榨菜腌制过程中微生物区系多样性分析及发酵剂研制
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摘要
榨菜是中国特产,与欧洲酸黄瓜、西德甜酸甘蓝一起被誉为世界三大名腌菜,深受世界人们的喜爱。近年来,酱腌菜朝着“低盐、健康、绿色”的方向发展,已在生产工艺改进、新产品研制等方面开展了大量的研究工作,使榨菜的生产工艺日趋成熟,产品也不断更新,对产品质量控制发挥了重要作用。
微生物的发酵在榨菜腌制过程中具有十分重要的作用,了解榨菜腌制中的微生物发酵类型是有效控制发酵进程的基础。目前,仅杨珺等通过传统微生物培养法,对四川榨菜后熟期间的微生物种类进行了一定的分析,但各腌制时期的微生物群落的变化规律仍不明确。
本研究以涪陵榨菜为原料,结合传统分离培养方法和分子生物学方法(DGGE),初步探明两种腌制工艺榨菜发酵不同时期的菌相组成及微生物菌系的消长变化规律,确定了榨菜发酵体系中的主要微生物及其性能,以及不同发酵时期的菌种种类及比例。在此基础上,研究接种微生物对榨菜品质及发酵特性的影响,为多菌种榨菜发酵剂的研制提供理论参考。本研究旨在通过对多菌种发酵剂的研究和使用,提高榨菜品质,简化加工工艺,缩短榨菜加工周期,为榨菜加工的现代化探索路径。研究结论如下:
①榨菜基本完成后熟转化过程中随着乳酸类细菌的发酵,乳酸累积,pH值一路降低,而细菌和酵母数量的变化呈现先增加后降低的趋势,至25-30 d左右达到峰值。这反应微生物的生命活动与榨菜腌制成熟有密切联系。
传统的分离纯化培养方法从盐脱水和风脱水两种腌制工艺中分离的微生物纯培养形态学及生理生化分析显示,盐脱水和风脱水榨菜中主要优势微生物一致,乳杆菌属(Lactobacillus)、鲁氏酵母(Saccharomyces rouxii)是两种腌制过程中的最主要优势菌群,同时风脱水还分离到盐杆菌、气球菌两株高耐盐性菌,并发现风脱水榨菜微生物多样性明显低于盐脱水榨菜中的微生物。
②榨菜腌制过程中细菌16S rRNA V3区间的序列与现有的数据库中的细菌序列有很高的相似性,都在97%以上,对分离得到的21条优势条带测序结果显示,优势细菌主要属于肠膜明串珠菌(Leuconostoc citreum)、猎隼乳酸菌(Lactobacillus sakei)和乳酸乳球菌(Lactococcus lactis subsp. Lactis),同时,混淆威斯菌和不可培养菌在图谱显现出较高的荧光强度。
③接种发酵榨菜产品在风味、色泽、香气等方面表现出与自然发酵产品相似的感官指标,无明显差异,但接种发酵能够使榨菜亚硝酸盐的含量得到明显的降低。表明,接种发酵对榨菜风味、品质无明显影响,但加快榨菜风味的形成,缩短了榨菜的生产周期,降低了产品的成本,同时提高榨菜产品的安全性。
Pickled of mustard tuber (Brassica juncea var. tsatsai) is the Chinese special product cultivated locally, which has been recognized as one of the most famous pickling vegetables in the world. For the past years, sauerkraut has head for the“low-salt, healthy, green”. Many researches have been done for updating the crafts and producing new variety of products, so the crafts optimized and the products presented more variety gradually. The modified crafts have simplified the process, and play an important role in the quality control of products.
The fermentation process of microorganism is important for the pickling of Brassica juncea. To make clear microbial fermented type in the period of pickling is the basic for controlling the crafts process. Now, Yang et. al, has analyzed the category of microorganism in after ripening time of Szechwan preserved vegetable. However, the variational regulation of microflora is indefinite in different periods.
In this research, Fuling Brassica juncea was chosed as experimental object. combined culture-dependent and culture-independent methods ( DGGE ) , the composition and the variational regulation of the different periods and different crafts were proved up primaryly, the principal microorganism and the function were determined, and the proportion of strain in different periods have also been researched. Moreover, the research of the reflection of inoculating microorganism on the quality and fermentated characteristic, provided theoretical and technical support for the the research of starter cultures. Depending on the research and use of starter culture, this article provide theoretical basis for development of suitable preparatory methods for enhancing quality of products, simplifying the crafts, and shortting the fermentation process.
The main results were as follows:
①Following the fermentation process, the characters of Brassica juncea have changed correspondly. The pH of the Brassica juncea decreased during fermentation, while the increase of lactic acid. The total microorganism experienced a course of growth crest-time, then stabilizated gradually, and get to the peak value at about 25-30 d during fermentation process. This reflects that the close relationship of vital movement of microorganism and the mature of Brassica juncea.
By the traditional culture method, the results of pure cultured morphology and physiology and biochemistry display that the main microorganism are the same, Lactobacillus and Saccharomyces rouxii are the main dominant flora in the immediate fermentation and the dehydration in air. Besides, the two saline tolerance strains of Halobacteria and Aerococcus were isolated only from the dehydration in air. We discovered the biodiversity of the dehydration in air is lower than that of the immediate fermentation.
②These 16S rDNA sequences exhibited more than 97% identity compared with sequences present in the databases. Twenty-one dominant bands from DGGE bands were sequenced, The result showed that the intense bands were the Leuconostoc citreum、Lactobacillus sakei and Lactococcus lactis subsp. Lactis, simultaneously, W. confuse and uncultured eubacteria were also found in DGGE profiles.
③The inoculated fermentated products has the similar oranoleptic indicator with the wild fermentated products, and the variety isn’t obviouse. However, the content of nitrite has reduced obviously in the inoculated fermentated products. It indicate that starter culture does not influence on the flavor and the quality of Brassica juncea, but speeds up the formation of flavor, shortens the period of production, cuts down the cost, and rises the security of the Brassica juncea products.
微生物的发酵在榨菜腌制过程中具有十分重要的作用,了解榨菜腌制中的微生物发酵类型是有效控制发酵进程的基础。目前,仅杨珺等通过传统微生物培养法,对四川榨菜后熟期间的微生物种类进行了一定的分析,但各腌制时期的微生物群落的变化规律仍不明确。
本研究以涪陵榨菜为原料,结合传统分离培养方法和分子生物学方法(DGGE),初步探明两种腌制工艺榨菜发酵不同时期的菌相组成及微生物菌系的消长变化规律,确定了榨菜发酵体系中的主要微生物及其性能,以及不同发酵时期的菌种种类及比例。在此基础上,研究接种微生物对榨菜品质及发酵特性的影响,为多菌种榨菜发酵剂的研制提供理论参考。本研究旨在通过对多菌种发酵剂的研究和使用,提高榨菜品质,简化加工工艺,缩短榨菜加工周期,为榨菜加工的现代化探索路径。研究结论如下:
①榨菜基本完成后熟转化过程中随着乳酸类细菌的发酵,乳酸累积,pH值一路降低,而细菌和酵母数量的变化呈现先增加后降低的趋势,至25-30 d左右达到峰值。这反应微生物的生命活动与榨菜腌制成熟有密切联系。
传统的分离纯化培养方法从盐脱水和风脱水两种腌制工艺中分离的微生物纯培养形态学及生理生化分析显示,盐脱水和风脱水榨菜中主要优势微生物一致,乳杆菌属(Lactobacillus)、鲁氏酵母(Saccharomyces rouxii)是两种腌制过程中的最主要优势菌群,同时风脱水还分离到盐杆菌、气球菌两株高耐盐性菌,并发现风脱水榨菜微生物多样性明显低于盐脱水榨菜中的微生物。
②榨菜腌制过程中细菌16S rRNA V3区间的序列与现有的数据库中的细菌序列有很高的相似性,都在97%以上,对分离得到的21条优势条带测序结果显示,优势细菌主要属于肠膜明串珠菌(Leuconostoc citreum)、猎隼乳酸菌(Lactobacillus sakei)和乳酸乳球菌(Lactococcus lactis subsp. Lactis),同时,混淆威斯菌和不可培养菌在图谱显现出较高的荧光强度。
③接种发酵榨菜产品在风味、色泽、香气等方面表现出与自然发酵产品相似的感官指标,无明显差异,但接种发酵能够使榨菜亚硝酸盐的含量得到明显的降低。表明,接种发酵对榨菜风味、品质无明显影响,但加快榨菜风味的形成,缩短了榨菜的生产周期,降低了产品的成本,同时提高榨菜产品的安全性。
Pickled of mustard tuber (Brassica juncea var. tsatsai) is the Chinese special product cultivated locally, which has been recognized as one of the most famous pickling vegetables in the world. For the past years, sauerkraut has head for the“low-salt, healthy, green”. Many researches have been done for updating the crafts and producing new variety of products, so the crafts optimized and the products presented more variety gradually. The modified crafts have simplified the process, and play an important role in the quality control of products.
The fermentation process of microorganism is important for the pickling of Brassica juncea. To make clear microbial fermented type in the period of pickling is the basic for controlling the crafts process. Now, Yang et. al, has analyzed the category of microorganism in after ripening time of Szechwan preserved vegetable. However, the variational regulation of microflora is indefinite in different periods.
In this research, Fuling Brassica juncea was chosed as experimental object. combined culture-dependent and culture-independent methods ( DGGE ) , the composition and the variational regulation of the different periods and different crafts were proved up primaryly, the principal microorganism and the function were determined, and the proportion of strain in different periods have also been researched. Moreover, the research of the reflection of inoculating microorganism on the quality and fermentated characteristic, provided theoretical and technical support for the the research of starter cultures. Depending on the research and use of starter culture, this article provide theoretical basis for development of suitable preparatory methods for enhancing quality of products, simplifying the crafts, and shortting the fermentation process.
The main results were as follows:
①Following the fermentation process, the characters of Brassica juncea have changed correspondly. The pH of the Brassica juncea decreased during fermentation, while the increase of lactic acid. The total microorganism experienced a course of growth crest-time, then stabilizated gradually, and get to the peak value at about 25-30 d during fermentation process. This reflects that the close relationship of vital movement of microorganism and the mature of Brassica juncea.
By the traditional culture method, the results of pure cultured morphology and physiology and biochemistry display that the main microorganism are the same, Lactobacillus and Saccharomyces rouxii are the main dominant flora in the immediate fermentation and the dehydration in air. Besides, the two saline tolerance strains of Halobacteria and Aerococcus were isolated only from the dehydration in air. We discovered the biodiversity of the dehydration in air is lower than that of the immediate fermentation.
②These 16S rDNA sequences exhibited more than 97% identity compared with sequences present in the databases. Twenty-one dominant bands from DGGE bands were sequenced, The result showed that the intense bands were the Leuconostoc citreum、Lactobacillus sakei and Lactococcus lactis subsp. Lactis, simultaneously, W. confuse and uncultured eubacteria were also found in DGGE profiles.
③The inoculated fermentated products has the similar oranoleptic indicator with the wild fermentated products, and the variety isn’t obviouse. However, the content of nitrite has reduced obviously in the inoculated fermentated products. It indicate that starter culture does not influence on the flavor and the quality of Brassica juncea, but speeds up the formation of flavor, shortens the period of production, cuts down the cost, and rises the security of the Brassica juncea products.
引文
陈灏,唐小树,林洁,等. 2002.不经培养的农田土壤微生物种群构成及系统分类的初步研究[J].微生物学报, 42(4): 78-83.
冯作山,热合曼,杨静. 2000.袋装酱腌菜的防腐保藏技术研究[J].新疆农业大学学报, 23(2): 60-62.
宫曼丽,任南琪,邢德峰. 2004. DGGE/TGGE技术及其在微生物分子生态学中的应用[J].微生物学报, 44(6): 845-848.
胡明方. 1992.食品分析[M].重庆:西南师范大学出版社.
蒋高强. 2006.榨菜腐败微生物的分离、鉴定及其特性的研究[D].浙江:浙江大学生物系统科学与食品科学学院硕士研究生学位论文.
焦振泉,刘秀梅. 2001.细菌分类与鉴定的新热点: 16S-23S rRNA间区[J].微生物通报, 21(1): 12-16.
鲁绯. 2005.腐乳发酵机理、品质改进和模式识别研究[D].北京:中国农业大学食品化学与营养学院博士论文.
刘佩英,刘心恕. 1994.中国芥菜[M].北京:中国农业出版社.
李沁元,崔晓龙,张东华,等. 2004.云南腾冲热海两热泉菌藻席细菌多样性的研究[J].微生物学报, 4: 21-25.
刘炜,马晓军,侯书贵,等. 2007.东天山地区庙儿沟雪坑中微生物多样性、群落结构与环境关系研究[J].微生物学报, 47(6): 1019-1026.
李学贵. 2004.对腌渍榨菜酸败变质的探讨[J].中国调味品, 3: 31-32.
李学贵. 2003.对榨菜在腌制过程中主要成分变化的探讨[J].中国酿造, 3: 9-12.
史幼波,古亚东. 2007.世界名腌之首的前世今生[J].中国西部, 4: 29-35.
王敏. 2003.高盐酱腌菜坯致腐微生物的分离鉴定[J].中国调味品, 5: 19-26.
王敏. 2004.非发酵性豆制品(豆腐丝)主要腐败细菌的分离鉴定及其防腐研究[D].河北农业大学硕士学位论文.
项锦欣. 2004.直投式发酵剂生产橄榄技术研究[D].西南农业大学硕士学位论文.
许玫英,曾国驱,任随周,等. 2003.分子检测技术对活性污泥中氨氧化细菌的比较研究[J].微生物学报, 43(3): 372-378.
杨珺,吴永娴,曾凡坤. 1999.四川榨菜后熟时期微生物区系初探[J].食品科学, 20(12): 50-53.
杨洁彬,郭兴华,张虝,等. 1996.乳酸菌-生物学基础及应用[M].北京:中国轻工业出版社, 31-38.
袁志辉,蓝希钳,杨廷,等. 2006.家蚕肠道细菌群体调查与分析[J].微生物学报, 2: 16-23.
朱伟云,姚文,毛胜勇,等. 2003.变性梯度凝胶电泳法研究断奶仔猪粪样细菌区系变化[J].微生物学报, 43(4): 503-509.
张宗显,王华全,田世润. 1992.乳酸发酵蔬菜制作[J].湖北商检科技, 1: 34-38.
Altaf Ahmad, Abdin MZ. 1999. NADH: nitrate reductase and NAD(P)H: nitrate reductase activities in mustard seedlings[J]. Plant Science, 143: 1-8.
Amann RI, Ludwig W, Schleifer KH. 1995. Phylogenetic identification and in-situ detection of individual Microbial-cells without cultivation[J]. Microbiology Review, 59(1): 143-169.
Caplice E, Fitzgerald GF. 1999. Food fermentations’role of microorganisms in food production and preservation[J].Internationnal Jounal of Food Microbiology, 50: 131-149.
Chen KH. 1983. Fermentation characteristics of heterolactic acid bacteria in green bean juice[J]. Food Science. 48: 962-966.
Cocolin L, Manzano M, Cantoni C, et al. 2001. 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[J]. Applied and Environmental Microbiology, 67(11): 5113-5121.
Daeschel MA. 1989. Antimicrobial Substances from Lactic Bacteria for Use as Food Preservatives[J]. Food Technology. 1: 164-167.
Fasoli S, Marzotto M, Rizzotti L, et al. 2003. Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis[J]. International Journal of Food Microbiology. 82(1): 59-70.
Fischer SG, Lerman LS. 1979. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis[J]. Cell. 16(1): 191-200.
Fromin N, Hamelin J, Tarnawski S, et al. 2002. Statistical analysis of denaturing gel electrophoresis (DGGE)fingerprinting patterns[J]. Environmental Microbiology, 4(11): 634-643.
Jenny SK, Backman, Anna H, et al. 2003. Liming induces growth of a diverse flora of ammonia-oxidising bacteria in acid spruce forest soil as determined by SSCP and DGGE[J]. Applied and Environmental Microbiology. 36(2): 584-588.
John GH., Nobel RK., Peter HA, et a1. 1994. Bergey’s Manual of determinative bacteriology[M]. 9th ed. Baltimore: WilliaIns&Wilkins Press.
Micheller J, Sandra J, Robert M, et al. 1999. Toward functional genomics in bacteria : analysis of gene expression in Es-cherichia coli from a bacterial artificial chromosome library of Bacilluscereu[J]. Proceedings of the National Academy Sciences, 96: 6451-6455.
Morra MJ, Kirkegaard JA. 2002. Isothiocyanate release from soil-incorporated Brassica tissues[J]. Plant, 34(11): 1683-1690.
Muyzers RM, Fischer SG, Lerman LS, et al. 1985. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis[J]. Nucleic Acids Research, 13(9): 3131-3145.
Muyzer G, Waal EC, Uittrlinden AG. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoreses analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J]. Applied Environment Microbiology, 59(3): 695-700.
Nickel JC, Downey J, Johnston B, et al. 2001. Predictors of patient response to antibiotic therapy for the chronic prostatitis/chronic pelvic pain syndrome: A prospective multicenter clinical trial[J]. Journal of Urology, 165(5): 1539-1544.
Omar NB, Ampe F. 2000. Microbial community dynamics during production of the Mexican fermented maize dough pozol[J]. Applied and Environmental Microbiology, 66(9): 3664-3673.
Pace NR, Stahl DA, Lane DJ, et al. 1986. The analysis of natural microbial populations by ribosomal RNA sequence[J]. Advances Microbial Ecology, 9: 1-5.
Qiao ZW, Zhang WX , Xiang WL, et al. 2004. Development of PCR Technique and Application Researches on Microbial Ecology to Chinese Cellar[J]. Sichuan Food and Fermentation, 40(suppl): 143-146.
Rose AH. 1982. Fermented Foods: Economic Microbiology Series[M]. New York: Academic Press.
Rafael Font, Mercedes del Rio-Celestino, Antonio de Haro-Bailon. 2006. The use of near-infrared spectroscopy (NIRS) in the study of seed quality components in plant breeding programs[J]. Industrial Corps and Products, 24: 307-313.
Schwieger F, Tebbe CC. 1998. A new approach to utilize PCR-single-strand-conformation polymorphism for 16s rRNA gene-based microbial community analysis[J]. Applied and Environmental Microbiology, 64(12): 4870-4876.
Suau A, Bonnet R, Sutren M, et al. 1999. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut[J]. Applied and Environmental Microbiology, 65(11): 4799-4807.
Stahnke LH. 1995. Dried sausages fermented with staphylococcus xylosus at different temperatures and with different ingredient levels-PartⅡ[J]. Volatile components.Meat Science, 41(2): 192-209.
Temmerman R, Scheirlinck l,Huys G, et al. 2003. Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis[J]. Applied and Environmental Microbiology, 69(1): 220-226.
Tian YK, Wang CH, Zhang JP, et al. 2003. Optimization of factors affecting RAPD amplification in apple genome[J]. Laiyang Agriculture College, 20(3): 157-161.
Vallaeys T, Topp E, Muyzer G, et al. 1997. Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs[J]. Microbiology Ecology. 24: 279-285.
Vergin KL, Rappe MS, Giovannoni SJ. 2001. Streamlined method to analyze 16S rRNA gene clone libraries[J]. Biotechniques, 30(5): 938-945.
Ward DM, Weller R, Batson MM, et al. 1990. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community[J]. Nature, 345: 63-65.
Watanabe T,Asakama S ,NaKamura A ,et al. 2004. DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil [J]. Federation of European Microbiological Societies (Microbiology Letters). 232:153-163.
Whitcomb, RF, French, FE, Tully, JG, et al. 1997. Spiroplasma species, groups, and subgroups from North American Tabanidae[J]. Microbiology, 35:287-293.
Wood BJ. 1985. Microbiology of Fermented Foods[M].
冯作山,热合曼,杨静. 2000.袋装酱腌菜的防腐保藏技术研究[J].新疆农业大学学报, 23(2): 60-62.
宫曼丽,任南琪,邢德峰. 2004. DGGE/TGGE技术及其在微生物分子生态学中的应用[J].微生物学报, 44(6): 845-848.
胡明方. 1992.食品分析[M].重庆:西南师范大学出版社.
蒋高强. 2006.榨菜腐败微生物的分离、鉴定及其特性的研究[D].浙江:浙江大学生物系统科学与食品科学学院硕士研究生学位论文.
焦振泉,刘秀梅. 2001.细菌分类与鉴定的新热点: 16S-23S rRNA间区[J].微生物通报, 21(1): 12-16.
鲁绯. 2005.腐乳发酵机理、品质改进和模式识别研究[D].北京:中国农业大学食品化学与营养学院博士论文.
刘佩英,刘心恕. 1994.中国芥菜[M].北京:中国农业出版社.
李沁元,崔晓龙,张东华,等. 2004.云南腾冲热海两热泉菌藻席细菌多样性的研究[J].微生物学报, 4: 21-25.
刘炜,马晓军,侯书贵,等. 2007.东天山地区庙儿沟雪坑中微生物多样性、群落结构与环境关系研究[J].微生物学报, 47(6): 1019-1026.
李学贵. 2004.对腌渍榨菜酸败变质的探讨[J].中国调味品, 3: 31-32.
李学贵. 2003.对榨菜在腌制过程中主要成分变化的探讨[J].中国酿造, 3: 9-12.
史幼波,古亚东. 2007.世界名腌之首的前世今生[J].中国西部, 4: 29-35.
王敏. 2003.高盐酱腌菜坯致腐微生物的分离鉴定[J].中国调味品, 5: 19-26.
王敏. 2004.非发酵性豆制品(豆腐丝)主要腐败细菌的分离鉴定及其防腐研究[D].河北农业大学硕士学位论文.
项锦欣. 2004.直投式发酵剂生产橄榄技术研究[D].西南农业大学硕士学位论文.
许玫英,曾国驱,任随周,等. 2003.分子检测技术对活性污泥中氨氧化细菌的比较研究[J].微生物学报, 43(3): 372-378.
杨珺,吴永娴,曾凡坤. 1999.四川榨菜后熟时期微生物区系初探[J].食品科学, 20(12): 50-53.
杨洁彬,郭兴华,张虝,等. 1996.乳酸菌-生物学基础及应用[M].北京:中国轻工业出版社, 31-38.
袁志辉,蓝希钳,杨廷,等. 2006.家蚕肠道细菌群体调查与分析[J].微生物学报, 2: 16-23.
朱伟云,姚文,毛胜勇,等. 2003.变性梯度凝胶电泳法研究断奶仔猪粪样细菌区系变化[J].微生物学报, 43(4): 503-509.
张宗显,王华全,田世润. 1992.乳酸发酵蔬菜制作[J].湖北商检科技, 1: 34-38.
Altaf Ahmad, Abdin MZ. 1999. NADH: nitrate reductase and NAD(P)H: nitrate reductase activities in mustard seedlings[J]. Plant Science, 143: 1-8.
Amann RI, Ludwig W, Schleifer KH. 1995. Phylogenetic identification and in-situ detection of individual Microbial-cells without cultivation[J]. Microbiology Review, 59(1): 143-169.
Caplice E, Fitzgerald GF. 1999. Food fermentations’role of microorganisms in food production and preservation[J].Internationnal Jounal of Food Microbiology, 50: 131-149.
Chen KH. 1983. Fermentation characteristics of heterolactic acid bacteria in green bean juice[J]. Food Science. 48: 962-966.
Cocolin L, Manzano M, Cantoni C, et al. 2001. 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[J]. Applied and Environmental Microbiology, 67(11): 5113-5121.
Daeschel MA. 1989. Antimicrobial Substances from Lactic Bacteria for Use as Food Preservatives[J]. Food Technology. 1: 164-167.
Fasoli S, Marzotto M, Rizzotti L, et al. 2003. Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis[J]. International Journal of Food Microbiology. 82(1): 59-70.
Fischer SG, Lerman LS. 1979. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis[J]. Cell. 16(1): 191-200.
Fromin N, Hamelin J, Tarnawski S, et al. 2002. Statistical analysis of denaturing gel electrophoresis (DGGE)fingerprinting patterns[J]. Environmental Microbiology, 4(11): 634-643.
Jenny SK, Backman, Anna H, et al. 2003. Liming induces growth of a diverse flora of ammonia-oxidising bacteria in acid spruce forest soil as determined by SSCP and DGGE[J]. Applied and Environmental Microbiology. 36(2): 584-588.
John GH., Nobel RK., Peter HA, et a1. 1994. Bergey’s Manual of determinative bacteriology[M]. 9th ed. Baltimore: WilliaIns&Wilkins Press.
Micheller J, Sandra J, Robert M, et al. 1999. Toward functional genomics in bacteria : analysis of gene expression in Es-cherichia coli from a bacterial artificial chromosome library of Bacilluscereu[J]. Proceedings of the National Academy Sciences, 96: 6451-6455.
Morra MJ, Kirkegaard JA. 2002. Isothiocyanate release from soil-incorporated Brassica tissues[J]. Plant, 34(11): 1683-1690.
Muyzers RM, Fischer SG, Lerman LS, et al. 1985. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis[J]. Nucleic Acids Research, 13(9): 3131-3145.
Muyzer G, Waal EC, Uittrlinden AG. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoreses analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J]. Applied Environment Microbiology, 59(3): 695-700.
Nickel JC, Downey J, Johnston B, et al. 2001. Predictors of patient response to antibiotic therapy for the chronic prostatitis/chronic pelvic pain syndrome: A prospective multicenter clinical trial[J]. Journal of Urology, 165(5): 1539-1544.
Omar NB, Ampe F. 2000. Microbial community dynamics during production of the Mexican fermented maize dough pozol[J]. Applied and Environmental Microbiology, 66(9): 3664-3673.
Pace NR, Stahl DA, Lane DJ, et al. 1986. The analysis of natural microbial populations by ribosomal RNA sequence[J]. Advances Microbial Ecology, 9: 1-5.
Qiao ZW, Zhang WX , Xiang WL, et al. 2004. Development of PCR Technique and Application Researches on Microbial Ecology to Chinese Cellar[J]. Sichuan Food and Fermentation, 40(suppl): 143-146.
Rose AH. 1982. Fermented Foods: Economic Microbiology Series[M]. New York: Academic Press.
Rafael Font, Mercedes del Rio-Celestino, Antonio de Haro-Bailon. 2006. The use of near-infrared spectroscopy (NIRS) in the study of seed quality components in plant breeding programs[J]. Industrial Corps and Products, 24: 307-313.
Schwieger F, Tebbe CC. 1998. A new approach to utilize PCR-single-strand-conformation polymorphism for 16s rRNA gene-based microbial community analysis[J]. Applied and Environmental Microbiology, 64(12): 4870-4876.
Suau A, Bonnet R, Sutren M, et al. 1999. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut[J]. Applied and Environmental Microbiology, 65(11): 4799-4807.
Stahnke LH. 1995. Dried sausages fermented with staphylococcus xylosus at different temperatures and with different ingredient levels-PartⅡ[J]. Volatile components.Meat Science, 41(2): 192-209.
Temmerman R, Scheirlinck l,Huys G, et al. 2003. Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis[J]. Applied and Environmental Microbiology, 69(1): 220-226.
Tian YK, Wang CH, Zhang JP, et al. 2003. Optimization of factors affecting RAPD amplification in apple genome[J]. Laiyang Agriculture College, 20(3): 157-161.
Vallaeys T, Topp E, Muyzer G, et al. 1997. Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs[J]. Microbiology Ecology. 24: 279-285.
Vergin KL, Rappe MS, Giovannoni SJ. 2001. Streamlined method to analyze 16S rRNA gene clone libraries[J]. Biotechniques, 30(5): 938-945.
Ward DM, Weller R, Batson MM, et al. 1990. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community[J]. Nature, 345: 63-65.
Watanabe T,Asakama S ,NaKamura A ,et al. 2004. DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil [J]. Federation of European Microbiological Societies (Microbiology Letters). 232:153-163.
Whitcomb, RF, French, FE, Tully, JG, et al. 1997. Spiroplasma species, groups, and subgroups from North American Tabanidae[J]. Microbiology, 35:287-293.
Wood BJ. 1985. Microbiology of Fermented Foods[M].