传统分离培养结合PCR-DGGE技术分析广式腊肠中优势菌

食品科学 ›› 2013, Vol. 34 ›› Issue (4): 157-160.

传统分离培养结合PCR-DGGE技术分析广式腊肠中优势菌

刘国庆,谢科

合肥工业大学生物与食品工程学院

收稿日期:2011-10-31 修回日期:2013-01-15 出版日期:2013-02-25 发布日期:2013-01-29
通讯作者: 谢科 E-mail:xieke_25899258@163.com
基金资助:
出入境口岸航空食品食源性致病微生物污染风险分析、快速检验及监管体系的研究;肉制品安全快速检测及危害分析关键技术研究

Analysis of Dominating Microbial Species in Cantonese Sausage by the Culture-independent and PCR-DGGE Methods

Received:2011-10-31 Revised:2013-01-15 Online:2013-02-25 Published:2013-01-29

摘要/Abstract

摘要：

摘要：采用传统培养方法结合聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)指纹技术对广式腊肠中微生物种群结构进行研究，分析广式腊肠发酵过程的优势微生物群落。结果表明：在传统分离培养中共得到葡萄球菌19株，乳酸菌12株，通过生理生化鉴定得到4株葡萄球菌(P1～P4)和2株乳酸菌(L1、L2)。DGGE指纹图谱显示广式腊肠混合菌群中有3条条带无对应的纯菌株而纯菌株中有1株在混合菌群的图谱上没有相应的条带。进一步的序列分析和相似性比较得出，P1、P4为木糖葡萄球菌，P2、P3、L1、L2分别为孔氏葡萄球菌、阿尔莱特葡萄球菌、格氏乳球菌、乳杆菌属。确定广式腊肠细菌菌群方面的主要优势菌为腐生葡萄球菌、乳杆菌属、木糖葡萄球菌。传统分离法与PCR-DGGE技术结合能够更有效、更全面地分析广式腊肠中微生物群落结构及优势菌，为广式腊肠发酵剂的研发奠定基础。

Abstract:

In order to analysis bacteria community structure overally and systematically in Cantonese Sausage, and to find out the dominating microbial species, which provided a basis for the research of culture starter.The traditio- nal culture method in combination and PCR-DGGE fingerprint technology was applied to study bacteria populati- on structure in Cantonese Sausage, and fermentation advantage bacterial community of Cantonese sausage has been analysised. The results showed that 19 strains of staphylococcus, 12 strains of lactic acid bacteria has been obtained by the traditional culture method, four strains of staphylococcus (named P1 to P4) and two strains of lactic acid bacteria (named L1 and L2) was identified by using physiological-biochemical characteristics. Three bands in the DGGE profiles of total bacterial DNA of Cantonese Sausage were not recovered by cultivation. Conversely, one bacterial species did not exhibit bands in DGGE fingerprints of total bacterial DNA. sequence analysis and similarity comparison showed that the P1 and P4 pertain to Staphylococcus xylosus, P2、 P3、 L1、L2 respectively pertain to Staphylococcus cohnii, Staphylococcus epidermidis, Lactococcus garvieae, Lactobacill- ales bacterium. The main culturable predominant bacteria in Cantonese Sausage were Staphylococcus saprophyti- cus, Lactobacillales bacterium, Staphylococcus xylosus. Conclusion：a combination of traditional separation and PCR-DGGE method could been applied to analysis of the bacteria community structure and advantage bacterium in Cantonese Sausage effectively and comprehensively, Screened advantage strains, layed the foundation for the research of developing of culture starter.

中图分类号:

Q503

刘国庆谢科. 传统分离培养结合PCR-DGGE技术分析广式腊肠中优势菌[J]. 食品科学, 2013, 34(4): 157-160.

参考文献

[1]吕兵,张国农.分离自传统腊肠中的乳酸菌的特性研究[J].食品与发酵工业,2004,30(8):64-67
[2]MARTA M，JOSEP M.Bacteria starter cultures for meat fermentation[J].Food Chemistry,1997,(59):457-554
[3]AMANN R I, LUDWIG W, SCHLEIFER K H.Phylogenetic identification and in situ detection of microbial cells without cultivation[J].Micro- biological Reviews,1995,59:143-169
[4]HUGENHOLTZ P, GOEBEL B M, PACE N R.Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity[J].Journal of Bacteriology,1998,180:4765-4774
[5]周剑忠,董明盛,江汉湖.PCR-DGGE指纹技术与分离技术结合筛选藏灵菇奶发酵过程的优势菌[J].中国农业科学,2006,39(8):1632-1638
[6]FONTANA C, VIGNOLO G, COCCONCELLI P S.PCR–DGGE analysis for the identification of microbial populations from Argentinean dry fermented sausages[J].Journal of Microbiological Methods,2005,63:254-263
[7]JOHN GH，NOBEL RK, PETER HA, et al. Bergey’s Manual ofdeterminative bacteriology. 9th ed[J].Baltimore: WilliaIns＆Wilkins Press,1994,:97-116
[8]东秀珠，蔡妙英,王宝玲,等.常见细菌系统鉴定手册[M],[J].蔡妙英王宝玲等常见细菌系统鉴定手册第一版北京科学出版社,2001,:216-635
[9]MUYZER G, DE WAAL E C, UITTERLINDEN A G.Profiling of complex microbial populations by denaturing gradient gel electrophoreses analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J].Applied of Environment Microbiology,1993,59(3):695-700
[10]HEUER H K, HARTUNG K, WIELANG G, KRAMER I, SMALLA K.Polynucleotide probes that target a hypervariable region of 16S rRNAgenes to identify bacterial isolates corresponding to bands of community fingerprints[J].Applied and Environmental Microbiology,1999,65:1045-1049
[11]李宗军.中国传统酸肉中葡萄球菌的分离鉴定与应用研究[J].生物技术通报,2006,3:77-80

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