JP3398897B2 - Hop-resistant DNA of lactic acid bacteria and method for detecting hop-resistant lactic acid bacteria - Google Patents
Hop-resistant DNA of lactic acid bacteria and method for detecting hop-resistant lactic acid bacteriaInfo
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- JP3398897B2 JP3398897B2 JP2000248358A JP2000248358A JP3398897B2 JP 3398897 B2 JP3398897 B2 JP 3398897B2 JP 2000248358 A JP2000248358 A JP 2000248358A JP 2000248358 A JP2000248358 A JP 2000248358A JP 3398897 B2 JP3398897 B2 JP 3398897B2
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- Prior art keywords
- lactic acid
- acid bacteria
- hop
- resistant
- beer
- Prior art date
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Description
【0001】[0001]
【産業上の利用分野】本発明は、特定の配列を有するD
NAに関するものである。FIELD OF THE INVENTION The present invention relates to D having a specific sequence.
It is about NA .
【0002】[0002]
【従来の技術と発明が解決しようとする課題】ビール
は、アルコールの含有、炭素源の枯渇、低pH、嫌気状
態という条件であることに加えて、抗菌活性をもつホッ
プを含むことから、微生物の生育抑制効果がある。しか
し、ある種のラクトバチルス(Lactobacillus)属菌
は、ホップ中の成分であるisoα酸に対して耐性を持つ
ことから、ビール製品に混入し生育することがある。BACKGROUND OF THE INVENTION Beer is a microorganism containing hops having antibacterial activity in addition to the conditions of alcohol content, carbon source depletion, low pH and anaerobic condition. Has the effect of suppressing the growth of. However, some Lactobacillus bacteria are resistant to isoα acid, which is a component in hops, and thus may be mixed with beer products to grow.
【0003】このビールの品質に影響するラクトバチル
ス属菌の検出、判定については従来から検討されてお
り、例えば、抗原抗体反応を利用したビール醸造有害細
菌の検出法(特開昭57−59166)、ビール乳酸菌
に対するモノクローナル抗体を用いた検出法(特開平0
6−105698)、乳酸菌からDNAを抽出して特定
のオリゴヌクレオチドをプライマーとしてPCRを行い
配列を増幅させた後乳酸菌の有無を判定する方法(特開
平06−141899等)がある。The detection and determination of Lactobacillus, which affects the quality of beer, have been studied in the past. For example, a method for detecting harmful beer-brewing bacteria using an antigen-antibody reaction (Japanese Patent Laid-Open No. 57-59166). , A detection method using a monoclonal antibody against beer lactic acid bacteria
6-105698), DNA is extracted from lactic acid bacteria, PCR is performed using a specific oligonucleotide as a primer to amplify the sequence, and then the presence or absence of lactic acid bacteria is determined (Japanese Patent Laid-Open No. 06-141899, etc.).
【0004】しかしながら、これらの方法は、主として
ラクトバチルス ブレビスのような特定の乳酸菌の判定
を行う手法であり、このため、ホップ耐性のないブレビ
スの検出が行われる一方、ホップ耐性のある他の種の菌
を検出できないので、ビール混濁乳酸菌を確実に検出で
きないという問題点があった。[0004] However, these methods are mainly for determining specific lactic acid bacteria such as Lactobacillus brevis. Therefore, while brevis not having hop resistance is detected, other hop-resistant species are detected. However, there is a problem that the beer-cloudy lactic acid bacterium cannot be reliably detected.
【0005】[0005]
【課題を解決するための手段】そこで本発明では、ビー
ル混濁乳酸菌のホップ耐性に関係するDNAを特定し、
そのDNAをプローブとして利用してホップ耐性乳酸菌
を迅速に検出するものである。Therefore, in the present invention, DNA relating to hop resistance of beer-cloudy lactic acid bacteria was identified,
The DNA is used as a probe to rapidly detect hop-resistant lactic acid bacteria.
【0006】ホップ耐性プラスミドは次の方法により得
られ、ホップ耐性乳酸菌を検出することができる。すな
わち、(1)ビール混濁乳酸菌を徐々にホップ濃度の高
い培地に植え継いで行き馴化し、ビール混濁乳酸菌のホ
ップ耐性を強化する。(2)この馴化操作を繰り返して
いくことによって、ビール混濁乳酸菌の中に増加したホ
ップ耐性に関係するプラスミドを確認する。(3)確認
されたプラスミドの一部または全部のDNAを標識して
判定用プローブをつくる。(4)ホップ耐性乳酸菌か否
かを判定したい乳酸菌を(3)で得たプローブで判定す
る。(5)(4)でビール混濁乳酸菌を検出できたプロ
ーブに含まれるDNA断片の塩基配列を決定する。 [0006] hops resistance plasmid obtained by the following method, it is possible to detect hop resistant lactic acid bacteria. That is, (1) the beer cloudy lactic acid bacterium is gradually subcultured in a medium having a high hop concentration to be acclimatized to enhance the hop resistance of the beer cloudy lactic acid bacterium. (2) By repeating this acclimation operation, a plasmid associated with increased hop resistance in beer cloudy lactic acid bacteria is confirmed. (3) A part of or the entire DNA of the confirmed plasmid is labeled to prepare a determination probe. (4) The lactic acid bacterium for which it is desired to determine whether it is a hop-resistant lactic acid bacterium is determined with the probe obtained in (3). (5) A professional who was able to detect beer cloudy lactic acid bacteria in (4)
The nucleotide sequence of the DNA fragment contained in the probe is determined.
【0007】ビール混濁乳酸菌は、ビール製造において
耐酸性と耐アルコール性を有する有害菌であり、具体的
には、ラクトバチルス ブレビス(Lactobacillus brev
is)、ラクトバチルス プランタラム(Lactobacillus p
lantarum)、ラクトバチルスカゼイ(Lactobacillus cas
ei) 等がある。Beer cloudy lactic acid bacteria are harmful bacteria having acid resistance and alcohol resistance in the production of beer, and specifically, Lactobacillus brevis ( Lactobacillus brev
is ), Lactobacillus p
lantarum) , Lactobacillus cas
ei ) etc.
【0008】以下に、本発明をさらに詳しく説明する。
ビール混濁乳酸菌のホップ馴化を行う培地は乳酸菌が生
育できる培地であればいずれでもよいが、例えばMRS
液体培地等が使用できる。培地に添加するホップエキス
としては、イソ化ホップエキスが利用できる。The present invention will be described in more detail below.
Any medium can be used as a medium for acclimatizing beer-turbid lactic acid bacteria to hops, as long as the lactic acid bacteria can grow. For example, MRS
A liquid medium or the like can be used. Isolated hop extract can be used as the hop extract added to the medium.
【0009】ホップエキスを添加する濃度は、試験に供
するビール混濁乳酸菌が生育できる最大濃度から開始
し、その濃度から50〜100マイクロモル(イソα酸
換算)ずつ上昇し、最大濃度の3倍以上まで達成させ
る。The concentration of hop extract to be added starts from the maximum concentration at which beer-turbid lactic acid bacteria to be subjected to the test can grow, and increases by 50 to 100 micromol (in terms of iso-alpha acid) from that concentration, and is at least 3 times the maximum concentration. To achieve.
【0010】培養条件は、ビール混濁乳酸菌が生育でき
る通常の適温で嫌気条件であればよい。培養時間は、各
ホップエキス含有培地で生育するまでであるが、最大5
日程度内である。Cultivation conditions may be any suitable anaerobic conditions at an ordinary temperature at which beer cloudy lactic acid bacteria can grow. Cultivation time is up to 5 until it grows in each hop extract-containing medium.
It is within a day.
【0011】ホップエキス馴化したビール混濁乳酸菌か
らホップ耐性プラスミドを取得する方法は以下のとおり
である。培養したビール混濁乳酸菌を公知のアルカリS
DS法(文献:D. G. Ardersen & L. L. Mckay, Appl.
Env. Microbiol., 46, 549 (1983))で乳酸菌内の全プラ
スミドを得る。得られた各プラスミドをアガロースゲル
電気泳動にかけ、馴化前に比べて馴化後にコピー数の増
加したプラスミドのバンド部分を切り出し、アガロース
を除去する。 The method for obtaining a hop resistant plasmid from beer cloudy lactic acid bacteria acclimated to hop extract is as follows. Known beer S containing cultured beer cloudy lactic acid bacteria
DS method (reference: DG Ardersen & LL Mckay, Appl.
Env. Microbiol., 46, 549 (1983)) obtains all plasmids in lactic acid bacteria. Each resulting plasmid was subjected to agarose gel electrophoresis, excised increased band portion of the plasmid copy number after habituation than before habituation, remove the agarose.
【0012】この得られたホップ耐性プラスミドをプロ
ーブに利用するには、プラスミド全体を用いてもよく、
あるいはこのうちの一部を用いても良い。特に本発明の
実施例で得られたORF5のホップ耐性プラスミドは、
ATP binding casette transporter によるホップ排
出ポンプ遺伝子をコードしたプラスミドと推定され、非
常にビール混濁乳酸菌の検出に有効なものである。この
プローブは通常これらを利用して検出することができる
公知の方法によって標識するのが好ましく、例えば、ビ
オチン−アビジン、ジゴキシゲニン等の標識、あるいは
放射性標識等が利用できる。In order to utilize the obtained hop resistance plasmid as a probe, the entire plasmid may be used,
Alternatively, a part of them may be used. In particular, the hop resistance plasmid of ORF5 obtained in the examples of the present invention is
It is presumed to be a plasmid encoding the hop efflux pump gene by the ATP binding casette transporter, and is very effective in detecting beer-cloudy lactic acid bacteria. It is preferable to label this probe by a known method that can usually be used for detection, and for example, a label such as biotin-avidin, digoxigenin, or a radioactive label can be used.
【0013】この標識されたホップ耐性プローブを用
い、ビール混濁乳酸菌を判定する方法としては、一般に
利用されているコロニーハイブリダイゼーション法、サ
ザンハイブリダイゼーション法、PCR法等が利用でき
る。The colony hybridization method, the Southern hybridization method, the PCR method or the like which is generally used can be used as a method for determining beer cloudy lactic acid bacteria using this labeled hop resistance probe.
【0014】[0014]
【実施例】以下、実施例により本発明を説明する。
(1)ラクトバチルス ブレビス(Lactobacillus brev
is)ABBC45株のホップ耐性プラスミドの取得
ラクトバチルス ブレビスABBC45株をイソ化ホッ
プエキスを150マイクロモル含有したMRS液体培地
5mlに106 セル/mlの割合で添加し、30℃で3
日間培養した。その後、ホップ馴化をすすめるために、
50マイクロモルずつイソ化ホップエキスを増加してい
き、最終濃度が900マイクロモルになるまで上記の条
件で同様の操作を行った。EXAMPLES The present invention will be described below with reference to examples. (1) Lactobacillus brev
is ) Acquisition of hop-resistant plasmid of ABCBC45 strain Lactobacillus brevis ABCBC45 strain was added at a rate of 10 6 cells / ml to 5 ml of MRS liquid medium containing 150 micromoles of isolated hop extract, and the mixture was mixed at 30 ° C. for 3 hours.
Cultured for a day. After that, in order to promote hop acclimatization,
The isotopic hop extract was increased in increments of 50 μmol, and the same operation was performed under the above conditions until the final concentration reached 900 μmol.
【0015】馴化前後の乳酸菌を集菌、洗浄し、次いで
アルカリSDS法によって全プラスミドを取得した。馴
化前後の乳酸菌から得られた全プラスミドをそれぞれア
ガロースゲル電気泳動にかけ、馴化後にコピー数が増加
したプラスミドのバンド部分を切り出し、アガロースを
除去した。Lactic acid bacteria before and after acclimatization were collected and washed, and then all plasmids were obtained by the alkaline SDS method. All the plasmids obtained from the lactic acid bacteria before and after acclimation were subjected to agarose gel electrophoresis, and the band portion of the plasmid whose copy number increased after acclimation was excised to remove agarose.
【0016】得られたプラスミド(以下、pRH45と
いう)は14.5kbであり、制限酵素地図は図1のと
おりである。本プラスミドのORF5断片を、PCR法
により増幅させ、ベーリンガーマンハイム社製DIG
DNA標識及び検出キットを使用し、ジゴキシゲニン−
dUTPを用いたランダムプライムシステムによるDN
A標識を行った。The obtained plasmid (hereinafter referred to as pRH45) is 14.5 kb, and the restriction map is shown in FIG. The ORF5 fragment of this plasmid was amplified by the PCR method to obtain DIG manufactured by Boehringer Mannheim.
Digoxigenin-using a DNA labeling and detection kit
DN by a random prime system using dUTP
A labeling was performed.
【0017】表1に示した乳酸菌からそれぞれプラスミ
ドを分離し、アガロース電気泳動にかけた後、ナイロン
メンブランに転写した。次に標識したプローブをハイブ
リダイズさせ、前記キットを用いたELISAによる検出を
行った。Plasmids were isolated from the lactic acid bacteria shown in Table 1, subjected to agarose electrophoresis, and then transferred to a nylon membrane. Next, the labeled probe was hybridized and detected by ELISA using the above kit.
【0018】[0018]
【表1】 [Table 1]
【0019】(2)標識されたホップ耐性プローブを用
いたビール混濁乳酸菌の判定
表2に示した乳酸菌をそれぞれ102cells/mlを懸濁し
た、ビール500ml をメンブラン濾過し、MRS平板培地上
にのせ、25℃で嫌気培養した。出現したコロニーをナイ
ロンメンブランにうつしとり、溶菌させDNAをメンブ
ラン上に固定した。次に実施例(1)で得られた標識し
たプローブをハイブリダイズさせ前記キットを用いたEL
ISAによる検出を行った。(2) Judgment of beer-turbid lactic acid bacteria using labeled hop-resistant probe 500 ml of beer in which 10 2 cells / ml of the lactic acid bacteria shown in Table 2 were suspended was subjected to membrane filtration and placed on an MRS plate medium. And anaerobically cultured at 25 ° C. The colonies that appeared were transferred to a nylon membrane, lysed, and the DNA was immobilized on the membrane. Next, the labeled probe obtained in Example (1) was hybridized and the above-mentioned EL was used.
Detection by ISA was performed.
【0020】[0020]
【表2】
(3)pRH45のORF5断片のDNA配列
公知の方法により分析したpRH45のORF5断片の
DNA配列を図2および配列表の配列番号:1に示し
た。[Table 2] (3) DNA sequence of ORF5 fragment of pRH45 The DNA sequence of the ORF5 fragment of pRH45 analyzed by a known method is shown in Fig. 2 and SEQ ID NO: 1 in the sequence listing.
【0021】[0021]
【発明の効果】本発明は、ビール混濁乳酸菌のホップ耐
性に関係するDNAを特定し、このDNAをプローブと
して利用するため、ホップ耐性乳酸菌を迅速に検出する
ことができる。これによりビールの品質に影響するラク
トバチルス属菌の検出、判定を確実に迅速に行うことが
できる。INDUSTRIAL APPLICABILITY According to the present invention, DNA relating to hop resistance of beer-turbid lactic acid bacteria is specified, and this DNA is used as a probe. Therefore, hop-resistant lactic acid bacteria can be rapidly detected. As a result, it is possible to surely and promptly detect and judge the Lactobacillus bacterium that affects the quality of beer.
【0022】[0022]
【配列表】 SEQUENCE LISTING <110> Asahi Breweries, Ltd. <120> Plasmid having a novel nucleic acid sequence <130> 96T3-22BU <160> 1 <210> 1 <211> 1749 <212> DNA <213> Lactobacillus brevis <400> 1 atgcaagctc agtccaagaa caataccaag tttaacttta aaacatttat gggcctaatc 60 aaccgaattc acccccgtta ctggcaactg ctgcttggct tttttctagg agttgtcgca 120 acggcgatgc aattgatggt tcccggcatc gccaagggga tcatcaactc aatcggtcat 180 tcaatggatg tcggcctaat cgttgccgtc attttactat tcgttttcag taccattatt 240 ggagcctttt ccggcagtat tttaggcttc ttcggtgaag acgtcgtcta taagctgcga 300 acaacacttt gggataaaat cttaaccctg ccggtgggtt attttgacca aaccaaatct 360 ggcgaaataa cgtccaggtt ggtcaatgat tccacacagg tcaaggaact gttggccaat 420 tcggttccca aaaccgcaac ttcgattctg caactggttg gcgcattggt cttaatgctc 480 atcatggact ggcggatgac tatcattatg tttatcgccg ttccgctcgt cttgatctgc 540 ctgctgccaa ttgtccgcca atcccacaaa gttgccagag cgagacagga cgcactggca 600 gatctcaatg gtaaagccgg tgaaatgctg ggcgaagtcc gtctagtcaa atcgtctacc 660 gcagaaaact tagaacgaac agccggcgat aaacggatgt atcgccttta tcgcatcggg 720 ttaaaagaag cgatctatga ttcaattgcc ggacctgtaa tgggcatggt catgatggcc 780 atggtcctgg aaattctggg ctatggtgcg atccgggttc gggaaggtgc cattgatatt 840 gggaccttat tttcatttct gatgtacctg gttcaaatga ttagtccatt tgcggttctc 900 ggccaattca tgtctgatgt tgccaaggca agtggctcaa ccactcgaat ccaggcatta 960 ttgcaaactc atgaagaaga tcgtctgact ggaacggatt tggatattgg cgatcaaaca 1020 cttcagatga accacgtcag tttttcttat gatcagcatc accccatttt atccggcgtg 1080 tcgtttacgg cagaacccaa ttcggtcatt gcctttgccg gaccatccgg cggtggcaaa 1140 tcaaccattt tcagcttaat tgaacgtttt tatgaaccta acgagggcag catcacgatt 1200 ggcaatacca atattactga tattcaactt gccgattggc gccagcaaat cggcctggtc 1260 ggccaagacg ctgcgatcat gtctggaacg attcgttaca atttaaccta tggtttgccg 1320 gggcattttt ccgatgaaca gctttggcat gtcttggaaa tggctttacg caacgaattt 1380 gtccagaaga tgcctcgggg cttggacacg gaagtcggtg agcgtggagt caaggtatcg 1440 gggggccaac gccaacgatt ggcgattgcc cgggccttcc tgcgtaatcc aaaaatatta 1500 atgttggatg aagcaacggc gagcctggat tccgagtccg aaatgatggt ccaaaaagcg 1560 ctggaccagt tgatggccaa tcgaacaaca ttggtgatcg cccacaggct aagcacaatt 1620 accaacgccg acgaaattta tttcatagaa aacggcaggg taacgggcca gggaacccac 1680 caacagttag tgaaaacgac tcctttgtat agggagtatg tgaaaaatca gagcgcgacg 1740 agcaacggg 1749[Sequence list] SEQUENCE LISTING <110> Asahi Breweries, Ltd. <120> Plasmid having a novel nucleic acid sequence <130> 96T3-22BU <160> 1 <210> 1 <211> 1749 <212> DNA <213> Lactobacillus brevis <400> 1 atgcaagctc agtccaagaa caataccaag tttaacttta aaacatttat gggcctaatc 60 aaccgaattc acccccgtta ctggcaactg ctgcttggct tttttcctagg agttgtcgca 120 acggcgatgc aattgatggt tcccggcatc gccaagggga tcatcaactc aatcggtcat 180 tcaatggatg tcggcctaat cgttgccgtc attttactat tcgttttcag taccattatt 240 ggagcctttt ccggcagtat tttaggcttc ttcggtgaag acgtcgtcta taagctgcga 300 acaacacttt gggataaaat cttaaccctg ccggtgggtt attttgacca aaccaaatct 360 ggcgaaataa cgtccaggtt ggtcaatgat tccacacagg tcaaggaact gttggccaat 420 tcggttccca aaaccgcaac ttcgattctg caactggttg gcgcattggt cttaatgctc 480 atcatggact ggcggatgac tatcattatg tttatcgccg ttccgctcgt cttgatctgc 540 ctgctgccaa ttgtccgcca atcccacaaa gttgccagag cgagacagga cgcactggca 600 gatctcaatg gtaaagccgg tgaaatgctg ggcgaagtcc gtctagtcaa atcgtctacc 660 gcagaaaact tagaacgaac agccggcgat aaacggatgt atcgccttta tcgcatcggg 720 ttaaaagaag cgatctatga ttcaattgcc ggacctgtaa tgggcatggt catgatggcc 780 atggtcctgg aaattctggg ctatggtgcg atccgggttc gggaaggtgc cattgatatt 840 gggaccttat tttcatttct gatgtacctg gttcaaatga ttagtccatt tgcggttctc 900 ggccaattca tgtctgatgt tgccaaggca agtggctcaa ccactcgaat ccaggcatta 960 ttgcaaactc atgaagaaga tcgtctgact ggaacggatt tggatattgg cgatcaaaca 1020 cttcagatga accacgtcag tttttcttat gatcagcatc accccatttt atccggcgtg 1080 tcgtttacgg cagaacccaa ttcggtcatt gcctttgccg gaccatccgg cggtggcaaa 1140 tcaaccattt tcagcttaat tgaacgtttt tatgaaccta acgagggcag catcacgatt 1200 ggcaatacca atattactga tattcaactt gccgattggc gccagcaaat cggcctggtc 1260 ggccaagacg ctgcgatcat gtctggaacg attcgttaca atttaaccta tggtttgccg 1320 gggcattttt ccgatgaaca gctttggcat gtcttggaaa tggctttacg caacgaattt 1380 gtccagaaga tgcctcgggg cttggacacg gaagtcggtg agcgtggagt caaggtatcg 1440 gggggccaac gccaacgatt ggcgattgcc cgggccttcc tgcgtaatcc aaaaatatta 1500 atgttggatg aagcaacggc gagcctggat tccgagtccg aaatgatggt ccaaaaagcg 1560 ctggaccagt tgatggccaa tcgaacaaca ttggtgatcg cccacaggct aagcacaatt 1620 accaacgccg acgaaattta tttcatagaa aacggcaggg taacgggcca gggaacccac 1680 caacagttag tgaaaacgac tcctttgtat agggagtatg tgaaaaatca gagcgcgacg 1740 agcaacggg 1749
【図1】本発明の実施例において得られたプラスミドの
制限酵素地図を示す図である。FIG. 1 is a diagram showing a restriction enzyme map of plasmids obtained in Examples of the present invention.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 北本 勝ひこ 東京都文京区弥生1−1−1 東京大学 大学院農学生命科学研究科応用生命工学 専攻内 (72)発明者 佐見 学 東京都大田区大森北2−13−1 アサヒ ビール株式会社酒類開発研究所内 (56)参考文献 特許3057552(JP,B2) 国際公開95/7289(WO,A1) (58)調査した分野(Int.Cl.7,DB名) C12N 15/09 ZNA C12Q 1/68 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Katsuhiko Kitamoto 1-1-1 Yayoi, Bunkyo-ku, Tokyo Within the Department of Applied Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo (72) Inami Sami Ota-ku, Tokyo 2-13-1, Omorikita Asahi Breweries, Ltd. Liquor Development Laboratory (56) References Patent 3055552 (JP, B2) International Publication 95/7289 (WO, A1) (58) Fields investigated (Int.Cl. 7 , DB name) C12N 15/09 ZNA C12Q 1/68
Claims (2)
るDNA。1. A sequence listing SEQ ID NO: 1 nucleic acid sequence Tona
DNA .
中のホップ耐性乳酸菌の検出方法。2. A method for detecting hop-resistant lactic acid bacteria in beer using the DNA according to claim 1.
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