JP3057552B2 - Hop resistance plasmid and hop resistance determination method of lactic acid bacteria - Google Patents
Hop resistance plasmid and hop resistance determination method of lactic acid bacteriaInfo
- Publication number
- JP3057552B2 JP3057552B2 JP17969095A JP17969095A JP3057552B2 JP 3057552 B2 JP3057552 B2 JP 3057552B2 JP 17969095 A JP17969095 A JP 17969095A JP 17969095 A JP17969095 A JP 17969095A JP 3057552 B2 JP3057552 B2 JP 3057552B2
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- Prior art keywords
- lactic acid
- hop
- plasmid
- acid bacteria
- beer
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Description
【０００１】[0001]
【産業上の利用分野】本発明は、ホップ耐性を有する乳
酸菌の判定法に関し、さらに詳しくは、ビールの品質に
影響するラクトバチルス属乳酸菌のホップ耐性判定法に
関するものである。The present invention relates to a method for determining lactic acid bacteria having hop resistance, and more particularly to a method for determining hop resistance of lactic acid bacteria belonging to the genus Lactobacillus, which affects beer quality.
【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。2. Description of the Related Art Beer is a microorganism containing hops having an antibacterial activity in addition to the conditions of alcohol content, depletion of carbon source, low pH and anaerobic condition. It has a growth inhibitory effect. However, certain Lactobacillus spp. Are resistant to iso-α-acid, a component in hops, and may be mixed into beer products and 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。[0003] The detection and determination of Lactobacillus spp. That affect the quality of beer have been studied in the past, for example, a method for detecting harmful bacteria in beer brewing using an antigen-antibody reaction (Japanese Patent Laid-Open No. 57-59166). , A detection method using a monoclonal antibody against lactic acid bacteria of beer
6-105698), a method of extracting DNA from a lactic acid bacterium, performing PCR using a specific oligonucleotide as a primer to amplify the sequence, and then determining the presence or absence of a lactic acid bacterium (JP-A-06-141899).
【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 methods for determining a specific lactic acid bacterium such as Lactobacillus brevis. Therefore, while brevis having no hop resistance is detected, other species having hop resistance are detected. Therefore, there was a problem that the lactic acid bacteria in the beer turbidity could not 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プラスミドを特定
し、そのプラスミド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。また、ビール混
濁乳酸菌を徐々にホップエキス濃度を高めた培地で培養
を繰り返しホップ耐性を強化した該乳酸菌からホップ耐
性プラスミドを取得する方法に関するものである。Therefore, in the present invention, a plasmid relating to hop resistance of beer turbid lactic acid bacteria is specified, and hop-resistant lactic acid bacteria are rapidly detected by using the plasmid as a probe. Also, the present invention relates to a method for obtaining a hop-resistant plasmid from a lactic acid bacterium having enhanced hop resistance by repeatedly culturing beer-turbid lactic acid bacteria in a medium in which the concentration of hop extract is gradually increased.
【０００６】本発明は、次の行程によって実施されるホ
ップ耐性プラスミドの取得方法および得られたホップ耐
性プラスミドである。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。The present invention relates to a method for obtaining a hop-resistant plasmid and a hop-resistant plasmid obtained by the following steps. That is, (1) Beer-turbid lactic acid bacteria are gradually transferred to a medium having a high hop concentration and adapted to the medium, thereby enhancing the hop resistance of the beer-turbid lactic acid bacteria. (2)
By repeating this adaptation operation, a plasmid associated with increased hop resistance in the beer turbid lactic acid bacteria is confirmed. (3) A part of or all of the DNA of the confirmed plasmid is labeled to prepare a determination probe.
(4) A lactic acid bacterium to be determined as a hop-resistant lactic acid bacterium is determined by the probe obtained in (3).
【０００７】また、本発明はこれらの行程で得られたホ
ップ耐性プラスミドも含む。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（Lact
obacillus brevis)、ラクトバチルス プランタラム（L
actobacillus plantarum)、ラクトバチルスカゼイ（Lac
tobacillus casei) 等がある。[0007] The present invention also includes hop-resistant plasmids obtained in these steps. Beer turbid lactic acid bacteria are harmful bacteria having acid resistance and alcohol resistance in beer production, and specifically, Lactobacillus brevis ( Lact.
obacillus brevis ), Lactobacillus plantarum ( L
actobacillus plantarum ), Lactobacillus casei ( Lac
tobacillus casei ).
【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。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。Hereinafter, the present invention will be described in more detail.
The medium for hop acclimation of the beer-turbid lactic acid bacteria may be any medium as long as the lactic acid bacteria can grow.
A liquid medium or the like can be used. As the hop extract to be added to the medium, an isomerized hop extract can be used. The concentration at which the hop extract is added starts from the maximum concentration at which the beer turbid lactic acid bacteria to be tested can grow, and is 50 to
The concentration is increased by 100 μmol (in terms of iso-α acid) to achieve at least 3 times the maximum concentration.
【０００９】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。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。[0009] The culturing conditions may be any anaerobic conditions at a normal suitable temperature at which beer turbid lactic acid bacteria can grow. The cultivation time is up to growth in each hop extract-containing medium.
Within about a day. The method for obtaining a hop-resistant plasmid from beer turbid lactic acid bacteria adapted to hop extract is as follows.
【００１０】培養したビール混濁乳酸菌を公知のアルカ
リＳＤＳ法（文献：D. G. Ardersen& L. L. Mckay, App
l. 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。得られたプラスミドが、本発明のホッ
プ耐性プラスミドである。[0010] The cultured beer turbid lactic acid bacteria are subjected to a known alkaline SDS method (Reference: DG Ardersen & LL Mckay, App.
l. Env. Microbiol., 46 , 549 (1983)) to obtain all plasmids in lactic acid bacteria. Each of the resulting plasmids is subjected to agarose gel electrophoresis, and the band portion of the plasmid whose copy number has been increased after acclimation as compared with before the acclimation is cut out to remove agarose. The resulting plasmid is the hop-resistant plasmid of the present invention.
【００１１】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。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 use the obtained hop-resistant plasmid as a probe, the entire plasmid may be used.
Alternatively, some of them may be used. It is preferable that the probe is usually labeled by a known method that can be detected by using these. For example, biotin-
Labels such as avidin and digoxigenin, or radioactive labels can be used.
【００１２】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等が利用できる。場合に
より、ＰＣＲ法により判定する乳酸菌のプラスミドをあ
らかじめ増幅しておいてもよい。As a method for judging turbid lactic acid bacteria of beer by using the labeled hop-resistant probe, generally used colony hybridization, southern hybridization and the like can be used. In some cases, the lactic acid bacteria plasmid determined by the PCR method may be amplified in advance.
【００１３】[0013]
【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。The present invention will be described below with reference to examples. (1) Lactobacillus brev
is ) Acquisition of Hop-Resistant Plasmid of ABCC45 Strain Lactobacillus brevis strain ABCC45 was added to 5 ml of MRS liquid medium containing 150 μmol of isomerized hop extract at a rate of 10 6 cells / ml, and the mixture was added at 30 ° C. for 3 hours.
Cultured for days.
【００１４】その後、ホップ馴化をすすめるために、５
０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。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。Thereafter, in order to promote hop acclimation, 5
The same operation was performed under the above-mentioned conditions until the final concentration reached 900 micromol, while increasing the isomerized hop extract by 0 micromol. Lactic acid bacteria before and after acclimation were collected, washed, and then all plasmids were obtained by alkaline SDS method. All plasmids obtained from 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 cut out,
Agarose was removed.
【００１５】得られたプラスミド（以下、ｐＲＨ４５と
いう）は１４．５ｋｂｐであり、制限酵素地図は図１の
とおりである。ｐＲＨ４５のBamHI-BamHI ２kb断片を、
大腸菌と乳酸菌のシャトルベクター上にクローニングす
ることにより増幅させ、ベーリンガーマンハイム社製Ｄ
ＩＧＤＮＡ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
ＤＮＡ標識を行った。The resulting plasmid (hereinafter, referred PRH45) is 14.5 kb p, restriction map is shown in Figure 1. The BamHI-BamHI 2 kb fragment of pRH45 was
Amplified by cloning on a shuttle vector of E. coli and lactic acid bacteria, Boehringer Mannheim D
Using an IG DNA labeling and detection kit, DNA labeling was performed by a random prime system using digoxigenin-dUTP.
【００１６】表１に示した乳酸菌から前記の方法で、同
様にして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 in the same manner as described above, subjected to agarose electrophoresis, and transferred to a nylon membrane. Next, the labeled probe was hybridized and detected by ELISA using the kit.
【００１７】[0017]
【表１】 [Table 1]
【００１８】（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 Lactic Acid Bacteria in Beer Using a Labeled Hop-Resistant Probe The lactic acid bacteria shown in Table 2 were suspended at 10 2 cells / ml, and 500 ml of beer was filtered through a membrane and placed on an MRS plate medium. It was anaerobically cultured at 25 ° C. The emerged colonies were transferred to a nylon membrane, lysed, and the DNA was fixed on the membrane. Next, the labeled probe obtained in Example (1) was hybridized and EL using the kit was used.
Detection by ISA was performed.
【００１９】[0019]
【表２】 [Table 2]
【００２０】[0020]
【発明の効果】本発明は、ビール混濁乳酸菌のホップ耐
性に関係するプラスミドを特定し、このプラスミドをプ
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。According to the present invention, a hop-resistant lactic acid bacterium can be rapidly detected by specifying a plasmid relating to hop resistance of beer turbid lactic acid bacteria and using this plasmid as a probe. Thereby, detection and determination of Lactobacillus bacteria affecting beer quality can be performed promptly and reliably.
【図１】本発明の実施例において得られたプラスミド
（ｐＲＨ４５）の制限酵素地図を示す図である。FIG. 1 is a diagram showing a restriction map of a plasmid (pRH45) 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 (58)調査した分野(Int.Cl.7，ＤＢ名) C12N 15/74 C12Q 1/68 ＢＩＯＳＩＳ（ＤＩＡＬＯＧ) ＷＰＩ（ＤＩＡＬＯＧ)──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Katsuhiko Kitamoto 1-1-1 Yayoi Bunyo-ku, Tokyo Graduate School of Agricultural and Life Sciences, Department of Applied Biotechnology, University of Tokyo (72) Inventor Manabu Sami Ota-ku, Tokyo 2-13-1 Omorikita Asahi Breweries, Ltd. Alcoholic Beverage Research Laboratory (58) Field surveyed (Int. Cl. 7 , DB name) C12N 15/74 C12Q 1/68 BIOSIS (DIALOG) WPI (DIALOG)
Claims (11)
ホップ耐性プラスミドであって、 【化１】 で示される制限酵素地図を有し、ラクトバチルス ブレ
ビス由来のプラスミド。1. A beer turbid lactic acid bacterium can be detected.
A hop-resistant plasmid comprising : In shown to have a restriction enzyme map is, Lactobacillus shake
Bis-derived plasmid.
請求項１記載のホップ耐性プラスミド。2. The hop-resistant plasmid according to claim 1, wherein the lactic acid bacterium is a Lactobacillus lactic acid bacterium.
ス濃度を高めた培地で培養を繰り返し、ホップ耐性を強
化した該乳酸菌からホップ耐性プラスミドを取得するこ
とを特徴とするホップ耐性プラスミドの取得方法。3. A method for obtaining a hop-resistant plasmid, comprising repeatedly culturing beer turbid lactic acid bacteria in a medium in which the concentration of hop extract is gradually increased, and obtaining a hop-resistant plasmid from the lactic acid bacteria having enhanced hop resistance.
請求項３記載の取得方法。4. The method according to claim 3, wherein the lactic acid bacterium is a Lactobacillus lactic acid bacterium.
取得した請求項１記載のホップ耐性プラスミド。5. The hop-resistant plasmid according to claim 1, obtained by the method according to claim 3 .
るプラスミドを特定し、そのプラスミドをプローブとし
て利用してホップ耐性乳酸菌を検出することを特徴とす
る乳酸菌のホップ耐性判定法。6. A method for determining hop resistance of lactic acid bacteria, comprising identifying a plasmid associated with hop resistance of beer turbid lactic acid bacteria and detecting the hop resistant lactic acid bacteria using the plasmid as a probe.
請求項６記載の判定法。7. The method according to claim 6, wherein the lactic acid bacterium is a Lactobacillus lactic acid bacterium.
得られた請求項１記載のホップ耐性プラスミド。8. The hop-resistant plasmid according to claim 1 , which is obtained by the method according to claim 6 or 7.
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し、（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し、（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り、（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
定することを特徴とする乳酸菌のホップ耐性判定法。9. (1) Beer-turbid lactic acid bacteria are gradually transferred to a medium having a high hop concentration to acclimate, and the hop resistance of the beer-turbid lactic acid bacteria is strengthened. (2) By repeating this acclimating operation, Confirmation of plasmids related to increased hop resistance in beer turbid lactic acid bacteria, (3)
A part of or all of the DNA of the confirmed plasmid is labeled to prepare a determination probe, and (4) a lactic acid bacterium to be determined to be a hop-resistant lactic acid bacterium is determined using the probe obtained in (3). A method for determining hop resistance of lactic acid bacteria.
る請求項９記載の判定法。10. The method according to claim 9, wherein the lactic acid bacterium is a lactic acid bacterium belonging to the genus Lactobacillus.
いて得られた請求項１記載のホップ耐性プラスミド。11. The hop-resistant plasmid according to claim 1 , which is obtained by the method according to claim 9 or 10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17969095A JP3057552B2 (en) | 1995-06-23 | 1995-06-23 | Hop resistance plasmid and hop resistance determination method of lactic acid bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17969095A JP3057552B2 (en) | 1995-06-23 | 1995-06-23 | Hop resistance plasmid and hop resistance determination method of lactic acid bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09260A JPH09260A (en) | 1997-01-07 |
| JP3057552B2 true JP3057552B2 (en) | 2000-06-26 |
Family
ID=16070175
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17969095A Expired - Lifetime JP3057552B2 (en) | 1995-06-23 | 1995-06-23 | Hop resistance plasmid and hop resistance determination method of lactic acid bacteria |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3057552B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3338924B2 (en) * | 1997-07-07 | 2002-10-28 | アサヒビール株式会社 | Oligonucleotide for detecting lactic acid bacteria and determination method using the same |
| TW483287B (en) | 1999-06-21 | 2002-04-11 | Semiconductor Energy Lab | EL display device, driving method thereof, and electronic equipment provided with the EL display device |
-
1995
- 1995-06-23 JP JP17969095A patent/JP3057552B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH09260A (en) | 1997-01-07 |
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