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[1]马俊孝,孔健,季明杰.利用PCR-DGGE技术分析乳制品中的乳酸菌[英文][J].应用与环境生物学报,2009,15(04):534-539.[doi:10.3724/SP.J.1145.2009.00534]
　MA Junxiao,KONG Jian,JI Mingjie.Detection of the Lactic Acid Bacteria in Commercial Yoghurts by PCR-denaturing Gradient Gel Electrophoresis[J].Chinese Journal of Applied & Environmental Biology,2009,15(04):534-539.[doi:10.3724/SP.J.1145.2009.00534]
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利用PCR-DGGE技术分析乳制品中的乳酸菌[英文]()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
15卷
期数:
2009年04期
页码:
534-539
栏目:
研究论文
出版日期:
2009-08-25

文章信息/Info

Title:
Detection of the Lactic Acid Bacteria in Commercial Yoghurts by PCR-denaturing Gradient Gel Electrophoresis
作者:
马俊孝; 孔健; 季明杰
山东大学微生物技术国家重点实验室 济南 250100
Author(s):
MA Junxiao;  KONG Jian;  JI Mingjie
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
关键词:
乳酸菌; 聚合酶链式反应–变性梯度凝胶电泳; 酸奶; 16S rRNA基因可变区; 益生菌; 发酵乳制品
Keywords:
lactic acid bacteria;  PCR-DGGE;  yoghurt;  variable region of 16S rRNA gene;  probiotics;  fermented dairy product
分类号:
TS201.3
DOI:
10.3724/SP.J.1145.2009.00534
文献标志码:
A
摘要:
在食品发酵行业，需要一种简捷有效的方法检测发酵产品中的有益微生物. 聚合酶链式反应–变性梯度凝胶电泳(Ploymerase chain reaction and denaturing gredient gel electrophoresis，PCR-DGGE)技术广泛应用于微生物生态学的研究. 采用PCR-DGGE对5种含有乳酸菌的乳制品进行了菌群组成分析，并通过传统培养方法和核酸序列分析进行了验证. 结果表明，所有测试样品中的活菌数量都在106~109 cfu/mL之间，通过平板培养法分离出了形态明显的杆状菌和链球状菌，对其进行16S rDNA核酸序列测定及同源性分析，将其鉴定为德氏乳杆菌(Lactobacillus delbrueckii)和嗜热链球菌(Streptococcus thermophilus). 建立了由已知乳酸菌组成的DGGE参考阶梯(Reference ladder)，可用其对相应菌株进行DGGE鉴定. 用两对不同引物进行PCR-DGGE分析，除样品1外，试样中均检测到德氏乳杆菌和嗜热链球菌. 对DGGE参考阶梯未能直接鉴定的样品1的电泳条带进行回收、序列分析，分别鉴定为卷曲乳杆菌(L. crispatus)和鼠李糖乳杆菌(L. rhamnosus). 同时发现，引物R518-F357对嗜热链球菌模板的识别效率高于引物Lac1GC-HDA2. 以上结果表明，RCR-DGGE技术可以对样品中的乳酸菌进行快速的分析和鉴定. 图4 表1 参30
Abstract:
For food fermentation industry, it is often desirable to monitor the probiotic microorganisms present in commercial products for quality control. Simple, reliable and rapid detection methods with low cost are required to achieve this goal. Denaturing gradient gel electrophoresis analysis of 16S rDNA has been widely used in identification of microbial communities without any culturing step. We have modified and tailored this technique, independently confirmed by sequencing and culture analyses, for detecting the lactic acid bacteria in commercial yoghurt products. All sampled yoghurt products contained bacteria ranging from 106 to 109-cfu/mL. Two strains, further identified as Streptococcus thermophilus and Lactobacillus delbrueckii, with typical morphological characters of Bacillus sp. and Streptococcus sp. were isolated. A reference ladder derived from amplification products of known lactic acid bacteria was constructed and characterized. L. delbrueckii and S. thermophilus were detected in four out of five yoghurt products by comparison of the reference ladder, while L. crispatus and L. rhamnosus were identified in another product by sequencing analysis. Furthermore, the preferential amplification was readily found between primers R518-F357 and Lac1GC-HDA2. Those results suggested that DGGE could be considered a reliable approach for rapid detection of bacteria in commercial yoghurt products. Fig 4, Tab 1, Ref 30

参考文献/References:

1 Temmerman R, Pot B, Huys G, Swings J. Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Intern J Food Microbiol, 2003, 81: 1~10
2 Shah NP, Lankaputhra WEV, Britz ML, Kyle WSA. Survival of Lactobacillus acidophilus and Bifidobacterium bifidum in commercial yoghurt during refrigerated storage. Intern Dairy J, 1995, 5: 515~521
3 Masco L, Huys G, De Brandt E, Temmerman R, Swings J. Culture-dependent and culture-independent qualitative analysis of probiotic products claimed to contain bifidobacteria. Intern J Food Microbiol, 2005, 102: 221~230
4 Theunissen J, Britz TJ, Torriani S, Witthuhn RC. Identification of probiotic microorganisms in South African products using PCR-based DGGE analysis. Intern J Food Microbiol, 2005, 98: 11~21
5 Hamilton-Miller JM, Shah S, Winkler JT. Public health issues arising from microbiological and labelling quality of foods and supplements containing probiotic microorganisms. Public Health Nutr, 1999, 2: 223~229
6 Hamilton-Miller JM, Shah S. Deficiencies in microbiological quality and labelling of probiotic supplements. Intern J Food Microbiol, 2002, 72: 175~176
7 Charteris WP, Kelly PM, Morelli L, Collins JK. Selective detection, enumeration and identification of potentially probiotic Lactobacillus and Bifidobacterium species in mixed bacterial populations. Intern J Food Microbiol, 1997, 35: 1~27
8 Silvi S, Rumney CJ, Rowland IR. An assessment of three selective media for bifidobacteria in faeces. J Appl Bacteriol, 1996, 81: 561~564
9 Stiles ME, Holzapfel WH. Lactic acid bacteria of foods and their current taxonomy. Intern J Food Microbiol, 1997, 36: 1~29
10 Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev, 1995, 59: 143~169
11 De Vuyst L, Vancanneyt M. Biodiversity and identification of sourdough lactic acid bacteria. Food Microbiol, 2007, 24: 120~127
12 Rantsiou K, Cocolin L. New developments in the study of the microbiota of naturally fermented sausages as determined by molecular methods: A review. Intern J Food Microbiol, 2006, 108: 255~267
13 Feng S (冯胜), Gao G (高光), Zhu GW (朱广伟), Zhang YL (张运林), Qin BQ (秦伯强). Bacterial community structure in different eco-restoration areas in a eutrophic lake based on 16S rDNA DGGE and FDC. Chin J Appl Environ Biol (应用与环境生物学报), 2007, 13 (4): 535~540
14 Walter J, Hertel C, Tannock GW, Lis CM, Munro K, Hammes WP. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Appl & Environ Microbiol, 2001, 67: 2578~2585
15 Flo’rez AB, Mayo B. PCR-DGGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese. Intern Dairy J, 2006, 16: 1205~1210
16 Muyzer G, Smalla K. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek, 1998, 73: 127~141
17 Muyzer G. DGGE/TGGE: A method for identifying genes from natural ecosystems. Curr Opin Microbiol, 1999, 2: 317~322
18 Cocolin L, Manzano M, Cantoni C, Comi G. Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages. Appl & Environ Microbiol, 2001, 67: 5113~5121
19 Dal Bello F, Water J, Hammes WP, Hertel C. Increased complexity of the species composition of lactic acid bacteria in human feces revealed by alternative incubation condition. Microbial Ecol, 2003, 45: 455~463
20 Temmerman R, Huys G, Swings J. Identification of lactic acid bacteria: culture-dependent and culture-independent methods. Trends Food Sci & Technol, 2004, 15: 384~359
21 Massana R, Murray AE, Preston CM, DeLong EF. Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel. Appl & Environ Microbiol, 1997, 63: 50~56
22 Meroth CB, Walter J, Hertel C, Brandt MJ, Hammes WP. Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis. Appl & Environ Microbiol, 2003, 69: 475~482
23 Omar NB, Ampe F. Microbial community dynamics during production of the Mexican fermented maize dough pozol. Appl & Environ Microbiol, 2000, 66: 3664~3673
24 Ma JX (马俊孝), Kong J (孔健). Study on detection conditions of lactic acid bacteria by PCR-DGGE. Food Sci (食品科学), 2008, 29 (4): 225~229
25 Von Wintzingerode F, Gobel UB, Stackebrandt E. Determination of microbial diversity in environmental samples: Pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev, 1997, 21: 213~229
26 Reysenbach AL, Giver LJ, Wickham GS, Pace NR. Differential amplification of rRNA genes by polymerase chain reaction. Appl & Environ Microbiol, 1992, 58: 3417~3418
27 Walter J, Tannock GW, Tilsala-Timisjarvi A, Rodtong S, Loach DM, Munro K, Alatossava T. Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl & Environ Microbiol, 2000, 66: 297~303
28 Nubel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI, Ludwig W, Backhaus H. Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacterioly, 1996, 178: 5636~5643
29 Fasoli S, Marzotto M, Rizzotti L, Rossi F, Dellaglio F, Torriani S. Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. Intern J Food Microbiol, 2003, 82: 59~70
30 Farrelly V, Rainey FA, Stackebrandt E. Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species. Appl & Environ Microbiol, 1995, 61: 2798~2801

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备注/Memo

备注/Memo:
Supported by the National High-tech R & D Program of China (No.2006AA10Z344) and the National Natural Science Foundation of China (No.30570043)
更新日期/Last Update: 2009-08-27