JP3398897B2 - Hop-resistant DNA of lactic acid bacteria and method for detecting hop-resistant lactic acid bacteria - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to D having a specific sequence.
It is about NA .

[0002]

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.

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] 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]

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] 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.

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.

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.

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.

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.

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.

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.

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]

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.

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.

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.

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]

[Table 1]

(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]

[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]

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]

[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

[Brief description of drawings]

FIG. 1 is a diagram showing a restriction enzyme map of plasmids obtained in Examples of the present invention.

─────────────────────────────────────────────────── ─── 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)

(57) [Claims] 1. A sequence listing SEQ ID NO: 1 nucleic acid sequence Tona
DNA . 2. A method for detecting hop-resistant lactic acid bacteria in beer using the DNA according to claim 1.