PCR技术用于啤酒中有害乳酸菌的检测和鉴定的研究
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摘要
乳酸菌是造成啤酒腐败的最常见微生物,对啤酒的危害很大。而纯生啤酒的厌氧菌污染危害更大,会使其质量在短时间内发生改变。目前常用的啤酒有害菌检测方法是厌氧平板培养法,即待长出菌落后通过菌落形态、细胞形态、革兰氏染色、过氧化氢酶阳性以及一系列生理生化实验来鉴定这些腐败菌。这种方法最大的缺陷是费时, 5～7d才可看到结果,不能够及时反馈微生物的污染状况。现代啤酒工业非常需要一种快速微生物检测技术,以解决传统微生物检测结果大大滞后于工艺生产的尴尬状况。PCR技术应用于啤酒厌氧菌的鉴定,不仅快速、准确,而且可以预测污染菌对啤酒的腐败能力,及时为生产提供指导性信息,具有一定的推广价值。
PCR技术在快速鉴定啤酒污染菌中的应用,需要对样品中的菌体细胞进行增殖培养以提高检测的灵敏度,为此,本课题比较了目前广泛用于啤酒有害菌检测的选择性培养基对啤酒有害菌检出的效果和菌体在培养基上的生长速度,为啤酒污染乳酸菌选择合适的富集培养基。本文比较了短乳杆菌、林氏乳杆菌、布氏乳杆菌、植物乳杆菌和四联球菌在NBB、MRS和自制UBA培养基上的菌落生量。同时比较了几种DNA提取的方法的提取效果。根据乳酸菌16s rDNA序列的特征,设计合成了针对啤酒有害乳酸菌的通用引物,同时根据乳酸菌16s rDNA序列的高变区设计了5种乳酸菌的特征引物,PCR技术验证结果表明该5种引物能够准确鉴定乳酸菌。
在以上研究的基础上,本文建立了PCR快速检测啤酒有害菌的新方法,用基于16s rDNA部分序列的特征引物对啤酒污染乳酸菌进行PCR检测,灵敏度可达到15个细胞(CFU)/250mL,样品的预培养需12～18h。啤酒有害菌检测所需时间从传统的5d减少到24h。
Microbial contaminants of the brewing process cause product spoilage. The recent increase in the consumption of draft beer as opposed to pasteurized beer has made the biological control of beer spoilage microorganisms even more important. The plate count method for enumerating microbiological contamination has remained unchanged for over a century, but it requires several days before the microorganisms are detected.The PCR method compares favorably in sensitivity with the present plate count methods (which require several days) for the detection of lactic acid bacteria and with many rapid detection methods.
Colony growth of Lactobacillus brevis,Lactobacillus lindneri,Lactobacillus buchneri, Lactobacillus plantarum,Pediococcus damnosus on five culture media (NBB ,MRS and self-made UBA) was compared. Furthermore, The effects of different factors ,including DNA templates and primers , on the quality and reproducibility of PCR were investigated. According to the characteristic of 16S ribosomal RNA gene (16s rDNA),a pair of universal primers were designed for beer spoilage lactic acid bacteria (LAB). Furthermore, 5 other special primers were designed based on identifying results. It was showed that all the primers acted well in accurate identification of LAB by PCR.
A new method for rapid detecting beer spoilage microorganisms by PCR was developed,This method is based on the sequence of 16s rDNA, on which a pair of specific primers were designed .Using the primers , beer spoilage LAB species were detected by PCR,The sensitivity was reached to 15CFU/250mL, The pre-enrichment of samples was needed only 12-18h,The duration for beer spoilage determination by PCR (24h) was markly shorter than by conventional method (5-7d) .
PCR技术在快速鉴定啤酒污染菌中的应用,需要对样品中的菌体细胞进行增殖培养以提高检测的灵敏度,为此,本课题比较了目前广泛用于啤酒有害菌检测的选择性培养基对啤酒有害菌检出的效果和菌体在培养基上的生长速度,为啤酒污染乳酸菌选择合适的富集培养基。本文比较了短乳杆菌、林氏乳杆菌、布氏乳杆菌、植物乳杆菌和四联球菌在NBB、MRS和自制UBA培养基上的菌落生量。同时比较了几种DNA提取的方法的提取效果。根据乳酸菌16s rDNA序列的特征,设计合成了针对啤酒有害乳酸菌的通用引物,同时根据乳酸菌16s rDNA序列的高变区设计了5种乳酸菌的特征引物,PCR技术验证结果表明该5种引物能够准确鉴定乳酸菌。
在以上研究的基础上,本文建立了PCR快速检测啤酒有害菌的新方法,用基于16s rDNA部分序列的特征引物对啤酒污染乳酸菌进行PCR检测,灵敏度可达到15个细胞(CFU)/250mL,样品的预培养需12～18h。啤酒有害菌检测所需时间从传统的5d减少到24h。
Microbial contaminants of the brewing process cause product spoilage. The recent increase in the consumption of draft beer as opposed to pasteurized beer has made the biological control of beer spoilage microorganisms even more important. The plate count method for enumerating microbiological contamination has remained unchanged for over a century, but it requires several days before the microorganisms are detected.The PCR method compares favorably in sensitivity with the present plate count methods (which require several days) for the detection of lactic acid bacteria and with many rapid detection methods.
Colony growth of Lactobacillus brevis,Lactobacillus lindneri,Lactobacillus buchneri, Lactobacillus plantarum,Pediococcus damnosus on five culture media (NBB ,MRS and self-made UBA) was compared. Furthermore, The effects of different factors ,including DNA templates and primers , on the quality and reproducibility of PCR were investigated. According to the characteristic of 16S ribosomal RNA gene (16s rDNA),a pair of universal primers were designed for beer spoilage lactic acid bacteria (LAB). Furthermore, 5 other special primers were designed based on identifying results. It was showed that all the primers acted well in accurate identification of LAB by PCR.
A new method for rapid detecting beer spoilage microorganisms by PCR was developed,This method is based on the sequence of 16s rDNA, on which a pair of specific primers were designed .Using the primers , beer spoilage LAB species were detected by PCR,The sensitivity was reached to 15CFU/250mL, The pre-enrichment of samples was needed only 12-18h,The duration for beer spoilage determination by PCR (24h) was markly shorter than by conventional method (5-7d) .
引文
1 徐岩,张丽苹等. 啤酒酿造中腐败细菌的研究. 酿酒,2000 ,(6) :68～72
2 曹程节. 啤酒工业的微生物管理. 食品工业,2000 , (4) :13～14
3 左永泉. 啤酒生产过程中微生物的污染与防治. 中国酿造,1997 , (3) :8～9
4 Farrow,J. A. E. ,Philips, B. A and Collins, M. D. (1988)Nucleic acid studies on some heterofermentative lactobacilli: Description ofLactobacillus malefermentans sp. Nov. and Lactobacillus parabuchneri sp. Nov. FEMS Microbio1. Letts. 55. 163—168.
5 Schleifer. K. H. ,Leuteritz, M. , Weiss, N. , Ludwig, W. , Kirchhof. G. and Seidel— Rufer, H. (1990)Taxonomic study of anaerobic, Gram —negative, rod—shaped bacteria from breweries:emended description of Pectinatus cerevisiiphilus and description of Pectinatus frisingensis sp. Nov. , Selenomonas lacticifex sp. Nov. , Zymaphilus raffinosivorans gen. Nov. , sp. Nov. , and Zymophilus paucivorans sp. Nov. Int. J. Syst. Bacterio1. 40,19—27.
6 Haikara, A. and Lounatmaa. K. (1987)Characterization of Measphaera sp. , a new anaerobic beer spoilage coccus. Proc. Eur. Brew. Conv. , 473—480.
7 Lawrence, D. R. (1988)Spoilage organisms in beer. In: R. K. Robinson(editor), Developments in Food Microbiology. Elsevier, London, l一48.
8 van Vuuren, H. J. J. (1996)Gram—negative spoilage bacteria. In: F. G. Priest and I. Campbell(editors), Brewing Microbiology. 2nd edn. E1sevier, London. 163— 191.
9 Lawrence, D. R. and Priest, F. G. (1981)Identification of brewery cocci. Proc. Eur. Bur. Brew. Conv. 217—227.
10 顾国贤.酿造酒工艺学(第二版) [M].中国轻工业出版社.1996.b
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12 Manabu Sami , H.Yamashita J.Am.Soc [J].Brew.Chem.1997 ,55(4) :137-140.
13 Manabu Sami , H.Yamashita J.Of Fermantation And Bioengineering[J].1997 ,84 (1):1-6.
14 凌代文主编.乳酸细菌分类鉴定及实验方法[M].中国轻工出版社1999.
15 Evaluation Study of the Frequencyof Different Beer-Spoiling Bacteria Using the VIT Analysis. MBAA,2006(43):31-35
16 Ampe F,ben Omar N,Cuyot JP. Culture-independent quantification ofphysiologically-active bbmicrobial groups in fermented foods using rRNA-targeted oligonucleotide probes: application to pozol,a Mexican lactic acid fermented maize dough. J Appl Microbiol,1999,87(1):131-140.
17 Ferris MM, Muyzer G, Ward DM, et al. Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol, 1996,62(2):340-346.
18 Godon JJ,Zumstein E,Dabert P,et al.Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.Appl Environ Microbiol, 1997,63(7): 2802-2813.
19 Suau A,Bonnet R,Sutren M,et al.Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut.Appl Environ Microbiol,1999,65(11): 4799-4807
20 Norman BP,New Perspective on the Natural Microbial World: Molecular Microbial Ecology.ASM General Meeting in New Orleans,1996.
21 Pace NR,Stahl DA,Lane DJ,et al.The analysis of natural microbial populations by ribosomal RNA sequence.Advances Microbial Ecol,1986(9): 1-55.
22 焦振泉. 16S rRNA序列同源性分析与细菌系统分类鉴定. 国外医学卫生学分册,1998,25(1):12-16.
23 徐岩,张丽萍,顾国贤.聚合酶链式反应(PCR)技术鉴定啤酒腐败菌的最新进展[J].食 品与发酵工业,2001,(5):71-74.
24 孔庆新,张惟广.啤酒生产中腐败微生物鉴定新技术[J].酿酒科技,2003,116(2):26-27. 25 闫雪,姚卫容,钱和.国内外食品微生物快速检测技术应用进展[J].食品科学,2005,26(6):269-271.
26 吴红.啤酒的微生物检验技术[J].酿酒,2000,(5):34-38.
27 田小群,周世宁.聚合酶链式反应快速鉴定啤酒有害菌研究[J].微生物学通报,2006,(1).
28 DiMichele, L. J., and Lewis, M. J. Rapid, species-specific detection of lactic acid bacteria from beer using the polymerase chain reaction. J. Am. Soc. Brew. Chem. 51:63-66, 1993.
29 Satokari, R., Juvonen, R., Mallison, K., von Wright, A., and Haikara,A. Detection of beer spoilage bacteria Megasphaera and Pectinatus by PCR and colorimetric microplatehybridization. Int. J. Food Microbiol. 45:119-127,1998.
30 Satokari, R., Juvonen, R., von Wright, A., and Haikara, A. Detection of Pectinatus beer spoilage bacteria by using the polymerase chain reaction. J. Food Prot. 60:1571-1573, 1997.
31 Thompson, A. N., Wright, D. M., Pawson, E. C., and Meaden, P. G.Polymerase chain reaction tests for lactic acid bacteria. Proc. Fourth Aviemore Conf. Malting Brew. Distill. I. Campbell and F. G. Priest, eds. The Institute of Brewing, London: pp. 213-216, 1995.
32 Tsuchiya, Y., Kaneda, H., Kano, Y., and Koshino, S. Detection of beer spoilage organisms by polymerase chain reaction technology. J.Am. Soc. Brew. Chem. 50:64-67, 1992.
33 Tsuchiya, Y., Kano, Y., and Koshino, S. Detection of Lactobacillus brevis in beer using polymerase chain reaction technology. J. Am. Soc. Brew. Chem. 51:40-41, 1993.
34 Vogeser, G., and Geiger, E. Microbiological analysis of beer samples with the PCR method. Brauwelt 24/25:1060-1062, 1998.
35 Yasui, T., Okamoto, T., and Taguchi, H. A specific oligonucleotide primer for the rapid detection of Lactobacillus lindneri by PCR. Can. J. Microbiol. 43:157-163, 1997.
36 Auli Haikara, Riikka Juvonen, Teija Koivula (FIN), Thomas Schuhbeck, Andreas Brandl, Gudrun Vogeser (D) & Erna Storg?rds (FIN). Microbiological quality control in breweries by PCR - the BREWPROC approach .EBC PCR techniques, 2003 .
37 Riikka Juvonen, Teija Koivula & Auli Haikara (FIN). PCR detection of Gram-negative brewery contaminants - present state . EBC PCR techniques, 2003 .
38 M. Voetz & F. Rath (D). A novel quantitative real-time PCR method for analysing the varietal purity of barley and malt samples in a batch mode . EBC PCR techniques, 2003 .
39 Antje Braune & Andreas Eidtmann (D) . First experiences using realtime-PCR as a rapid detection method for brewery process control at Beck & Co . EBC PCR techniques, 2003 .
40 Youichi Tsuchiya, Yasukazu Nakakita, Yasunobu Nara & Masachika Takashio (J) Real-Time PCR and melting curve analysis for rapid detection and identification of beer spoilage microorganisms . EBC PCR techniques, 2003 .
41 Tore Hage & Kari Wold (N) . Practical experiences on the combat of a major Pectinatus and Megasphera infection with the help of TaqMan Realtime-PCR . EBC PCR techniques, 2003 .
42 S.L. Walker, J.R.M. Hammond, K.M.D. Pawlowsky & T. M-J. Maugueret(UK) .A PCR-ELISA assay for the detection of beer spoilage organisms . EBC PCR techniques, 2003 .
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2 曹程节. 啤酒工业的微生物管理. 食品工业,2000 , (4) :13～14
3 左永泉. 啤酒生产过程中微生物的污染与防治. 中国酿造,1997 , (3) :8～9
4 Farrow,J. A. E. ,Philips, B. A and Collins, M. D. (1988)Nucleic acid studies on some heterofermentative lactobacilli: Description ofLactobacillus malefermentans sp. Nov. and Lactobacillus parabuchneri sp. Nov. FEMS Microbio1. Letts. 55. 163—168.
5 Schleifer. K. H. ,Leuteritz, M. , Weiss, N. , Ludwig, W. , Kirchhof. G. and Seidel— Rufer, H. (1990)Taxonomic study of anaerobic, Gram —negative, rod—shaped bacteria from breweries:emended description of Pectinatus cerevisiiphilus and description of Pectinatus frisingensis sp. Nov. , Selenomonas lacticifex sp. Nov. , Zymaphilus raffinosivorans gen. Nov. , sp. Nov. , and Zymophilus paucivorans sp. Nov. Int. J. Syst. Bacterio1. 40,19—27.
6 Haikara, A. and Lounatmaa. K. (1987)Characterization of Measphaera sp. , a new anaerobic beer spoilage coccus. Proc. Eur. Brew. Conv. , 473—480.
7 Lawrence, D. R. (1988)Spoilage organisms in beer. In: R. K. Robinson(editor), Developments in Food Microbiology. Elsevier, London, l一48.
8 van Vuuren, H. J. J. (1996)Gram—negative spoilage bacteria. In: F. G. Priest and I. Campbell(editors), Brewing Microbiology. 2nd edn. E1sevier, London. 163— 191.
9 Lawrence, D. R. and Priest, F. G. (1981)Identification of brewery cocci. Proc. Eur. Bur. Brew. Conv. 217—227.
10 顾国贤.酿造酒工艺学(第二版) [M].中国轻工业出版社.1996.b
11 Jacqueline L.Fernandez and W.J.Simpson Journal of Bacteriology[M]. 1994 ,77 ,635-638.
12 Manabu Sami , H.Yamashita J.Am.Soc [J].Brew.Chem.1997 ,55(4) :137-140.
13 Manabu Sami , H.Yamashita J.Of Fermantation And Bioengineering[J].1997 ,84 (1):1-6.
14 凌代文主编.乳酸细菌分类鉴定及实验方法[M].中国轻工出版社1999.
15 Evaluation Study of the Frequencyof Different Beer-Spoiling Bacteria Using the VIT Analysis. MBAA,2006(43):31-35
16 Ampe F,ben Omar N,Cuyot JP. Culture-independent quantification ofphysiologically-active bbmicrobial groups in fermented foods using rRNA-targeted oligonucleotide probes: application to pozol,a Mexican lactic acid fermented maize dough. J Appl Microbiol,1999,87(1):131-140.
17 Ferris MM, Muyzer G, Ward DM, et al. Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol, 1996,62(2):340-346.
18 Godon JJ,Zumstein E,Dabert P,et al.Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.Appl Environ Microbiol, 1997,63(7): 2802-2813.
19 Suau A,Bonnet R,Sutren M,et al.Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut.Appl Environ Microbiol,1999,65(11): 4799-4807
20 Norman BP,New Perspective on the Natural Microbial World: Molecular Microbial Ecology.ASM General Meeting in New Orleans,1996.
21 Pace NR,Stahl DA,Lane DJ,et al.The analysis of natural microbial populations by ribosomal RNA sequence.Advances Microbial Ecol,1986(9): 1-55.
22 焦振泉. 16S rRNA序列同源性分析与细菌系统分类鉴定. 国外医学卫生学分册,1998,25(1):12-16.
23 徐岩,张丽萍,顾国贤.聚合酶链式反应(PCR)技术鉴定啤酒腐败菌的最新进展[J].食 品与发酵工业,2001,(5):71-74.
24 孔庆新,张惟广.啤酒生产中腐败微生物鉴定新技术[J].酿酒科技,2003,116(2):26-27. 25 闫雪,姚卫容,钱和.国内外食品微生物快速检测技术应用进展[J].食品科学,2005,26(6):269-271.
26 吴红.啤酒的微生物检验技术[J].酿酒,2000,(5):34-38.
27 田小群,周世宁.聚合酶链式反应快速鉴定啤酒有害菌研究[J].微生物学通报,2006,(1).
28 DiMichele, L. J., and Lewis, M. J. Rapid, species-specific detection of lactic acid bacteria from beer using the polymerase chain reaction. J. Am. Soc. Brew. Chem. 51:63-66, 1993.
29 Satokari, R., Juvonen, R., Mallison, K., von Wright, A., and Haikara,A. Detection of beer spoilage bacteria Megasphaera and Pectinatus by PCR and colorimetric microplatehybridization. Int. J. Food Microbiol. 45:119-127,1998.
30 Satokari, R., Juvonen, R., von Wright, A., and Haikara, A. Detection of Pectinatus beer spoilage bacteria by using the polymerase chain reaction. J. Food Prot. 60:1571-1573, 1997.
31 Thompson, A. N., Wright, D. M., Pawson, E. C., and Meaden, P. G.Polymerase chain reaction tests for lactic acid bacteria. Proc. Fourth Aviemore Conf. Malting Brew. Distill. I. Campbell and F. G. Priest, eds. The Institute of Brewing, London: pp. 213-216, 1995.
32 Tsuchiya, Y., Kaneda, H., Kano, Y., and Koshino, S. Detection of beer spoilage organisms by polymerase chain reaction technology. J.Am. Soc. Brew. Chem. 50:64-67, 1992.
33 Tsuchiya, Y., Kano, Y., and Koshino, S. Detection of Lactobacillus brevis in beer using polymerase chain reaction technology. J. Am. Soc. Brew. Chem. 51:40-41, 1993.
34 Vogeser, G., and Geiger, E. Microbiological analysis of beer samples with the PCR method. Brauwelt 24/25:1060-1062, 1998.
35 Yasui, T., Okamoto, T., and Taguchi, H. A specific oligonucleotide primer for the rapid detection of Lactobacillus lindneri by PCR. Can. J. Microbiol. 43:157-163, 1997.
36 Auli Haikara, Riikka Juvonen, Teija Koivula (FIN), Thomas Schuhbeck, Andreas Brandl, Gudrun Vogeser (D) & Erna Storg?rds (FIN). Microbiological quality control in breweries by PCR - the BREWPROC approach .EBC PCR techniques, 2003 .
37 Riikka Juvonen, Teija Koivula & Auli Haikara (FIN). PCR detection of Gram-negative brewery contaminants - present state . EBC PCR techniques, 2003 .
38 M. Voetz & F. Rath (D). A novel quantitative real-time PCR method for analysing the varietal purity of barley and malt samples in a batch mode . EBC PCR techniques, 2003 .
39 Antje Braune & Andreas Eidtmann (D) . First experiences using realtime-PCR as a rapid detection method for brewery process control at Beck & Co . EBC PCR techniques, 2003 .
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