摘要
苹果酸-乳酸发酵是酿造优质红葡萄酒的一个重要环节。经酒精发酵后的葡萄酒都会形成一个相对较恶劣的环境(低pH值、高酒精浓度和高SO2浓度、营养物质匮乏等),从而影响苹果酸-乳酸发酵的启动和完成。利用抗性较强的乳酸菌菌株进行苹果酸-乳酸发酵就成为解决这个问题的关键。进行苹果酸-乳酸发酵的乳酸菌经研究发现主要是酒酒球菌。因此,为了筛选优良的发酵菌株,对酒酒球菌抗逆性方面的研究是非常必要的。
但目前关于酒酒球菌抗性方面的研究多集中在蛋白质水平,从其一级信息源—基因方面考虑其抗性的研究国内外鲜见报道。本研究旨在从DNA分子水平上了解耐酸性菌株与酸敏性菌株之间的差异,筛选与酒酒球菌耐酸性相关的RAPD分子标记,为优良酒酒球菌的筛选奠定基础。
1、从宁夏地区分离得到30株苹果酸-乳酸发酵菌,经过初步鉴定,其生长特征基本上与酒酒球菌的生长特征相同。通过葡萄酒乳酸菌属的鉴定表和明串珠菌属细菌(Leuconostoc)种内鉴定双歧式检索表对所分离的菌种做进一步鉴定,结果表明,所分离得到的菌株均为酒酒球菌。利用这30株菌进行了抗酸性的筛选,结果得到10株抗酸性菌株和9株酸敏性菌株。
2、以学院保藏的酒酒球菌SD-2a为试验材料,研究了酒酒球菌基因组DNA提取方法,结果表明,采用改良的传统细菌基因组DNA提取方法,所提取的DNA质量较高,提取的DNA含量在170ng/μl左右,A260/A280比值均介于1.75-1.90之间。经电泳检测条带清晰,无杂带,而且没有蛋白质,糖类的污染,能够满足RAPD反应的需要。
3、以提取的酒酒球菌SD-2a基因组DNA和随机引物S333为试验材料,对影响RAPD-PCR反应体系的主要因素进行了优化,最终建立了适合本实验室的RAPD反应体系:25μl反应体积、1.0UTaq酶、160μmol/L dNTP、0.4μmol/L随机引物、3.0mmol/L Mg2+、10ng的DNA模板用量;PCR反应程序为:94℃预变性5min;94℃变性1min,36℃退火1min,72℃延伸1.5min,45个循环;72℃延伸5min。
4、在RAPD-PCR反应体系建立的基础上以33株抗酸性酒酒球菌和9株酸敏性菌株为试材。采用BSA法和RAPD相结合的技术,通过对45个随机引物的筛选以及在单菌株中的验证,结果两条随机引物在单菌株中表现稳定的多态性,出现了较为稳定的有规律的三条特异性条带,这两条随机引物分别为:S40、S333。三条特异性条带命名如下:S40—1400;S333—2500;S333—650。这样就标记出了与酒酒球菌抗酸相关基因相连锁的三条特异性条带。证明抗酸性菌株和酸敏性菌株基因组之间确实存在显著的差异性,同时也证明酒酒球菌的抗酸性状是多基因控制的。为建立快速筛选酒酒球菌优良菌株的方法提供了理论依据。
Malolactic fermentation (MLF) is an indispensable processing technology for red wine. The poor environmental conditions for lactic acid bacteria growth will be created after wine alcoholic fermentation, such as low pH, high alcohol, SO2 concentration and lack of nutrients. In this poor condition MLF is often sluggish and even bring about fall of inoculation. Using strong resistance lactic acid bacteria strains in Malolactic fermentation have been becoming the key to solve this problem. It is found that Oenococcus oeni is the main lactic acid bacteria in the processing of Malolactic fermentation. Therefore, it is necessary to study the resistance of the Oenococcus oeni to pH, alcohol and SO2, in order to screen excellent to carry out the Malolactic fermentation.
However, at present, the research about resistance of Oenococcus oeni concentrates on the protein level. As a source of information, genetic study about its resistance is scarcely at home and abroad. The purpose of this study is to learn the difference between acid-resistant strains and acid-sensitive strains based on DNA of the Oenococcus oeni. RAPD specific molecular markers linked to acid resistance gene in Oenococcus oeni will be discovered in this study. These molecular markers will lay a foundation for screening of the superior Oenococcus oeni.
1. With selective culture media, we have isolated 30 strains malolactic bacteria from NingXia region wines of China. Preliminary identification of the isolates was performed. The Growth characteristics of the isolates are the same as Oenococcus oeni. Further identification of the isolates was performed according to the criteria established by Roger and Villani. The results show that 30 isolates are also Oenococcus oeni. The work that selected acid-resistant strains and acid-sensitive strains in 30 Oenococcus oeni strains has been done. As a result, we obtained 10 Oenococcus oeni strains of acid-resistance and 9 Oenococcus oeni strains of acid-sensitive.
2. In this study, Oenococcus oeni SD-2a was used as experimental material. The method of genome DNA extraction was studied in Oenococcus oeni. The results showed that high grade genome DNA was obtained by the revised traditional method. The genome DNA extracted by the revised traditional method concentration is about 170ng/μl. The ratios of A260/A280 are from 1.75 to 1.90. With detection of bands by electrophoresis, we obtain a clear, non-hybrid band. These bands have not been polluted by protein or carbohydrate. This genome DNA can accomplish the reaction of RAPD.
3. In this study, genome DNA of Oenococcus oeni SD-2a and random primers S333 were used as experimental material. The primary conditions of the RAPD-PCR were optimized and RAPD reacting system that is appropriate for our laboratory was established. The optimal PCR system for RAPD analysis was as follows: 1.0UTaq polymerase, 160μmol/L dNTP, 0.4μmol/L random primer, 3.0mmol/L Mg2+, 10 ng DNA template, in 25μL reaction system; The reaction program of PCR was devised at 94℃for 5minutes and followed by 45 cycles, each with l minute at 94℃, l minute at 36℃, l.5minutes at 72℃, and a final extension at 72℃for 5 minutes.
4. Using the optimal PCR system for RAPD analysis, 33 Oenococcus oeni strains of acid-resistance and 9 Oenococcus oeni strains of acid-sensitive were used as experimental material for screening the RAPD markers linked to the gene resistant to acid. The methods of random amplified polymorphic DNA (RAPD) markers and bulked segregate analysis (BSA) were employed in this study. Finally, three markers S40—1400, S333—2500 and S333—650 linked to Acid in Oenococcus oeni were obtained through a selection among 45 random primers and confirmation in single Oenococcus oeni strain. This study proved that there was a significant difference between Oenococcus oeni strains of acid-resistance and Oenococcus oeni strains of acid-sensitive in their genome. At the same time, the research also proved that the trait of acid-resistance is controlled by multiple genes in Oenococcus oeni. This work may lay a theoretical basis for the establishment of the rapid screening of fine Oenococcus oeni strains.
引文
[1]杨洁彬,郭兴华等.乳酸菌—生物学基础及应用[M].中国轻工业出版社.1996.
[2]张春晖,李华.葡萄酒微生物学[M].陕西人民出版社.2003:119.
[3] Dicks L M T, Dellaglio F and Collins M D. Proposal to rechssify Leuconostoc oenos as Oenococcus oeni [corrig] gen. nov,comb. nov. Int. J Syst. Bacterial.1995,45(2):395-397.
[4] Yoann Augagneur, Jean-Franc?ois Ritt, Daniel M. L. Dual effect of organic acids as a function of external pH in Oenococcus oeni[J]. Arch Microbiol 2007,188:147–157.
[5] G.Zhao and G.Zhang. Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze- drying[J]. Journal of Applied Microbiology, 2005, 99(2): 333.
[6] S. Lafon-Lafourcade, A. Lonvaud-Funel and E. Carre.Lactic acid bacteria of wines: stimulation of growth and malolactic fermentation [J]. Antonie van Leeuwenhoek.1983,49(3): 349-352.
[7] Lafon-Lafourcade S, Carre E, Ribéreau-Gayon P. Occurrence of Lactic Acid Bacteria During the Different Stages of Vinification and Conservation of Wines [J]. Appl Environ Microbiol. 1983,46(4):874-880.
[8]张春晖.中国优良酒类酒球菌(Oenococcus oeni )的分离筛选及苹果酸一乳酸发酵研[D].西北农林科技大学博士学位论文.2001.
[9]郭永亮.苹—乳发酵管理是优质葡萄酒酿造工艺中极其重要的一环[J].中外葡萄与葡萄酒.2002, 3: 49-51.
[10] Beelman R B and Gallander J F. Wine deacidification advances. Food Research, 1979, 25: 1-53.
[11] Isabel López, Carmen Tenorio, and Myriam Zarazaga, Evidence of mixed wild populations of Oenococcus oeni strains during wine spontaneous malolactic fermentations[J]. European Food Research and Technology.2007,226(1): 215-223.
[12] Boulton R B, Singleton V L, Bisson L F, et al. Principles and practices of winemaking. New York: Chapman&Hall, 1995.
[13] Garcia-Quitans N, Magni C and Diego de Mendoza et al. The citrate transport system of Gactococcus lactis subsp. lactis biovar diacetylactis is induced by acid stress[J]. Appl. Environ. Microbiol., 1998, 3: 850-857
[14] Maicas S, Gil J V, Patdo I and Fewer S. Improvement of volatile composition of wines by controlled addition of molactic bacteria [J]Food Research International. 1999,32 (7): 491-496.
[15] Nielsen J C and Richelieu M. Control of flavor development in wine during and after malolactic fermentation by Oenococcus oeni[J]. Appl. Environ. Microbiol., 1999, 2: 740-745.
[16] Revel G, Martin N, Pripis-Nicolau L, Lonvaud-Funel A and Bertrand A. Contribution to the knowledge of malolactic fermentation influence on wine aroma.[J]. Agic Food Chem. 1999, 47(10): 4003-4008.
[17]权英,王颉,张伟.葡萄酒中的乳酸菌[J].酿酒科技.2002 ,2 : 59-61.
[18] Wibowo D, Eschenbruch R and Davis C R. et al. Occurrence and growth of lactic acid bacteria in wine:a review[J].Enol Vitic. 1985,4:302-313.
[19] Nancy N, Florence M, Patricia T. Quantitative study of interactions between Saccharomyces cerevisiae and Oenococcus oeni strains[J]. Ind Microbiol Biotechnol. 2008,35:685–693.
[20] Henick-kling T, Lee T, Hand Nicholas D J D. Inhibition of bacterial growth and malolactic fermentation in wine by bac-teriophage[J]. Appl Bacterial. 1986,(61):287~293.
[21]赵文英,李华,王华.酒酒球菌(Oenococcus oeni)胁迫适应性反应机制[J].微生物学报.2008,48(4):556~561.
[22] Beelman R B, McArdel F J and Duke G R. Comparison of Ieuconostoc oenos strains ML-34 and PSU-1 to induce malolactic fermentation in Pennsylvanis red table wine[J]. Am. J Enol. Vitic.1980, 31: 269~276.
[23]张春晖,夏双梅,莫海珍.微生物降酸技术在葡萄酒酿造中的应用[J].酿酒科技.2000,2: 66-70.
[24] Briz T J and Tracey R P. The combination effect of pH, SO2, ethanol and temperature on the growth of leuconostoc oenos[J].Appl Bacterial. 1990,68:23-31.
[25]张浩,莫海珍.葡萄酒苹果酸-乳酸发酵细菌生理特性研究进展[J].安徽农业科学.2006,34(17):4213-4218.
[26] Carbo.R et al. Influence of degree of alcohol in malolactic fermentation[J]. Alimentacion Equiposy Technology. 1998,17(2): 71-74.
[27]赵文英,李华,王爱莲等.不同培养基对酒酒球菌SD-2a存活率及膜脂肪酸组分的影响[J].微生物学报.2008,48(10):1319~1323.
[28] Guzzo J, Cavin J-F, Divies C. Induction of stress proteins in Leuconostoc oenos to perform direct inoculation of wine. Bio-technology Letters, 1994,16:1189-1194.
[29] Chu-Ky S, Tourdot-Marechal R, Marechal PA, et al. Combined cold, acid, ethanol shocks in Oenococcus oeni: Effects on mem-brane fluidity and cell viability. Biochimica et Biophysica Acta,2005, 17: 118?124.
[30] Beltramo C, Desroche N, Tourdot-Maréchal R. Real-time PCR for characterizing the stress response of Oenococcus oeni in a wine-like medium. Research in Microbiology, 2006, 157:267-274.
[31] Guzzo J, Jobin MP, Diviés C. Increase of sulfite tolerance in Oe-nococcus oeni by means of acidic adaptation. FEMS Microbiol-ogy Letters, 1998, 160: 43-47.
[32]王滨,张国政,路福平等.酵母酒精耐性机制的研究进展[J].天津轻工业学院学报.2001,1: 18-23.
[33] Vincent R, Lou Cadet V, Olivier C. Genetic and phenotypic evidence for two groups of Oenococcus oeni strains and their prevalence during winemaking[J]. Applied Microbiology and Biotechnology. 2009,83(1): 85-97.
[34] Carbo.R et al. Influence of degree of alcohol in malolactic fermentation[J]. Alimentacion Equiposy Technology. 1998,17(2): 71-74.
[35] Edwards.C G. et al. Evaluation of processing methods to control the Growth of Lactobacillus kunkeei ,a micro-organism implicated in sluggish alcoholic fermentation of grape musts[J]. South African Journal of Enology and Viticulture.1999,20(1): 11-18.
[36] Hua Li, Chunhui Zhang , Yanlin Liu. Species attribution and distinguishing strains of Oenococcus oeni isolated from Chinese wines[J].World Journal of Microbiology and Biotechnology.2006,22(5):515-518.
[37]张翠英,孙健.葡萄酒中M L F的研究及技术应用.四川食品与发酵[J].2006, 42(2):24-27.
[38] Edwards C G, Beelman R B and Bartley C E, et al. Production of decanoic acid and other volatile compounds and the growth of yeast and malolactic bacteria during vinification. Am. J Enol. V itic., 1990, 41(1):48-56
[39] Caridi, A. et al. Inhibition of malolactic fermentation by cryotolerant yeasts[J]. Biotechnology Letters,1997,19(8): 723-726.
[40] Caridi,A. et al. Inhibition of the production yeast strainon the development of malolactic fermentation in white wine[J]. Food Technology andBiotechnology, 1998,36,(1): 63-68.
[41] Guilloux-Benatier,M. et al. Enayme activity: glycosidase and peptidase activities in Leuconostoc oenos during growth. Effects of yeast macromolecules[J]. Vitis, 1993, 32,(1): 51-57.
[42] Gonzalez ,M.C. et al. Study of induction of fermentation with selected in Navarra winesⅢ.Trials of induction of malolactic fermentation with selected clones of yeasts and lactic bacteria[J].Alimentaria.1995,(264):49-56.
[43]张如金,李华等.酒类酒球菌SD- 2a活性干粉与国外干粉生产性能比较[J].酿酒.2008,35(4):74-77.
[44]Tourdot-Marechal, R. Fortier, L C Guzzo, J, Lee B, Divie`s C,1999. Acid sensitivity of neomycin-resistant mutants of Oenococcus oeni: a relationship between reduction of ATPase activity and lack of malolactic activity. FEMS Microbiol. Lett. 178,319– 326.
[45]张春晖,李华.苹果酸—乳酸发酵能量产生机理[J].生物工程进展.2001, 21(5):72-74.
[46] Sanders JW, Leenhouts K, Burghoorn J, Brands JR, et al. A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation[J].Mol microbial. 1998,27:299-310.
[47] Frees D, Vogensen FK, Ingmer H. Identifications of protein are low pH in Lactococcus lactis[J]. Food Microbial. 2003,87:293-300.
[48] Blankenhorn D, Phillips J,Slonczewski JL. Acid-and Base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis[J].Bacteriol. 1999,181:2209-2216.
[49] Schauder S, Shokat K, Surette MG, Bassler BL. The LuxS family of bacteria autoinducers: biosynthesis of a novel quorum-sensing signal transduction molecule[J]. Mol Microbiol. 2001,41:463-467.
[50] Kilstrap M, Jacobsen S, Hammer K, Vogensen FK. Induction of heat shock proteins Dnak, GroEL, and GroES by salt stress in Lactococcus lactis[J]. Appl Environ Microbial 1997,63:1826-1837.
[51] Ingmer H, Vogensen FK, Hammer K, Kilstrup M. Disruption and analysis of the clpB, cipC, andcipE genes in Lactococcus lactis: ClpE, a new CIp family in gram-positive bacteria[J].Bacteria.1999,181:2075-2083.
[52] Free D, Ingmer H. CipP participates in the degradation of misfolded protein in Lactococcus lactis[J]. Mol Microbial. 1999,31:79-87.
[53] Jean Guzzo, Michel-Philippe Jobin, et al. Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase[J]. International Journal of Food Microbiology 2000, 55 :27–31.
[54] Carrete R, Vidal MT, Bordons A, Constanti M.. Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni[J]. FEMS Microbiol Letters. 2002,211:155– 159.
[55] Delmas F, Pierre F, Coucheney F, Divie’s C, Guzzo J, Biochemical and physiological studies of the small heat shock protein Lo18 from the lactic acid bacterium Oenococcus oeni [J].Mol Microbiol Biotechnol. 2001, 3: 601–610.
[56] Francoise C, Nicolas D, Magali B et al. A new approach for selection of Oenococcus oeni strains inorder to produce malolactic starters [J]. Food Microbiology. 2005,105:463– 470.
[57] Clegg M T, Kaheler A L P. Molecular diversity in plant population[M].Asot: sinauer. 1989: 98-115.
[58]白建荣,郭秀荣,侯变英.分子标记的类型特点及在育种中的应用[J].山西农业科学. 1999, 27(4):33-38.
[59] Grozdicker T, Williams J, Sharps P, et al. Physical mapping of temperature sensitive mutations of adenovirus. Cold Spring Harbor Syrup Quant Biol, 1974,39: 439.
[60] Hamada H, Petrino M, Kakunaga T.A novel repeated element with Z-DNA-forming potential is widely found in evolutionarily diverse eukaryotic genomes. Proc Natl Acad Sci, 1982, 79:646-649.
[61] Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers [J]. Nucleic Acids Research.1990, 18: 7213~7218.
[62] Williams J G , Kubelik A R, Livak K J, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic marker [J]. Nucleic Acids Research..1990, 8: 6531~6535.
[63] Admas M D, Kelley J M, Gocayne J D, et al. Complementary DNA sequencing: expressed sequence tags human genome project. Science, 1991, 252(5013):1651-1656.
[64] Vos Dieter, Hogers Rene, Bleeker Marjo, et al. AFLP:a new technique for DNA fingerprinting. Nucleic Acids Research,1995, 23(21):4407-4414.
[65] Zietkiewicz E. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase reaction amplification.Genomes ,1994,20:176~183.
[66] Hu J,Vick B A.target region amplied polymorphism:A novel marker technique for plant genotyping .Plant molecular biology reporter,2003,21:289~294.
[67] W. A. van Haeringen,L H P van de Goor, H Panneman et al. Detection of Universal Variable Fragments as Markers for Genetic Studies A Novel Technology for DNA Fingerprinting [J]. Molecular Biotechnology. 2003, (23)2: 117–125.
[68]周延清.DNA分子标记技术在植物研究中的应用[M].化学工业出版社.2005:79-94.
[69]唐立俊. RAPD的研究与应用进展[J].遵义师范学院学报. 2001,3(4):64-66.
[70]闰华超,高岚,李桂兰.分子标记技术的发展及应用[J].生物学通报.2006,41(2): 17-19.
[71]聂书萍,代林远.分子标记的发展及应用[J].河南职工医学院学报.2006,18(6): 526-528.
[72]古瑜,王春国,孙德岭等. RAPD分子标记方法及在蔬菜研究中的应用[J].天津农学院学报.2003,10(3): 47-51.
[73]吴少慧,张成刚,张忠泽. RAPD技术在微生物生物多样鉴定中的应用[J].微生物学杂志.2000,20(2): 44-47.
[74]焦锋,楼程富. RAPD技术应用中的一些问题及对策[J].西北农业学报.2000,9(4):98-102.
[75] Paran I, Michelmore R W. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce [J]. TAG Theoretical and Applied Genetics. 1993,85(8): 985-993.
[76] Michelmore R W, Paran I, Kesseli R V. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapaid method to detect markers in specific genomic region by using segregating populations[J]. Proceedings of the National Academy of Sciences of the United States of America. 1991,88(21):9828-9832.
[77]白凤虎,李德芳,陈安国,唐慧娟.基于BSA分析法的分子标记基因定位技术在农作物中的应用[J].中国麻业科学.2006,28(6): 282-288.
[78]邹继军,董伟,杨庆凯,等大豆灰斑病7号生理小种抗性的遗传分析及抗病基因的RAPD标记[J].科学通报.1998 ,43(21):2302一2307
[79]索广力,黄占景,何聪芬等.利用RAPD-BSA技术筛选小麦耐盐突变位点的分子标记[J].植物学报. 2001,43(6):598-602.
[80] YANG Qing-chuan, HAN Jian-guo, SUN Yan,et al. Identification of Molecular Marker Linked to Salt Tolerance Gene in Alfalfa[J]. Agricultural Science in China.2005,4(10) :781-787
[81]江启沛,冀宏,朱朝晖,贾英民. RAPD技术及其在食(药)用菌研究中的应用[J].河北省科学院学报2003,20(2):59-64.
[82]赵福宽,杨瑞,林成等.番茄耐冷性RAPD分子标记的筛选及特异片段的克隆[J].中央民族大学学报(自然科学版).2004,13(1): 70~74.
[83]赵福宽,周辉,高遐虹.南瓜抗CMV基因的RAPD分子标记筛选[J].华北农学报2007,22(1):90-94.
[84]杨青川,韩建国,孙彦.康俊梅苜蓿耐盐基因分子标记的筛选[J].作物学报.2005, 31(9): 1157-1161.
[85]王跃进,Lamikanra Olusola.检测葡萄无核基因DNA探针的合成与应用[J].西北农林科技大学学报(自然科学版).2002,30(3): 42~45.
[86]杨英军,王跃进,周鹏,王西平,张剑侠.葡萄无核基因的SCAR标记及Southern blot分析[J].西北农林科技大学学报(自然科学版) .2002, 30(6): 77~80.
[87]王跃进,Lamikanra O,卢江,Ramming D.葡萄无核基因的RAPD遗传标记[J].西北农业大学学报.1996, 24 (5): 1~9.
[88]梁利群,高俊生,李绍戊等.与鲤鱼抗寒性状相关的RAPD分子标记的筛选及其克隆[J].中国水产科学,2006, 13(3): 360~364.
[89]杜立新,万海伟,王爱华,李宏滨.用RAPD技术筛选中国荷斯坦牛产奶量性状遗传标记[J].畜牧兽医学报,2005,36(9),882~886.
[90]刘晓勇,姚勤,陈克平.利用RAPD技术筛选家蚕抗核型多角体病分子标记[J].江苏大学学报(自然科学版). 2004, 25(1):17~20.
[91] Du, Plessis E M, Dicks L M. Evaluation of random amplified polymorphic DNA (RAPD)- PCR as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus Amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus jihnsonii [J].Current Microbiology, 1995, 31: 114- 118.
[92] Booysen C, Dicks L M , Meijering I, et al. Isolation, identification and changes in the composition of lactic acid bacteria during the malting of two different barely cultivars [J]. International Journal of Food Microbiology, 2002,76:63- 73.
[93] Hayford A E, Petersen A, Vogensen F K et al. Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of Lactobacillus fermented in Ghanaian Fermented Maize Dough [J].Applied and Environmental Microbiology, 1999, 65: 3213- 3221.
[94] Antonsson M, Molin G, Ardo Y. Lactobacillus strains isolated from Danbo cheese as adjunct cultures in a cheese model system[J]. International Journal of Food Microbiology, 2003, 85: 159- 169.
[95] Holt S M and Cote G L. Differentiation of dextran-producing Leuconostoc strains by a modified randomlyamplified Polymorphic DNA protocol. [J].Environ. Microbiol.1998, 64: 3096-3098.
[96] Zavaleta A J, Martinez-Murcia A J and Rodriguez-Valera E, et al. Intraspecific genetic diversity of Oenococcus oeni as derived from DNA fingerprinting and sequence analyses[J] Environ.Microbiol.1997, 634: 1261一1267.
[97] H.W. du Plessisa,b, L.M.T. Dicksc, I.S. Pretorius ,et al. Identification of lactic acid bacteria isolated from South African brandy base wines[J]. International Journal of Food Microbiology. 2004,91: 19– 29.
[98]Tourdot-Mare′chal, R, Fortier, L-C, Guzzo, JLB, Divie`s, C, Acid sensitivity of neomycin-resistant mutants of Oenococcus oeni: a relationship between reduction of ATPase activity and lack of malolactic activity[J].FEMS Microbiol. Lett. 178, 319–326.
[99] Emilie Walling, Emmanuel Gindreau, Aline Lonvaud-Funel. A putative glucan synthase gene dps detected in exopolysaccharide-producing Pediococcus damnosus and Oenococcus oeni strains isolated from wine and cider[J].International Journal of Food Microbiology .2005,98:53– 62.
[100]凌代文.乳酸细菌分类鉴定及实验方法[M].中国轻工业出版社.1999.
[101]杨洁彬.乳酸菌—生物学基础及应用[M].中国轻工业出版社.1996.
[102]钱存柔,黄仪秀.微生物学实验教程[M].北京大学出版社.1999.
[103] Boulton R B, Singleton V L, Bisson L F, et al. Principles and practices of winemaking[M].Chapman & Hall, 1995
[104] Villani F Moschetti G and Blaiotta G. Characterisation of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA[J]. Appl. Microbiol. 1997, (82):578-588.
[105]奥斯伯F M等.精编分子生物学实验指南(第四版)[M].科学出版社, 2005:55-56.
[106]Clara G. Reyes-Gavilan, GaetanK.Y. Limsowtin, Patrick Tailliez. et. al. A Lactobacillus helveticus-Specific DNA probe detects restriction fragment length polymorphisms in this species[J]. Applied and Environmental Microbiology, 1992,(10):3429-3432.
[107]朱秀志,向成华,彭正松等.峨眉含笑基因组DNA提取及RAPD反应体系的优化[J].河北农业大学学报, 2006,29(2):46-49.
[108]孟现东,陈益泰.枫香DNA提取方法与PCR扩增程序的优化[J].林业科学研究.2004,17(1):42 -46.
[109]管晓庆,王奎玲,刘庆华等.野蔷薇的DNA提取和RAPD反应体系的建立[J].北方园艺, 2007(6): 220-222.
[110]易金鑫,侯喜林,冷月祥.茄子基因组DNA提取及RAPD- PCR体系的优化[J].江苏农业科学.2004, (3):50-52.
[111]王华,张春晖,李华.乳酸菌在葡萄酒酿造中的应用[J].西北农业大学报,1996(4): 92- 98.
[112]张春晖,李华,夏双梅.酒明串珠菌31-DH酿酒特性的研究[J].微生物学通报,1997(1): 92- 98.
[113] Booysen C, Dicks L M , Meijering I, et al. Isolation, identification and changes in the composition of lactic acid bacteria during the malting of two different barely cultivars [J]. International Journal of Food Microbiology, 2002,76:63- 73.
[114]丁红梅.红曲菌基因组DNA提取及RAPD反应体系优化[J].生物学杂志,2008,25(4):62-64.
[115]王尉,周文钊.剑麻DNA高效提取及RAPD反应体系优化[J].中国麻业科学,2008,30(4): 188-192.
[116]贾俊香,陶承光.大葱RAPD反应体系的优化[J].江苏农业科学, 2008, 3: 77-80.
[117]陈文炳,林媛等.双孢蘑菇RAPD-PCR反应体系的优化[J].食品科学,2008, 29(10):428-432.
[118]张春晖,夏双梅,李华.酒类酒球菌分离培养基研究[J].食品科学,2003,24(1):28~30.
[119]江志国,陶永胜.葡萄酒酒类酒球菌分离培养基设计[J].宁夏农学院学报,2004, 25(12):94~96.
[120] Sushanta Kumar Saha, L. Uma and G. Subramanian. An Improved Method for Marine Cyanobacterial DNA Isolation[J].World Journal of Microbiology and Biotechnology,2005,21(6-7): 877-881.
[121]A.G. Doulis, A.L. Harfouche and F.A. Aravanopoulos. Rapid, High Quality DNA Isolation from Cypress (Cupressus sempervirens L.) Needles and Optimization of the RAPD Marker Technique[J]. Plant Molecular Biology Reporter,1999,17(4):411-412.
[122]麻慧芳,王宙,黄晋玲.分子标记的类型与RAPD技术本身存在的问题及对策[J].山西师范大学学报(自然科学版), 2007,21(3):64-66.
[123]王丽,乔爱民等.菜心基因组DNA提取及RAPD反应体系的优化[J].西南师范大学学报(自然科学版), 2006,31(2):124-127.
[124] María Isabel Araque, Albert Bordons and Cristina Reguant. A multiplex PCR method for simultaneous species identification and strain typification of Oenococcus oeni[J]. World Journal of Microbiology and Biotechnology,2009,25(1): 15-18.
[125]汪小全,邹喻苹,张大明等.RAPD应用于遗传多样性和系统学研究中的问题[J].植物学报,1996,38 (12):954-962.
[126] Fortier LC, Tourdot-Marechal R, Divie′s C. Induction of Oenococcus oeni H+-ATPase activity and mRNA transcription under acidic conditions[J].FEMS Microbiology Letters, 2003, 222:165-169.
[127]Cox DJ, Henick-Kling T. Chemiosmotic energy from MLF[J].Journal of Bacteriology,1989,171: 5750-5752.
[128] Jobin MP, Garmyn D, Divies C, et al. The Oenococcus oeni clpX Homologue Is a Heat Shock Gene Preferentially Expressed in Exponential Growth Phase[J]. Journal of Bacteriology, 1999, 181:6634-6641.
[129] Guzzo J, Jobin MP, Delmas F, et al. Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase. Internationl[J] .Journal of Food Microbiology, 2000,55: 27-31.
[130] Bourdineaud JP, Nehme B, Tesse S, et al. The ftsH gene of the wine bacterium Oenococcus oeni is involved in protection against environmental stress[J]. Applied and Environmental Micro-biology, 2003, 69: 2511-2520.
[131] Beltramo C, Grandvalet C, Pierre F, et al. Evidence for multiple levels of regulation of Oenococcus oeni clpPclpL locus expres-sion in response to stress[J]. Journal of Bacteriology, 2004, 186:2200-2205.
[132] Bourdineaud JP, Nehme B, Tesse S, et al. A bacterial gene ho-mologous to ABC transporters protect Oenococcus oeni fromethanol and other stress factors in wine[J]. International Journal of Food Microbiology, 2004, 92: 1-14.
[133] Mills DA, Rawsthorne H, Parker C, et al. Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking[J]. FEMS Microbiology Reviews, 2005, 29: 465-475.
[134] Sabel López1, Carmen Torres, Fernanda Ruiz-Larrea1. Genetic typification by pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) of wild Lactobacillus plantarum and Oenococcus oeni wine strains[J].European Food Research and Technology. 2008, 227(2):547-555.