: TX33-S-6; 14: TX33-S-5. Streptococcus SHZ1-M-18 SHZ1-B-67 TX33-R-372 TX33-R-397 Lactobacillus SHZ1-M-18 SHZ1-B-67 L.

faecium YL21-B-428 Lactococcus Lactococcus lactis strain 17 (KT633922.

faecalis YL13 84.6% E. faecium YL21 Lactobacillus SHZ1 TX33 Lactobacillus Lactococcus 8 S.

5 2108 微生物学通报 Microbiol. China 2017, Vol.44, No.9 图 2 Lactobacilli sp. 扩增产物 Figure 2 PCR amplification products from Lactobacilli sp. Note: M: Marker 100 bp ladder; 1: SHZ1-M-6; 2: SHZ1-M-7; 3: SHZ1-M-8; 4: SHZ1-M-9; 5: SHZ1-R-11; 6: SHZ1-R-13; 7: SHZ1-M-18; 8: SHZ1-R-20; 9: TX33-M-8; 10: TX33-M-9; 11: TX33-B-7; 12: TX33-B-6; 13: TX33-S-6; 14: TX33-S-5. Streptococcus SHZ1-M-18 SHZ1-B-67 TX33-R-372 TX33-R-397 Lactobacillus SHZ1-M-18 SHZ1-B-67 L. gasseri TX33-R-372 TX33-R-397 Lactobacillus casei 99% YL13-M-413 YL13-B-422 YL21-R-438 YL21-R-462 Enterococcus YL13-M-413 YL13-B-422 E. faecalis YL21-R-438 YL21-R-462 E. faecium YL21-B-428 Lactococcus Lactococcus lactis strain 17 (KT ) 图 3 母乳中乳酸菌所占百分比 Figure 3 The percentage of lactic acid bacteria in human breast milk 100% YL64-M-456 YL67-M-410 Streptococcus S. salivarius 99% 2 Enterococcus 4 46% E. faecalis YL % E. faecium YL21 Lactobacillus SHZ1 TX33 Lactobacillus Lactococcus 8 S. salivarius 11 图 4 母乳中部分细菌菌株的 Rep-PCR 指纹图谱 Figure 4 Rep-PCR fingerprint patterns of partial bacterial strains in 2% agarose Note: M: Marker 5000 ladder; 1: SHZ1-M-7; 2: SHZ1-B-11; 3: SHZ1-R-13; 4: SHZ1-M-18; 5: SHZ1-R-20; 6: SHZ1-R-21; 7: SHZ1-M-22; 8: SHZ1-M-23; 9: SHZ1-M-27; 10: SHZ1-R-56; 11: SHZ1-R-60; 12: SHZ1-B-67; 13: SHZ1-R-87; 14: TX33-M-8; 15: TX33-M-9; 16: TX33-M-7; 17: TX33-M-6; 18: TX33-M-3; 19: TX33-M-5; 20: TX33-M-4; 21: TX33-B-7; 22: TX33-B-6; 23: TX33-B-8.

6 : 2109 图 5 基于 16S rrna 基因序列的菌株系统发育树 Figure 5 Phylogenetic tree of strains based on partial 16S rrna gene sequences Bootstrap (>50%) Note: The accession number is shown in parenthesis. Numbers at the branch points indicated the level of bootstrap support based on resampled data sets (>50%). The scale bar corresponds to 0.01 substitutions per nucleotide position. Bachu County Sample Qeshqer Shehiri Table 2 Tashikuergantajike 表 2 新疆喀什地区不同母乳样品中乳酸菌分布 The distribution of lactic acid bacteria in human breast milk from Kashi of Xinjiang Lactobacillus gasseri Lactobacillus casei Enterococcus faecalis Enterococcus faecium Streptococcus salivarius Lactococcus lactis SHZ SHZ YL YL YL YL TX TX TX TX TX Total Note: : No strain in sample. Total

7 2110 微生物学通报 Microbiol. China 2017, Vol.44, No.9 3 结论与讨论 30 [20] 11 Enterorococcus Streptococcus Lactobacillus Lactococcus Enterorococcus spp. (46%) Collado 50 Staphylococcus Streptococcus Lactobacillus Bifidobacterium 96% Clostridium clusters XIVa XIVb Enterorococcus [21] Bifidobacterium spp. Bifidobacterium spp. Bifidobacterium spp. [22] ( ) [23] Bifidobacterium spp. 11 S. salivarius S. salivarius [24] 22 Lactobacillus 12 L. gasseri 10 L. casei Lactobacillus 2006 Olivares Lactobacillus gasseri CECT5714 増 [25] Lactobacillus 参考文献 [1] Walker A. Breast milk as the gold standard for protective nutrients[j]. The Journal of Pediatrics, 2010, 156(S2): S3-S7 [2] Petherick A. Development: Mother s milk: A rich opportunity[j]. Nature, 2010, 468(7327): S5-S7 [3] Li SQ, Ma SQ, Yu KF, et al. Species and origin of microorganisms in breast milk and their effects on infants[j]. World Chinese Journal of Digestology, 2016, 24(12): (in Chinese),,,. [J]., 2016, 24(12): [4] Gavin A, Ostovar K. Microbiological characterization of human milk[j]. Journal of Food Protection, 1977, 40(9): [5] Dominguez-Bello MG, Costello EK, Contreras M, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns[j]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(26): [6] Jiménez E, Fernández L, Maldonado A, et al. Oral administration of Lactobacilli strains isolated from breast milk as an alternative for the treatment of infectious mastitis during lactation[j]. Applied and Environmental Microbiology, 2008, 74(15): [7] Arroyo R, Martín V, Maldonado A, et al. Treatment of infectious mastitis during lactation: antibiotics versus oral administration of Lactobacilli isolated from breast milk[j]. Clinical Infectious Diseases, 2010, 50(12): [8] Jiménez E, Delgado S, Fernández L, et al. Assessment of the bacterial diversity of human colostrum and screening of staphylococcal and enterococcal populations for potential virulence factors[j]. Research in Microbiology, 2008, 159(9/10): [9] Martín V, Maldonado-Barragán A, Moles L, et al. Sharing of bacterial strains between breast milk and infant feces[j]. Journal of Human Lactation, 2012, 28(1): [10] Albesharat R, Ehrmann MA, Korakli M, et al. Phenotypic and genotypic analyses of lactic acid bacteria in local fermented food,

8 : 2111 breast milk and faeces of mothers and their babies[j]. Systematic and Applied Microbiology, 2011, 34(2): [11] Cabrera-Rubio R, Collado MC, Laitinen K, et al. The human milk microbiome changes over lactation and is shaped by maternal weight and mode of delivery[j]. The American Journal of Clinical Nutrition, 2012, 96(3): [12] Heikkilä MP, Saris PEJ. Inhibition of Staphylococcus aureus by the commensal bacteria of human milk[j]. Journal of Applied Microbiology, 2003, 95(3): [13] Martin R, Langa S, Reviriego C, et al. Human milk is a source of lactic acid bacteria for the infant gut[j]. The Journal of Pediatrics, 2003, 143(6): [14] Chiu YH, Tsai JJ, Lin SL, et al. Characterisation of bifidobacteria with immunomodulatory properties isolated from human breast milk[j]. Journal of Functional Foods, 2014, 7: [15] Li MH. Isolation and identification of bifidobacteria in human faeces in parts of China and screening of the strains with potential probiotic properties[d]. Hohhot: Masterʼs Thesis of Inner Mongolia Agricultural University, 2012 (in Chinese). [D]. :, 2012 [16] Ni YW, Lan GW, Yang SJ, et al. Diversity of Lactobacillus isolates in fermented dairy products from different regions of Xinjiang[J]. Modern Food Science and Technology, 2016, 32(6): (in Chinese),,,. Lactobacillus [J]., 2016, 32(6): [17] Matsuki TK, Watanabe J, Fujimoto Y, et al. Development of 16S rrna-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces[j]. Applied and Environmental Microbiology, 2002, 68(11): [18] Ke DB, Picard FJ, Martineau F, et al. Development of a PCR assay for rapid detection of Enterococci[J]. Journal of Clinical Microbiology, 1999, 37(11): [19] Dubernet S, Desmasures N, Guéguen M. A PCR-based method for identification of Lactobacilli at the genus level[j]. FEMS Microbiology Letters, 2002, 214(2): [20] Pu ZY, Dobos M, Limsowtin GKY, et al. Integrated polymerase chain reaction-based procedures for the detection and identification of species and subspecies of the Gram-positive bacterial genus Lactococcus[J]. Journal of Applied Microbiology, 2002, 93(2): [21] Collado MC, Delgado S, Maldonado A, et al. Assessment of the bacterial diversity of breast milk of healthy women by quantitative real-time PCR[J]. Letters in Applied Microbiology, 2009, 48(5): [22] Quince C, Lanzén A, Curtis TP, et al. Accurate determination of microbial diversity from 454 pyrosequencing data[j]. Nature Methods, 2009: 6(9): [23] Huang WQ. Microbial diversity of human breast milk in four areas of China[D]. Hohhot: Masterʼs Thesis of Inner Mongolia Agricultural University, 2015 (in Chinese). [D]. :, 2015 [24] Wen YL, Wan HC. Research progresses in administer Streptococcus salivarius to halitosis[j]. International Journal of Stomatology, 2008, 35(5): (in Chinese),. [J]., 2008, 35(5): [25] Olivares M, Díaz-Ropero MP, Martín R, et al. Antimicrobial potential of four Lactobacillus strains isolated from breast milk[j]. Journal of Applied Microbiology, 2006, 101(1): 72-79

Microsoft Word tb 呼斯楞-研究报告

Microsoft Word tb 呼斯楞-研究报告微生物学通报 Microbiology China tongbao@im.ac.cn May 20, 2016, 43(5): 984 990 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150919 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 ( 010018)