Abstract:
The present invention provides oligonucleotides and a method for using same to confirm the presence or absence of a gene resistant to hop, which gene is an important factor for determining the beer-spoilage ability of lactic acid bacteria, and for rapidly assessing the likelihood for beer-spoilage by the presence or absence of lactic acid bacteria having a hop resistance gene, hopA.

Description:
FIELD OF THE INVENTION 
     The present invention relates to oligonucleotides for judging beer-spoilage ability of lactic acid bacteria, which influences beer quality, and a method for judging the beer-spoilage ability with the oligonucleotides. 
     DESCRIPTION OF THE PRIOR ART 
     Beer is effective for controlling bacteria growth, because it contains alcohol, exhausts a carbon source, and has low pH and anaerobic conditions, and further it contains a hop having antibiotic activity. However, since a part of bacteria such as Lactobacillus genera is resistant to iso-α-acid, which is an ingredient in the hop, the bacteria mix in beer products and give the products turbidity. 
     Hitherto, the detection and judgement of bacteria, which influence the quality of beer, have been studied, for example, there are a method for detecting noxious bacteria in beer brewing by using antigen-antibody reaction (Japanese Patent Kokai Publication No. 57-59166), a method for detecting with monoclonal antibody for beer lactic acid bacteria (Japanese Patent Kokai Publication No. 06-105698), and a method comprising extracting DNA from lactic acid bacteria, conducting PCR with primers designated based on a particular sequence, amplifying the sequence and judging the presence or absence of lactic acid bacteria (Japanese Patent Kokai Publication No. 06-141899). 
     However, these methods are substantially methods for judging particular bacteria such as Lactobacillus brevis. For this reason, when Lactobacillus brevis not having beer-spoilage ability or the other bacteria having beer-spoilage ability are detected from beer or half-finished goods during beer production, there are problems that the beer-spoilage ability is judged in error, so that the judgement whether detected bacteria have beer-spoilage ability or not is difficult. 
     SUMMARY OF THE INVENTION 
     The present invention provides a method for judging rapidly whether detected lactic acid bacteria have beer-spoilage ability, by limiting a part of sequence of hop-resistance gene on a plasmid contained in Lactobacillus brevis of beer-spoilage lactic acid bacteria (Japanese Patent Kokai Publication No. 8-186621) and the nucleotide in the sequence, and conducting Polymerase Chain Reaction (PCR) with the nucleotide as the primer. 
     The present invention is an oligonucleotide for judging beer-spoilage ability of lactic acid bacteria, wherein it has a part of the nucleotide sequence or the whole nucleotide sequence of SEQ ID NO:1 or has the corresponding complementary sequence. Further, the present invention is an oligonucleotide for judging beer-spoilage ability of lactic acid bacteria, characterized in that a part of the nucleotide sequence is 
     5&#39;-ATCCGGCGGTGGCAAATCA-3&#39;(SEQ ID NO:2) or 
     5&#39;-AATCGCCAATCGTTGGCG-3&#39;(SEQ ID NO:3). 
     Preferably, the oligonucleotide of the present invention contains at least 10 continuous bases in above-mentioned nucleotide sequence. 
     The present invention is further a method for amplifying a target nucleotide, the above nucleotide sequence functionate as a primer of a polymerase chain reaction. 
     Moreover, the present invention is a method for judging beer-spoilage ability of lactic acid bacteria, said method comprising separating the nucleotide amplified by the above method by using electrophoresis, and judging the presence or absence of the nucleotide having a sequence to be recognized in the resulting nucleotide. 
     In the present invention, noticing the point that the most important factor for determining the beer-spoilage ability of lactic acid bacteria is hop resistance, since it is able to judge whether detected lactic acid bacteria have a hop-resistance gene or not by using gene sequences relating to the hop resistance, it is possible to foreknow rapidly the beer-spoilage ability of bacteria without confirmation by the bacteria growth in beer and the like. 
     DETAILED DESCRIPTION OF THE INVENTION 
     In the present invention, when lactic acid bacteria is detected from beer or the half-finished goods produced during beer production, the lactic acid bacteria are cultured. When lactic acid bacteria can grow, any culture mediums may be used, for example, the bacteria can increase in a MRS liquid medium. The culture conditions are anaerobic conditions at normal temperature to grow the lactic acid bacteria. The culture times are 1-2 days. 
     Cultured and increased bacteria are collected, and DNA is extracted by a well-known DNA extract method, for example a method of D. G. Anderson &amp; L. L. Mackay et al. (App. Env. Microbiol. 46, p549, 1983) and is utilized in the judgement of the present invention. 
     To obtain two oligonucleotides for use as primers, a part of the sequence is determined from the hop-resistance gene described in Japanese Patent Application No. 8-186621, and it is synthesized chemically to obtain two primers opposite to each other. Especially, when the nucleotide sequences of SEQ ID NO:2 and 3 are used as primers for a polymerase chain reaction, if the lactic acid bacteria have the hop-resistance gene, DNA fragments of 342 bp are amplified by the judge method of the present invention. 
     Moreover, thermotolerance at 90° C. or more is enough for using the DNA polymerase. The temperature conditions of a PCR reaction are 90-98° C. in a thermal denaturation reaction for changing a double strand DNA into a one strand DNA, 37-68° C. in annealing of primers to a template DNA, and 50-75° C. in a chain elongation reaction to act polymerase. Using one cycle of these reactions, several tens cycle are conducted to amplify fragments of the hop-resistance gene. 
     After the PCR reaction, the reactant are separated by electrophoresis, nucleic acid stain with ethidium bromide or the like is conducted and the presence or absence of the amplification of DNA fragments is confirmed. When DNA fragments having a chain length specific for the primer designed, it is judged that the bacteria have the hop-resistance gene and beer-spoilage ability. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention is described by working examples in the following. After Lactobacillus strains bacteria 95 strains described in Table 1, to be concrete, Lactobacillus brevis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus lindneri, Lactobacillus collinoides and the like were cultured in MRS liquid medium for 24 hours, DNA was extracted by a method of Anderson &amp; MacKay. 
     Using the DNA as a template, and using two primers, 
     5&#39;-ATCCGGCGGTGGCAAATCA-3&#39;(SEQ ID NO:2) or 5&#39;-AATCGCCAATCGTTGGCG-3&#39;(SEQ ID NO:3), which are based on SEQ ID NO:1 
     PCR was conducted with TAKARA Ex Taq and Program Temp Control System PC-800 (ASTEC). 
     As to the PCR method, well-known technique may be used (Science 230, p 13, 1995). 
     Five μl of the PCR product was separated by electrophoresis with agarose gel (NuSieve GTG, TAKARA) in which TAE buffer (0.04M, Tris-acetate, 0.01M EDTA, pH8.0) is dissolved to be 3%, and a set of MUPID minigel electrophoresis (ADVANCE CO.), and the PCR product was stained with ethidium bromide. As a molecular weight marker, φX174 DNA (TAKARA) cut with HincII was used. 
     The temperature conditions of the judge method in the present invention are 94° C. for 3 minutes as one cycle, 98° C. for 30 seconds and 55° C. for one minute and 72° C. for 0.5 minutes as 25 cycles. 
     Moreover, as to 95 strains described in Table 1, beer-spoilage ability was tested by the following steps. 
     Firstly, after culturing in a MRS liquid medium, each bacterium was inoculated to be 10 5  cells/ml to degassed beer of bitterness value 20, which is adjusted to pH4.6, and cultured at 25° C. for two months under anaerobic conditions. After culturing, the strain, which was recognized the presence of beer-spoilage ability by the eye, was judged that it has beer-spoilage ability. 
     As shown in the results of Table 1, by the judge method (horA-PCR) of the present invention, the strains that the specific amplification of 342 bp DNA fragments was observed were 61 strains in 95 strains, and the strains that the specific amplification was not observed were 34 strains. In fact, compared with the results that turbidity was recognized after inoculating the bacteria to beer of pH4.6, two strains not having beer-spoilage ability were judged as positive by a horA-PCR method, while one strain having beer-spoilage ability was judged as negative by the horA-PCR method. Accordingly, it was recognized that the method was very accurate. 
     
                       TABLE 1______________________________________Bacterium    Test Bacterium               Judgement by the  Species No. Present Invention Beer-spoilage ability______________________________________L.brevis 1          +           +  L.brevis 2 + +  L.brevis 3 + +  L.brevis 4 + +  L.brevis 5 + +  L.brevis 6 + +  L.brevis 7 + +  L.brevis 8 + +  L.brevis 9 + +  L.brevis 10 + +  L.brevis 11 + +  L.brevis 12 + +  L.brevis 13 + +  L.brevis 14 + +  L.brevis 15 + +  L.brevis 16 + +  L.brevis 17 + +  L.brevis 18 + +  L.brevis 19 + +  L.brevis 20 + +  L.brevis 21 + +  L.brevis 22 + +  L.brevis 23 + +  L.brevis 24 + +  L.brevis 25 - -  L.brevis 26 - -  L.brevis 27 - -  L.brevis 28 - -  L.brevis 29 + +  L.brevis 30 + +  L.brevis 31 + +  L.brevis 32 + +  L.brevis 33 + +  L.brevis 34 + +  L.brevis 35 + +  L.brevis 36 + +  L.brevis 37 + +  L.brevis 38 + +  L.brevis 39 + +  L.brevis 40 + +  L.brevis 41 + +  L.brevis 42 + +  L.brevis 43 + +  L.brevis 44 - -  L.brevis 45 + +  L.brevis 46 + +  L.brevis 47 + +  L.casei 48 - -  L.casei 49 - -  L.casei 50 - -  L.casei 51 + +  L.casei 52 - -  L.casei 53 - -  L.casei 54 - -  L.casei 55 - -  L.casei 56 - -  L.casei 57 - -  L.casei 58 - -  L.plantarum 59 - -  L.plantarum 60 - -  L.plantarum 61 - -  L.plantarum 62 - -  L.lindneri 63 + +  L.lindneri 64 + +  L.lindneri 65 + +  L.collinoides 66 - -  L.collinoides 67 - -  L.collinoides 68 - -  L.delbrueckii 69 - -  L.fermentum 70 - -  L.rhamnosus 71 - -  L.sp. 72 - -  L.sp. 73 - -  L.sp. 74 - -  L.sp. 75 - -  L.sp. 76 + +  L.sp. 77 + +  L.sp. 78 + +  L.sp. 79 + +  L.sp. 80 + +  L.sp. 81 + +  L.sp. 82 + +  L.sp. 83 + +  L.sp. 84 + +  L.sp. 85 - -  L.sp. 86 - -  L.sp. 87 - -  L.sp. 88 - -  L.sp. 89 + +  L.sp. 90 + +  L.sp. 91 + +  L.sp. 92 + +______________________________________ 
    
     Merits of the invention are as follows. When bacteria having a high possibility of beer-spoilage ability, such as Lactobacillus brevis and Lactobacillus lindneri, are judged as beer-spoilage bacteria by many conventional identifiable methods, there are many error judges. When beer-spoilage ability is further actually tested by inoculation of bacteria, it takes several weeks to several months. 
     However, by using the oligonucleotide of the present invention and the method for judging beer-spoilage ability with the oligonucleotide, it is possible to accurately judge the beer-spoilage ability in a few hours. 
     
         __________________________________________________________________________#             SEQUENCE LISTING   - -  - - &lt;160&gt; NUMBER OF SEQ ID NOS: 3   - - &lt;210&gt; SEQ ID NO 1  &lt;211&gt; LENGTH: 396  &lt;212&gt; TYPE: DNA  &lt;213&gt; ORGANISM: Lactobacillus brevis   - - &lt;400&gt; SEQUENCE: 1   - - ggaccatccg gcggtggcaa atcaaccatt tctagcttaa ttgaacgttt tt -#atgaacct     60   - - aacgagggca gcatcacgat tggcaatacc aatattactg atattcaact tg -#ccgattgg    120   - - cgccagcaaa tcggcctggt cggccaagac gctgcgatca tgtctggaac ga -#ttcgttac    180   - - aatttaacct atggtttgcc ggggcatttt tccgatgaac agctttggca tg -#tcttggaa    240   - - atggcttacg caacgcaatt tgtccagaag atgcctcggg gcttggacac gg -#aagtcggt    300   - - gagcgtggag tcaaggtatc ggggggccaa cgccaacgat tggcgattgc cc -#gggccttc    360   - - ctgcgtaatc caaaaatatt aatgttggat gaagca      - #- #      396   - -  - - &lt;210&gt; SEQ ID NO 2  &lt;211&gt; LENGTH: 19  &lt;212&gt; TYPE: DNA  &lt;213&gt; ORGANISM: Lactobacillus brevis   - - &lt;400&gt; SEQUENCE: 2   - - atccggcggt ggcaaatca             - #                  - #   - # 19  - -  - - &lt;210&gt; SEQ ID NO 3 &lt;211&gt; LENGTH: 18 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Lactobacillus brevis  - - &lt;400&gt; SEQUENCE: 3  - - aatcgccaat cgttggcg             - #                  - # - #  18__________________________________________________________________________