JP4084949B2 - Method for determining beer turbidity of Lactobacillus brevis using gyrase gene - Google Patents

Description

【００２２】
上記の代表株は、いずれも菌株保存機関から容易に入手可能であり、それらの入手先は、菌株番号に示されている。
【００２３】
次いで、各グループの菌株について、イソα酸に対する耐性およびビール中での増殖能力を調べたところ、グループＩＩbに属するもののみに、ビール混濁性を認めた。従って、上記の制限酵素の組み合わせを使用すると、被検菌の制限酵素切断パターンがこの特定された分類（すなわち、グループＩＩb）に当てはまるかどうかを確認し、当てはまるとき、該被検菌をビール混濁性株であると判定することができる。
【００２４】
ビール工場等現場で、ビール混濁性を有するラクトバチルス・ブレビス菌株を検出・同定するには、対象となる製品ビールをメンブレンフィルターでろ過して、フィルター上に存在する菌類を乳酸菌選択増殖培地（例えば、ｍ−ＮＢＢ培地）で培養した後、集菌して本発明の判定方法に供する。
【００２５】
【実施例】
以下、実施例を挙げて本発明をさらに具体的に説明するが、本発明はこれらの実施例に限定されるものではない。
【００２６】
（実施例１） 供試菌のgyrB遺伝子の増幅
プライマーセット［GYPF（配列番号１）、GYPR（配列番号２）］は、gyrB遺伝子の一部約540bp［Staphylococcus aureus ATCC12600のgyrB遺伝子（GenBank登録No.D10489）の346番目の核酸から907番目の核酸に相当する］を増幅する。このプライマーセットによるラクトバチルス・ブレビスのgyrB遺伝子の増幅、およびその他の各種乳酸菌、さらに乳酸菌以外の供試菌のgyrB遺伝子の増幅について検討した。
【００２７】
（ゲノムＤＮＡ調製）
MRS寒天培地（Difco社）に供試菌を植菌し、30℃、嫌気ボックス（タバイエスペック社、N₂：CO₂：H₂＝90：5：5）中で、2〜5日間培養を行った菌を試験に供した。ＤＮＡ抽出液PrepMan(商標)Ultra（アプライド・バイオシステム社）を用いて菌体からゲノムＤＮＡを抽出した。
【００２８】
（ＰＣＲ増幅）
ＰＣＲアッセイはGeneAmp(商標)PCR System 9700（アプライド・バイオシステム社）を用いて行った。反応液TaKaRa Ex Taq(商標)（寶酒造社）に上記ＤＮＡ溶液、およびプライマーセットを加え、ＰＣＲ反応を行った。この液はゲノムＤＮＡ100ng以上を含み、ＤＮＡ変性は95℃、30秒、アニーリングは55℃、30秒、ＤＮＡ伸長反応は72℃、45秒で35サイクル繰り返した。
【００２９】
（ＰＣＲ産物の検出）
ＰＣＲ増幅に続いて、ＰＣＲ産物の検出はゲル電気泳動によって行った。ＰＣＲサンプル５μｌをポリアクリルアミドゲルに供した。ＤＮＡのバンドは、エチジウムブロマイド溶液で10分間染色後、紫外線照射して観察し、確認した。ＰＣＲアッセイで試験した菌株とそれらの増幅結果を表２に示す。
【００３０】
（ＰＣＲ産物の核酸配列）
増幅したgyrB遺伝子の核酸配列の決定は、増幅断片の５’、３’部分をGYPFとGYPRを用いて、ABI PRISM(商標)dRhodamine Terminator Cycle Sequencing Ready Reaction KitとジェネティックアナライザABI PRISM(商標)310（アプライド・バイオシステム社）で行った。
【００３１】
表２に示すように、GYPFとGYPRとのプライマーセットを用いたＰＣＲ法による約540bpの増幅断片は、ラクトバチルス・ブレビスの全ての株に確認できた。しかし、ラクトバチルス・ブレビス以外の幾種かの乳酸菌にも、約540bpの増幅断片が観察された。
【００３２】
さらに、ラクトバチルス・ブレビスJCM1061株、VTT-E-64028株、SBC8026株およびJCM1170株の540bp断片の核酸配列を決定し、５’側から452bpの核酸配列を比較したところ、菌株間で数核酸〜数十核酸が異なっていることが判明した。それぞれの核酸配列を配列表の配列番号３、配列番号４、配列番号５、および配列番号６に示す。
【００３３】
【表２】

【００３４】
（実施例２） 供試菌株のグループ分け
ラクトバチルス・ブレビス種の４菌株の核酸配列に相違がみられたことより、それぞれを識別できる制限酵素サイトを検索し、制限酵素Hin1Ｉ、ApaLＩ、HaeＩＩＩを選び出した。
【００３５】
（約540bp増幅断片の制限酵素による切断）
実施例１で示したＰＣＲ増幅条件で、各種菌株の約540bp断片を増幅した。この断片を含む反応液8.5μｌに、10倍濃度の緩衝液１μｌおよび酵素液0.5μｌを加え、37℃、4時間〜１晩の間、反応を行った。Hin1ＩおよびHaeＩＩＩには緩衝液M、ApaLＩには緩衝液Lを使用した（寶酒造社）。反応終了後、反応液５μｌをポリアクリルアミドゲルに供し、ＤＮＡのバンドはエチジウムブロマイド溶液で10分間染色後、紫外線照射して観察した。各制限酵素で切断したパターンを図１に示す。図１および表１に従い、菌株の制限酵素切断パターンをグループ分けすると表３のようになる。
【００３６】
【表３】

【００３７】
JCM1559株を除く、１５株のラクトバチルス・ブレビス菌はグループＩ、ＩＩa、ＩＩb、ＩＩＩの何れかのグループに分類された。また、ラクトバチルス・ブレビス以外の菌株で、約540bpの断片が増幅された株については、増幅断片がHin1ＩおよびApaLＩで切断されないか、されても切断長がラクトバチルス・ブレビスのものと明らかに異なっていたため、上記Ｉ〜ＩＩＩの何れのグループにも属さないことが確認された。
【００３８】
（実施例３） ビール混濁性株の識別
（イソα酸に対する耐性試験）
ラクトバチルス・ブレビス種に属する計１６株について、イソα酸に対する耐性試験を以下の手順で行った。
【００３９】
MRS液体培地（Difco社）で培養した培養液を滅菌生理食塩水にて、濁度OD660が約0.1になるように調製した。この菌液0.1mlを、試験管に分注した、40ppmのイソα酸（Versuchsstation Schweiz Breuereien社）を含むMRS液体培地（ｐH5.2）10mlに添加し、嫌気ボックス中、30℃、3〜10日間培養を行い、その間に増殖が目視で確認されたものを、イソα酸耐性を有する株と判定した。この耐性試験結果を各菌株について表４に示す。
【００４０】
（ビール混濁試験）
ラクトバチルス・ブレビス種に属する前記と同一の計１６株について、ビール混濁試験を以下の手順で行った。
【００４１】
MRS液体培地（Difco社）で培養した培養液を滅菌生理食塩水にて、濁度OD660が約0.1になるように調製した。この菌液0.1mlを、試験管に分注した市販のビール10mlに添加し、嫌気ボックス中、30℃、1〜4週間培養を行い、その間にビールの混濁等を目視で確認した。培養期間中に、ビールの混濁が確認されたものを、ビール混濁性を有する株と判定した。このビール混濁試験結果を各菌株について表４に示す。
【００４２】
【表４】

【００４３】
表４の結果から明らかなように、ビール混濁試験において、ビール混濁性を有する株と判定された株はすべてグループＩＩbに属している。なお、グループＩＩbに属するVTT-E-64029株は、ビール混濁試験において非混濁性株と判定された。しかし、本菌株は、乳酸菌のビール混濁性を判定する指標の1つとして考えられているイソα酸に耐性を示すことから、混濁性を示す生理状態に変化する可能性があると考えられる。したがって、グループＩＩbに属する菌株は、ビール混濁を起こす能力を有する可能性が非常に高い株である、と考えるのが妥当である。このように、実施例２において得られた制限酵素切断パターンに基づく分類とビール混濁性判定試験として認められているビール混濁試験やイソα酸耐性試験の結果に基づく分類が良好な一致を示すことから、本発明の判定方法は後者に劣らない信頼性のあるビール混濁性判定方法となる。しかも、本発明の判定方法は、菌株のＤＮＡさえ得られれば、約4〜10時間程度で完了し、数日以上の培養試験を必要とする従来技術の判定方法に比較して、判定時間の大幅な短縮をもたらす。
【００４４】
【発明の効果】
本発明によれば、ラクトバチルス・ブレビス種と同定された菌株がビール中で増殖する能力（ビール混濁性）を有するか否かの判定を、従来技術の方法（例えば、ビール混濁試験やイソα酸耐性試験）よりも迅速、かつ正確に行うことができる。したがって、本発明の判定方法をビール工場における工程管理の指標として導入することにより、ビール製造工程における微生物管理に寄与することができる。
【００４５】
【配列表】

【図面の簡単な説明】
【図１】実施例２において得られた、各菌株からの約540bp増幅断片の制限酵素切断パターンを示す電気泳動模式図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for determining beer turbidity of a subject Lactobacillus brevis strain. More specifically, a part of the DNA gyrase subunit B gene ( gyrB ) of the test Lactobacillus brevis strain is amplified by PCR, and using the restriction enzyme cleavage pattern of the amplified fragment as an index, the test Lactobacillus brevis is used. The present invention relates to a method for determining beer turbidity of a strain.
[0002]
[Prior art]
It is known that certain lactic acid bacteria may be mixed during the beer production process, causing turbidity, acidity, and the like to impair the quality of the produced beer. In particular, Lactobacillus brevis is a typical beer-turbid bacterium. However, not all strains belonging to these species have the property of causing turbidity in beer (hereinafter referred to as “beer turbidity”). Therefore, not only whether or not the test bacterium is Lactobacillus brevis species, but also whether or not the test strain is a beer turbidity strain is identified, and a countermeasure according to the strain is planned Need to be implemented. As one of the methods, a method of detecting Lactobacillus brevis having beer turbidity using an antiserum has been proposed (Japanese Patent Laid-Open No. 10-104238). However, in this method, there is a problem that it may cause an erroneous determination because it is considered that the expression of the surface antigen of Lactobacillus brevis is influenced by the influence of the grown medium components. Further, a method for determining the turbidity of beer such as lactic acid bacteria by examining the resistance of hops and hops to isoalpha acid (Japanese Patent Laid-Open No. 9-260, Fernandez, JLet al .; Letters in Applied Microbiol., 1992, 14: 13-16) has also been proposed, but in this method, the test conditions may affect the determination results, and it is difficult to accurately determine the growth of the test bacteria in beer. Furthermore, a method for determining whether or not the microorganism is a turbid group from the mobility of polyacrylamide electrophoresis of D-lactate dehydrogenase of Lactobacillus brevis is also proposed (Japanese Patent Laid-Open No. 9-900894). However, there is a problem that it takes time to make a decision. In addition, the 16S rRNA used for identification and detection of various strains at the gene level has not been able to perform an accurate molecular phylogenetic analysis of bacteria between Lactobacillus brevis species. That is, it was difficult to distinguish between a strain having beer turbidity and a strain having no beer turbidity.
[0003]
[Problems to be solved by the invention]
In view of the above problems, there is a development of a method for determining whether or not a strain capable of growing in beer for Lactobacillus brevis species, which is the most threat in beer production, from a viewpoint different from the conventional technique. It is desired. Therefore, the object of the present invention is to solve the above-mentioned problems, and for a test strain identified as Lactobacillus brevis, whether or not it has the ability to grow in beer, that is, whether or not its beer turbidity exists quickly and accurately. It is to provide a method of determination.
[0004]
[Means for Solving the Problems]
The present inventors have repeatedly studied to solve such problems, and focused on the DNA gyrase subunit B gene ( gyrB ) gene (hereinafter also referred to as gyrB gene) in the process. Furthermore, when a part of gyrB gene of a strain identified as Lactobacillus brevis was identified by PCR technique from physiological, biochemical and genetic techniques, the restriction fragment cleavage pattern of the amplified fragment was largely classified into four groups. In addition, the present inventors have found that strains having beer turbidity can be concentrated in one group, and have completed the present invention based on this finding.
[0005]
That is, the present invention is a method for determining the beer turbidity of a test Lactobacillus brevis strain, which comprises using the DNA gyrase subunit B gene ( gyrB ) specific primer, A part of the B gene region of Bacillus brevis strain is amplified, the obtained amplified DNA fragment is cleaved with a restriction enzyme, and beer turbidity is determined based on the obtained restriction enzyme cleavage pattern. The method is provided.
[0006]
In the above method, the DNA gyrase subunit B gene ( gyrB ) -specific primer is preferably composed of an oligonucleotide consisting of the nucleic acid sequence shown in SEQ ID NO: 1 of the sequence listing and a nucleic acid sequence shown in SEQ ID NO: 2 of the sequence listing. A set with oligonucleotides.
[0007]
In the above method, the restriction enzyme is preferably a combination of Hin 1 I, Apa LI, and Hae III. In this case, restriction enzyme cleavage patterns are classified into four.
[0008]
In the above method, when the restriction enzyme cleavage pattern of the test Lactobacillus brevis strain is applied to the classification to which the Lactobacillus brevis standard strain VTT-E64029 belongs, the test Lactobacillus brevis strain is a beer turbid strain. judge.
[0009]
In the above method, the present invention obtains a restriction enzyme cleavage pattern of a standard strain of Lactobacillus brevis containing at least one beer turbid strain in advance, and compares it with the restriction enzyme cleavage pattern of the Lactobacillus brevis strain to be tested. The beer turbidity is determined.
[0010]
Preferably, the method further comprises the step of classifying the restriction enzyme cleavage pattern of the obtained Lactobacillus brevis standard strain group and identifying the classification to which the beer turbid strain belongs. In this case, when the restriction enzyme cleavage pattern of the test Lactobacillus brevis strain is applicable to the specified classification, the test Lactobacillus brevis strain is determined to be a beer turbid strain.
[0011]
Furthermore, the present invention provides an oligonucleotide having a nucleic acid sequence selected from the group consisting of the nucleic acid sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing. These oligonucleotides are characterized by the nucleic acid sequence characteristic of each Lactobacillus brevis strain from which it is derived, ie, the nucleic acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
[0012]
In addition, the present invention provides a primer set for amplifying DNA gyrase subunit B gene ( gyrB ), which can be used in any of the above methods, which is an oligonucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. And an oligonucleotide consisting of the nucleic acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
As used herein, “beer turbidity” refers to the property that a test Lactobacillus brevis strain can grow in beer, and the strain causes turbidity of beer by growth.
[0014]
In order to determine the beer turbidity of Lactobacillus brevis strains to be tested, the present invention is a gene amplification method by PCR using oligonucleotide primers, and pattern analysis by acrylamide gel electrophoresis after cutting the amplified fragment with a restriction enzyme Is based on doing.
[0015]
As mentioned above, not all strains identified as Lactobacillus brevis grow in beer, but only some strains can grow in beer. In order to determine such a harmful strain, that is, whether or not the test strain is a harmful strain, the present invention uses a nucleic acid sequence of about 540 bp in the DNA gyrase subunit B gene region of the strain. Is.
[0016]
Gyrase is a kind of bacterial DNA topoisomerase and is said to be involved in the repair of higher-order structure of DNA. DNA gyrase is composed of two proteins, protein A having a molecular weight of 100 kDa and protein B having a molecular weight of 90 kDa or 70 kDa. In recent years, it has been reported that the gene gyrB encoding this protein B is very effective for the classification of various bacteria (for example, Watanabe, K., et al, Appl. Environ. Microbiol. 61, 1104-1109). (See 1995).
[0017]
According to the present invention, using a gyrB specific primer, a part of the gyrB gene region of the test Lactobacillus brevis strain is amplified, the obtained amplified DNA fragment is cleaved with a restriction enzyme, and the obtained restriction enzyme Based on the cutting pattern, beer turbidity can be determined. Here, the determination of beer turbidity is obtained in advance by obtaining a restriction enzyme cleavage pattern of a standard strain of Lactobacillus brevis containing at least one beer turbidity strain and comparing it with a restriction enzyme cleavage pattern of a test bacterium Lactobacillus brevis. By doing. In a preferred embodiment, first, the restriction enzyme cleavage pattern of the obtained Lactobacillus brevis standard strain group is classified, and the classification to which the beer turbid strain belongs is specified. Furthermore, it is confirmed whether or not the restriction enzyme cleavage pattern of the test Lactobacillus brevis strain is applicable to the specified classification, and when it is applied, the test Lactobacillus brevis strain is determined to be a beer turbid strain.
[0018]
The gyrB- specific primer is a primer that specifically anneals to a target nucleic acid sequence in the test bacteria gyrB gene, but one of the primer sets may be non-specifically annealed. For Lactobacillus brevis, preferably a primer comprising a set of an oligonucleotide consisting of the nucleic acid sequence shown in SEQ ID NO: 1 of the sequence listing and an oligonucleotide consisting of the nucleic acid sequence shown in SEQ ID NO: 2 of the sequence listing The set is used as a specific primer.
[0019]
A part of the gyrB gene region is PCR amplified with such a primer, but the PCR conditions and the like are not substantially different from the standard one. After amplification, the obtained amplified DNA fragment is cleaved with a restriction enzyme, and subjected to, for example, agarose electrophoresis and staining with ethidium bromide to detect DNA. The restriction enzyme cleavage patterns (DNA electrophoresis patterns) thus obtained are compared between strains.
[0020]
In a preferred embodiment, a restriction enzyme cleavage pattern of a Lactobacillus brevis standard strain group containing at least one beer turbid strain is obtained in advance. Specifically, a series of operations including PCR amplification, restriction enzyme cleavage, and agarose electrophoresis are performed with the primer set using the following standard strain. Further, a combination of Hin 1 I, Apa LI, and Hae III is preferably used as a restriction enzyme, but is not limited to this specific enzyme combination. If the restriction enzyme cleavage pattern of the Lactobacillus brevis standard strain group enables the discrimination and identification of the beer turbid strain contained therein, almost any combination is possible. The obtained restriction enzyme cleavage patterns can be roughly classified into four groups, and it was found that strains having beer turbidity are concentrated in one of them. Table 1 shows representative examples of standard strains, restriction enzymes, and group classifications used here.
[0021]
[Table 1]

[0022]
All of the above representative strains can be easily obtained from a strain preservation institution, and their sources are indicated by strain numbers.
[0023]
Next, when the strains of each group were examined for resistance to isoalpha acid and ability to grow in beer, beer turbidity was recognized only in those belonging to Group IIb. Therefore, using the above combination of restriction enzymes, it is confirmed whether or not the restriction enzyme cleavage pattern of the test bacterium applies to this specified classification (ie, group IIb), and when this is the case, the test bacterium is turbid in beer. It can be determined that it is a sex strain.
[0024]
In order to detect and identify a Lactobacillus brevis strain having beer turbidity at a site such as a beer factory, the target product beer is filtered with a membrane filter, and the fungi present on the filter are selected as a lactic acid bacteria selective growth medium (for example, , M-NBB medium), and then collected and used for the determination method of the present invention.
[0025]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these Examples.
[0026]
(Example 1) amplification primer sets gyrB gene of test bacteria [GYPF (SEQ ID NO: 1), GYPR (SEQ ID NO: 2)] is about a part of the gyrB gene 540 bp [Staphylococcus aureus gyrB gene of ATCC12600 (GenBank Registration No Corresponding to the 907th nucleic acid from the 346th nucleic acid of D10489)]. Amplification of gyrB gene of Lactobacillus brevis of this primer set, and other various lactic acid bacteria were further examined amplification of gyrB gene of test bacteria other than lactic acid bacteria.
[0027]
(Genomic DNA preparation)
Inoculate the test bacteria on MRS agar medium (Difco) and culture in anaerobic box (Tabaie specs, N ₂ : CO ₂ : H ₂ = 90: 5: 5) for 2-5 days at 30 ° C. The performed bacteria were used for the test. Genomic DNA was extracted from the cells using a DNA extract PrepMan ™ Ultra (Applied Biosystems).
[0028]
(PCR amplification)
PCR assays were performed using GeneAmp ™ PCR System 9700 (Applied Biosystems). The above DNA solution and primer set were added to the reaction solution TaKaRa Ex Taq (trademark) (Takara Shuzo Co., Ltd.) to carry out a PCR reaction. This solution contained 100 ng or more of genomic DNA, DNA denaturation was repeated at 35 ° C. for 30 seconds, annealing was performed at 55 ° C. for 30 seconds, and DNA elongation reaction was repeated at 35 ° C. at 72 ° C. for 45 seconds.
[0029]
(PCR product detection)
Following PCR amplification, detection of PCR products was performed by gel electrophoresis. 5 μl of PCR sample was subjected to polyacrylamide gel. The DNA band was confirmed by staining with ethidium bromide solution for 10 minutes and then irradiating with ultraviolet light. The strains tested in the PCR assay and their amplification results are shown in Table 2.
[0030]
(Nucleic acid sequence of PCR product)
The nucleic acid sequence of the amplified gyrB gene was determined using the ABI PRISM (trademark) dRhodamine Terminator Cycle Sequencing Ready Reaction Kit and the Genetic Analyzer ABI PRISM (trademark) 310 (using GYPF and GYPR on the 5 ′ and 3 ′ portions of the amplified fragment. Applied Biosystems).
[0031]
As shown in Table 2, an amplified fragment of about 540 bp by PCR using a primer set of GYPF and GYPR was confirmed in all strains of Lactobacillus brevis. However, an amplified fragment of about 540 bp was also observed in some lactic acid bacteria other than Lactobacillus brevis.
[0032]
Furthermore, when the nucleic acid sequences of 540 bp fragments of Lactobacillus brevis JCM1061 strain, VTT-E-64028 strain, SBC8026 strain and JCM1170 strain were determined and 452 bp nucleic acid sequences were compared from the 5 ′ side, several nucleic acids between the strains Dozens of nucleic acids were found to be different. The respective nucleic acid sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 in the sequence listing.
[0033]
[Table 2]

[0034]
(Example 2) Grouping of test strains Since there were differences in the nucleic acid sequences of 4 strains of Lactobacillus brevis species, restriction enzyme sites that can be distinguished from each other were searched, restriction enzymes Hin1 I, Apa LI, Hae III was selected.
[0035]
(Cleavage of about 540 bp amplified fragment with restriction enzyme)
Under the PCR amplification conditions shown in Example 1, approximately 540 bp fragments of various strains were amplified. To 8.5 μl of the reaction solution containing this fragment, 1 μl of 10-fold buffer solution and 0.5 μl of enzyme solution were added, and the reaction was carried out at 37 ° C. for 4 hours to overnight. Buffer M was used for Hin 1I and Hae III, and Buffer L was used for Apa LI (Takara Shuzo). After completion of the reaction, 5 μl of the reaction solution was applied to a polyacrylamide gel, and the DNA band was observed by irradiating with ultraviolet rays after staining with ethidium bromide solution for 10 minutes. The pattern cut with each restriction enzyme is shown in FIG. When the restriction enzyme cleavage patterns of strains are grouped according to FIG. 1 and Table 1, Table 3 is obtained.
[0036]
[Table 3]

[0037]
15 strains of Lactobacillus brevis, excluding JCM1559 strain, were classified into any of groups I, IIa, IIb and III. In addition, for strains other than Lactobacillus brevis, in which a fragment of about 540 bp was amplified, the amplified fragment was not cleaved with Hin 1I and Apa LI. Therefore, it was confirmed that they did not belong to any of the above groups I to III.
[0038]
(Example 3) Identification of beer turbidity strain (resistance test against iso-alpha acid)
A total of 16 strains belonging to the Lactobacillus brevis species were tested for resistance to iso-α acid by the following procedure.
[0039]
A culture solution cultured in an MRS liquid medium (Difco) was prepared with sterile physiological saline so that the turbidity OD660 was about 0.1. 0.1 ml of this bacterial solution was added to 10 ml of MRS liquid medium (pH 5.2) containing 40 ppm of isoalpha acid (Versuchsstation Schweiz Breuereien) dispensed into a test tube, and placed in an anaerobic box at 30 ° C., 3-10 Culturing was conducted for a day, and during which growth was visually confirmed during that period, it was determined as a strain having resistance to iso-α acid. The resistance test results are shown in Table 4 for each strain.
[0040]
(Beer turbidity test)
A beer turbidity test was conducted by the following procedure for the same 16 strains belonging to the Lactobacillus brevis species as described above.
[0041]
A culture solution cultured in an MRS liquid medium (Difco) was prepared with sterile physiological saline so that the turbidity OD660 was about 0.1. 0.1 ml of this bacterial solution was added to 10 ml of commercially available beer dispensed into a test tube, and cultured in an anaerobic box at 30 ° C. for 1 to 4 weeks, during which the beer turbidity was visually confirmed. Those in which turbidity of beer was confirmed during the culture period were determined to be strains having beer turbidity. The beer turbidity test results are shown in Table 4 for each strain.
[0042]
[Table 4]

[0043]
As is clear from the results in Table 4, all the strains determined to be strains having beer turbidity in the beer turbidity test belong to group IIb. The VTT-E-64029 strain belonging to Group IIb was determined as a non-turbid strain in the beer turbidity test. However, since this strain exhibits resistance to isoalpha acid, which is considered as one of the indicators for determining the turbidity of lactic acid bacteria, it is considered that the strain may change to a physiological state that exhibits turbidity. Therefore, it is reasonable to consider that the strains belonging to Group IIb are very likely to have the ability to cause beer turbidity. Thus, the classification based on the restriction enzyme cleavage pattern obtained in Example 2 and the classification based on the results of the beer turbidity test and the iso-α acid resistance test recognized as the beer turbidity determination test show good agreement. Therefore, the determination method of the present invention is a reliable beer turbidity determination method not inferior to the latter. In addition, the determination method of the present invention can be completed in about 4 to 10 hours as long as the strain DNA is obtained, compared with the conventional determination method that requires a culture test of several days or more. This leads to a significant shortening.
[0044]
【The invention's effect】
According to the present invention, a determination as to whether a strain identified as Lactobacillus brevis species has the ability to grow in beer (beer turbidity) is performed using conventional methods (eg, beer turbidity tests and iso-α It can be performed more quickly and accurately than the acid resistance test. Therefore, by introducing the determination method of the present invention as an index for process management in a beer factory, it can contribute to microorganism management in the beer production process.
[0045]
[Sequence Listing]

[Brief description of the drawings]
1 is a schematic electrophoretic diagram showing restriction enzyme cleavage patterns of about 540 bp amplified fragments from each strain obtained in Example 2. FIG.

Claims (9)

Test Lactobacillus brevis ( Lactobacillus brevis ) a method for determining beer turbidity of a strain, wherein a DNA gyrase subunit B gene ( gyrB ) specific primer is used to amplify a part of the B gene region of the Lactobacillus brevis strain to be tested The amplified DNA fragment is cleaved with a restriction enzyme, and the beer turbidity of the strain is determined based on the obtained restriction enzyme cleavage pattern, and the DNA gyrase subunit B gene (gyrB ) specific primers is a set of an oligonucleotide consisting of a nucleic acid sequence set forth in SEQ ID NO: 2 of the oligonucleotide sequence listing comprising a nucleic acid sequence shown in SEQ ID NO: 1, methods. The method according to claim 1 , wherein the restriction enzyme is a combination of Hin1I, ApaLI, and HaeIII. The method according to claim 2 , wherein the restriction enzyme cleavage patterns are classified into four. When the restriction enzyme cleavage pattern of the test Lactobacillus brevis strain is applicable to the classification to which the Lactobacillus brevis standard strain VTT-E64029 belongs, it is determined that the test Lactobacillus brevis strain is a beer turbid strain. 3. The method according to 3 .   The restriction enzyme cleavage pattern of the Lactobacillus brevis standard strain group containing at least one beer turbidity strain is acquired in advance, and the beer turbidity is determined by comparison with the restriction enzyme cleavage pattern of the Lactobacillus brevis strain to be tested. The method of claim 1. 6. The method according to claim 5 , further comprising the step of classifying a restriction enzyme cleavage pattern of the obtained Lactobacillus brevis standard strain group and identifying a classification to which a beer turbid strain belongs. The method according to claim 6 , wherein when the restriction enzyme cleavage pattern of the test Lactobacillus brevis strain is applied to the specified classification, the test Lactobacillus brevis strain is determined to be a beer turbid strain. SEQ ID NO: 3 in the sequence listing, SEQ ID NO: 4, SEQ ID NO: 5 and the nucleic acid sequences Tona Ru oligonucleotide selected from the group consisting of the nucleic acid sequence set forth in SEQ ID NO: 6.   A primer set for amplifying a DNA gyrase subunit B gene (gyrB) comprising a set of an oligonucleotide consisting of the nucleic acid sequence shown in SEQ ID NO: 1 of the sequence listing and an oligonucleotide consisting of the nucleic acid sequence shown in SEQ ID NO: 2 of the sequence listing .

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