基于宏基因组学技术的传统发酵泡菜中乳酸菌多样性研究
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摘要
泡菜类食品是一种独特而具有悠久历史的发酵制品。在广西泡菜制品多采用芥菜、豆角、竹笋、姜、辣椒、萝卜等蔬菜和草鱼、鸭肉、猪肉等肉类进行腌制而成。泡菜在发酵过程中,通过乳酸菌作用生成各种生理活性物质,从而成为具有综合功能的食品。然而目前对自然发酵泡菜制品中乳酸菌的组成、生物多样性、开发利用的可能性等方面的认识并不十分全面和系统。本研究运用宏基因组学原理借助16S rDNA、PCR-DGGE技术研究传统发酵泡菜食品中乳酸菌的生物多样性,同时,采用传统纯培养技术对相同的发酵泡菜制品中乳酸菌进行筛选、分离。系统的反映广西传统发酵泡菜食品中的有益菌并确立优势菌株,为揭示广西传统发酵食品的真实面目提供理论基础和技术支持,也为我国今后的传统发酵食品加工业提供的微生物资源。
利用培养法对从广西发酵泡菜(酸芥菜、酸蕌头、酸鱼、酸肉等)样品中分离出的乳酸菌进行了纯化。经表型特征和系统的生理生化实验鉴定,确定为乳杆菌属、链球菌属、四联球菌属3个属,植物乳杆菌、德氏乳杆菌、干酪乳杆菌、乳酸链球菌、盐水四联球菌5个种。
在抽提发酵泡菜中细菌基因组总DNA时采用了三种不同的方法：Beadbeater机械法、SDS反复冻融法和溶菌酶消化裂解法进行比较分析。从琼脂糖凝胶电泳的验证结果中可以看出溶菌酶消化裂解法效果比较好,而且其操作更快速、简便。将所得DNA样品作模板,用细菌通用引物进行PCR扩增检测,琼脂糖凝胶电泳检测显示片段长度与乳酸菌16Sr DNA的V3区长度相符,且清晰可辨。
将DGGE获得的10条带谱进行克隆测序得到7条不同的序列。测定的序列与乳酸菌16SrDNA部分序列相似性大多在99%以上。其中条带A与保加利亚乳杆菌(Lactobacillus delbrueck i i subsp)有较近的亲缘关系,相似性达到100%；条带B与乳酸链球菌(Lactobacillus sp.)有较近的亲缘关系,相似性达到96%；条带D与漫游球菌菌(Planococcaceae bacterium)有较近的亲缘关系,相似性达到100%；条带E与植物乳杆菌(Lactobacillus Versmoldensis)有较近的亲缘关系,相似性达到100%；条带F(LLactobacillus acetotolerans)与嗜酸乳杆菌有较近的亲缘关系,相似性达到100%。
Kimchi is a unique food with a long history. Mustard, beans, bamboo shoots, ginger, peppers, radishes and fish, duck, pork be made traditional fermented kimchi in Guangxi. In fermentation process, the lactic acid bacteria produce a variety of physiologically active substances, which make Kimchi become a comprehensive functional food. In this study, both culture-dependent and culture-independent methods based on the metagenomics technology were used to examine the lactic acid bacteria diversity of fermented kimchi. Purpose is to reflect the beneficial bacteria and established the dominant strains of in traditional fermented kimchi, to reveal the true face of traditional fermented foods and provide a strong theoretical foundation and technical support for Guangxi, but also for the future of the traditional fermentation of the food processing industry research important microbial resources. The bacteria were sifted and isolated from the samples of pickles juice and yogurt. According to the culture of phenotypic characteristics and under the physiological and biochemical characteristics, and the reference to "common bacteria identified manual system" to determine its biological categories. Strains were attributable to the Lactobacillus (5), Streptococcus (2), Tetraggenococcus (1), a total of five species. The effectives of three methods for extraction of the total bacterial DNA from different fermentation kimchi were compared.The result showed that the Beadbeater mechanical method, SDS repeated freezing method could obtain low DNA, the improved method of lysozyme combine digestion and pyrolysis could obtain highest DNA yield and was the best one for the DNA purity.According to the PCR-DGGE analysis, the DNA extracted by the lysozyme combine digestion and pyrolysis method presented more diverse lactic acid bacteria community than that of the others methods. 10 DGGE band spectrum were cloned and sequenced, obtained seven different sequences at last. Mostly sequences were similar to the 16SrDNA sequence of lactic acid bacteria. Which band A and Lactobacillus delbrueckii subsp have close genetic relationship,100% similarity; band B and Lactobacillus sp have close genetic relationship,96% similarity; bandsD and Planococcaceae bacterium have close genetic relationship,100% similarity; band E and Lactobacillus Versmoldensis have close genetic relationship,100% similarity; band F and Lactobacillus acidophilus have close genetic relationship,100% similarity
利用培养法对从广西发酵泡菜(酸芥菜、酸蕌头、酸鱼、酸肉等)样品中分离出的乳酸菌进行了纯化。经表型特征和系统的生理生化实验鉴定,确定为乳杆菌属、链球菌属、四联球菌属3个属,植物乳杆菌、德氏乳杆菌、干酪乳杆菌、乳酸链球菌、盐水四联球菌5个种。
在抽提发酵泡菜中细菌基因组总DNA时采用了三种不同的方法：Beadbeater机械法、SDS反复冻融法和溶菌酶消化裂解法进行比较分析。从琼脂糖凝胶电泳的验证结果中可以看出溶菌酶消化裂解法效果比较好,而且其操作更快速、简便。将所得DNA样品作模板,用细菌通用引物进行PCR扩增检测,琼脂糖凝胶电泳检测显示片段长度与乳酸菌16Sr DNA的V3区长度相符,且清晰可辨。
将DGGE获得的10条带谱进行克隆测序得到7条不同的序列。测定的序列与乳酸菌16SrDNA部分序列相似性大多在99%以上。其中条带A与保加利亚乳杆菌(Lactobacillus delbrueck i i subsp)有较近的亲缘关系,相似性达到100%；条带B与乳酸链球菌(Lactobacillus sp.)有较近的亲缘关系,相似性达到96%；条带D与漫游球菌菌(Planococcaceae bacterium)有较近的亲缘关系,相似性达到100%；条带E与植物乳杆菌(Lactobacillus Versmoldensis)有较近的亲缘关系,相似性达到100%；条带F(LLactobacillus acetotolerans)与嗜酸乳杆菌有较近的亲缘关系,相似性达到100%。
Kimchi is a unique food with a long history. Mustard, beans, bamboo shoots, ginger, peppers, radishes and fish, duck, pork be made traditional fermented kimchi in Guangxi. In fermentation process, the lactic acid bacteria produce a variety of physiologically active substances, which make Kimchi become a comprehensive functional food. In this study, both culture-dependent and culture-independent methods based on the metagenomics technology were used to examine the lactic acid bacteria diversity of fermented kimchi. Purpose is to reflect the beneficial bacteria and established the dominant strains of in traditional fermented kimchi, to reveal the true face of traditional fermented foods and provide a strong theoretical foundation and technical support for Guangxi, but also for the future of the traditional fermentation of the food processing industry research important microbial resources. The bacteria were sifted and isolated from the samples of pickles juice and yogurt. According to the culture of phenotypic characteristics and under the physiological and biochemical characteristics, and the reference to "common bacteria identified manual system" to determine its biological categories. Strains were attributable to the Lactobacillus (5), Streptococcus (2), Tetraggenococcus (1), a total of five species. The effectives of three methods for extraction of the total bacterial DNA from different fermentation kimchi were compared.The result showed that the Beadbeater mechanical method, SDS repeated freezing method could obtain low DNA, the improved method of lysozyme combine digestion and pyrolysis could obtain highest DNA yield and was the best one for the DNA purity.According to the PCR-DGGE analysis, the DNA extracted by the lysozyme combine digestion and pyrolysis method presented more diverse lactic acid bacteria community than that of the others methods. 10 DGGE band spectrum were cloned and sequenced, obtained seven different sequences at last. Mostly sequences were similar to the 16SrDNA sequence of lactic acid bacteria. Which band A and Lactobacillus delbrueckii subsp have close genetic relationship,100% similarity; band B and Lactobacillus sp have close genetic relationship,96% similarity; bandsD and Planococcaceae bacterium have close genetic relationship,100% similarity; band E and Lactobacillus Versmoldensis have close genetic relationship,100% similarity; band F and Lactobacillus acidophilus have close genetic relationship,100% similarity
引文
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[3]杨晓晖.泡菜中低温发酵乳酸菌的分离鉴定及发酵工艺的研究[D].北京.中国农业大学,2005.
[4]杨瑞,张伟,陈炼红.发酵条件对泡菜发酵过程中微生物菌系的影响[J].食品与发酵工业,2005,31(3)：90～92.
[5]沈国华.纯菌接种发酵技术在腌渍菜加工上的应用(二)[J].中国调味品,2002,(6)：24～27.
[6]陈忠军.内蒙古河套地区酸粥中发酵菌生物学特性研究[D].内蒙古.内蒙古农业大学,2001.
[7]罗欣,王海燕,张春江等.发酵香肠中的菌种分离及鉴定[J].山东农业大学学报,2002,1(12～16).
[8]张刚.乳酸菌——基础、技术和应用[M].北京：化学工业出版社,2007.
[9]刘屹峰.乳酸菌的生理特性和生物学功能[J].丹东纺专学报,2002,9(2)：6-7.
[10]武晋海,郝林,白瑞华.山西老陈醋大曲微生物生态分布[J].山西农业大学学报,2004,24(3)：279～282.
[11]邓启宝,张立新.新风型大曲生产工艺研究[J].酿酒科技,2004,(2)：32～34.
[12]李文茹,谢小保,施庆珊等.酱油中微生物及防腐体系的效能研究[J].中国调味品,2009,34(9)：43～46.
[13]付琳琳.应用PCR-DGGE技术分析泡菜中乳酸菌的多样性[D].南昌.南昌大学,2005.
[14]JW Veljo K. Combining culture-dependent and-independent methodologies for estimation of richness of esturiue bacterioplankton consuming[J]. Appl.Environ Microbiol,2003,69:3607-3616.
[15]R Jo Handelsman,,Brady SF,etal. Molecular biological access to the chemistry of unknown soil microbes:a new frontier for natural products[J]. Chem.Biol,1998, (5): 245-249.
[16]J Handelsman. Metagenomics:Application of genomics to uncultured microorganisms [J]. Microbiology and Molecular Biology Reviews,2004,68(4):669-685.
[17]KR Craigventer J, Heidelberg J. Environmental genome shotgun sequencing of the sargasso sea[J]. Science,2004,304(7):66-74.
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