JP5158669B2 - Method for discriminating yeast for brewing using YHR213W or YAR062W gene - Google Patents

Description

  The present invention relates to yeast discrimination, and more particularly to classification and discrimination of brewing yeast.

  As yeast for brewing, several types of yeast such as wine yeast, sake yeast, shochu yeast, etc. have been used, and among these yeasts, for example, wine yeast is 2236 yeast sold by Lallmand. And EC1118 yeast, etc. are used, and association 7 (K-7), 9 (K-9) yeast, etc. are used as sake yeast, and Kagoshima yeast K2, Miyazaki yeast MK, etc. are used as shochu yeast. ing.

  However, these brewing yeasts are all classified into Saccharomyces, and further, sake yeast and shochu yeast are classified into Saccharomyces cerevisiae. Thus, since brewing yeast belongs to the same genus or the same species, normal bacteriological properties are common, and it is extremely difficult to accurately distinguish these yeasts in a short time. Therefore, at present, the fact is that the yeast must be discriminated by a method of discriminating from the brewing characteristics, for example, by conducting a small preparation test on the yeast. However, these methods require considerable time for discrimination because it is necessary to actually cultivate and prepare yeast to confirm the brewing characteristics of the yeast, and this industry requires improvement. It has been.

  Thus, establishment of an accurate and simple yeast discrimination system in a short period of time has been an important issue in the field of actual liquor production and also in test and research institutions.

Therefore, in view of the present state of the art, the present inventors are able to accurately and easily discriminate between yeasts of the same use, such as association wine yeast Nos. 1, 3, and 4, for a short period of time. We have deliberately set up a very difficult technical issue of development of, and have conducted extensive research. And it succeeded in developing the discrimination | determination method of the yeast using a gene, and has just applied for a patent about the result (refer patent documents 1, 2, and 3).
JP 2003-245077 A JP 2004-329086 A JP 2006-340671 A

  The inventions according to the prior application are all excellent, but the present inventors have developed and improved the invention according to the prior application without satisfying these results.

The present invention has been made to achieve the above object.
First, the inventions of the prior application (Patent Document 1 and Patent Document 2) are obtained by amplifying part or all of the YIL169C gene, YOL155C gene, and MUC1 gene, and brewing them depending on the length and number of the amplified fragments. The basic technical idea is to discriminate the yeast for use.

  In the prior application invention, the genomic DNA of the brewing yeast is amplified by PCR using primers that amplify a part or all of the YIL169C gene, YOL155C gene, and MUC1 gene, and the amplified gene fragments are electrophoresed or After the restriction enzyme treatment, electrophoresis is performed, and the electrophoresis pattern can be observed. Therefore, the operation is easy and simple. In addition, the use of primers that amplify a specific gene (in this case, part or all of the YIL169C gene, YOL155C gene, and MUC1 gene) also stabilizes the type of DNA fragment obtained by PCR amplification. There are many excellent points.

  In the prior application, first, the YIL169C gene and the YOL155C gene are focused on. These genes are not essential genes for yeast growth, and the protein encoded by the gene has a short amino acid sequence repeated in the molecule. As a result of searching for genes having these characteristics from among genes not having homology with the YIL169C gene and YOL155C gene, focusing on the fact that they have common characteristics, the MUC1 gene was found. Confirmed that it is available.

  Then, as a result of further study on the above-mentioned invention of the prior application, the present inventors have found that the FLO gene can also be discriminated. Although the FLO gene itself is known (there is no particular difficulty in obtaining it since it has been published on the Internet (http: www.Yeastgenome.org)), it is possible to discriminate yeasts that focus on the FLO gene before filing the prior application. Has not been reported, and it has not been reported at all about the fact that a specific part of the FLO gene is amplified as a primer and yeast is identified using the amplified gene fragment. The invention is first (see Patent Document 3).

  In addition, ribosomal genes have been conventionally used for yeast discrimination, but as a result of detailed gene sequence studies, it was newly found that there are sites in the IGS region that have different numbers of repeats in units of 6 bp between brewing yeasts. . We have designed a primer that focuses on the sequence and found that the DNA fragments to be amplified by PCR reaction are different among brewing yeasts and can be used to discriminate brewing yeasts. (See Patent Document 3).

  By these methods, it was possible to accurately and easily distinguish between yeasts of the same use in a short period of time. However, although it is possible to discriminate between yeasts of different uses such as wine yeast and shochu yeast, wine yeast and sake yeast by the methods described in these patent documents, the difference in length of DNA fragments by electrophoresis is small. Therefore, it was necessary to calculate accurately.

  For this reason, it is considered that discrimination becomes easier if a significant difference is observed in the amplified DNA fragment between yeasts of different uses. Specifically, there is a difference in the presence or absence of amplified DNA fragments between yeasts with different uses, the length of the amplified DNA fragments differs by about 30%, and yeasts with different uses but with different uses Then, if a result such as the same can be obtained, the determination becomes easy.

  In the present invention, solving these points is newly set as a new technical problem.

  The present invention has been made for the purpose of solving this technical problem. In other words, the present invention has been made for the purpose of greatly improving the method for determining yeast using the gene developed by the present inventors and enhancing its effectiveness.

  In order to achieve the above object, the present inventors have paid attention to the FLO gene again as a result of examination from various directions. Then, the present inventors have continued further research on the FLO gene. By amplifying the FLO5 gene by the PCR method, sake yeast and shochu yeast can be further amplified, and further one of the FLO5 gene, YHR213W gene or YAR062W gene can be obtained. It was found for the first time that most of sake yeast, shochu yeast, and wine yeast show amplified DNA fragments characteristic for each use, and yeast can be easily and accurately distinguished for use by amplifying the portion.

  The present invention has been completed as a result of further research based on the above useful new findings, and was amplified by performing a PCR reaction using the DNA fragment derived from the gene as a primer and brewing yeast genomic DNA as a template. The basic technical idea is that the yeast for brewing is discriminated based on the presence or absence of a gene fragment, the difference in length or number, and the like.

  In practicing the present invention, it is first necessary to develop a primer. As a result of intensive studies, the present inventors have succeeded in developing primers A to H (the base sequences thereof are shown in SEQ ID NOs: 1 to 8 in the Sequence Listing and Table 1 below) for the first time.

  The base sequences of primers A to H are shown in Table 1 below.

  In practicing the present invention, as described above, the primers A to H (SEQ ID NOs: 1 to 8: Table 1), which were successfully developed for the first time by the present inventors as described above, were selected and used, and the yeast genomic DNA was selected. PCR is performed using a template, but the primer may be cut out from the gene, or the above-described primer may be synthesized and used. As a primer, a primer corresponding to the yeast to be discriminated may be appropriately selected and used. Depending on the yeast, a primer other than A to H may be amplified from the FLO5 gene or a DNA fragment containing the YHR213W or YAR062W gene. It may be designed and used. PCR may be performed according to a conventional method, and as a result, the intended DNA fragment can be obtained. In the present invention, the primer includes a DNA fragment comprising the base sequence shown by at least one of SEQ ID NOs: 1 to 8, and a DNA fragment containing the base sequence.

  The gene fragment obtained by amplification in this manner can be distinguished from yeast by electrophoresis of the gene fragment such as agarose gel electrophoresis and observing the migration pattern. By selecting the type and combination of primers, a clear migration pattern unique to yeast can be obtained. If desired, the gene fragment amplified by PCR can be cleaved with a restriction enzyme and electrophoresed to observe the electrophoresis pattern.

  Therefore, according to the present invention, it is possible to discriminate and identify various brewing yeasts by changing the kind of at least one of primers, genomic DNA used as a template, amplified DNA fragment, and restriction enzyme digests thereof. Or, conversely, in order to discriminate and identify the use or the like of a specific brewing yeast, a primer or the like may be selected.

  Thus, according to the present invention, brewing yeast can be classified by use, so that a kit for distinguishing, identifying, and classifying yeast can be assembled using at least one of the above-described primers and the like. Therefore, using, for example, primers A and B, it is possible to construct an association shochu yeast discrimination, identification and classification kit.

  According to the present invention, compared to conventional methods such as small preparation test, determination by changing drugs and culture conditions, etc., there is a remarkable effect that it can be determined in a short time, and it can be clearly distinguished and stable. Results are obtained, have reproducibility, and can be easily operated.

  Furthermore, the present invention can be applied to all yeasts belonging to Saccharomyces cerevisiae, for example, sake yeast and shochu yeast, wine yeast and shochu yeast, wine Differentiating between different brewing yeasts such as yeast and sake yeast, as well as distinguishing between very difficult and delicate same brewing yeasts, for example, between wine yeasts, shochu yeasts, and sake yeasts Is possible. Therefore, the present invention enables simple classification, identification, and determination of yeast in a short period of time in liquor manufacturing science, testing research institutions, and the like.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Example 1: Method for discriminating brewing yeast)
As yeast, sake yeast, shochu yeast, wine yeast and laboratory yeast belonging to Saccharomyces cerevisiae were used.

  Specifically, as sake yeast, K-6, K-7, K-9, K-10, K-11, K-13, K-14, K-1501, K-1601, K-1701, K -601, K-701, K-901, K-1001 and K-1401 (Japan Brewing Association) are used as shochu yeast, KF1, KF3 and CAN1 (Kuma Shochu Shuzo Association), MK021, I-33 and C14 (Miyazaki) Prefectural Food Development Center), C4, H5 and K2 (Kagoshima Prefectural Industrial Technology Center), Aw101 (Okinawa National Tax Office), S-2 and S-3 (Japan Brewing Association) No. (KyokaiNo1), No.3 (KyokaiNo3) and No.4 (KyokaiNo4) (Japan Brewing Association), L2323, BM45, 71B, K1V1116, CS2, CEG, SIHA7, L 7, EC1118, SIHA4, T73,43, BC, SIHA4, BDX (or Lallemand Inc.) and W-3 (the CRIEPI save strain), the S288C (Invitrogen) as a laboratory yeast, respectively, were used.

  Using “Jentori-kun” from Takara Shuzo Co., Ltd., the purified genomic DNA was subjected to PCR using a combination of each of primers A to H (SEQ ID NOs: 1 to 8) (PCR reaction conditions were 3 at 95 ° C.). 1 minute at 95 ° C., 1 minute at 61 ° C., 1 minute at 72 ° C., 25 cycles for 1 minute, 72 minutes at 3 minutes at 72 ° C., and 1 cycle at 72 ° C. with a Robocycler 96 (Stratagene). After amplification with agarose gel electrophoresis.

<Result 1>
FIG. 1 shows the results of electrophoresis of amplified DNA fragments using the primer A (SEQ ID NO: 1) and the primer B (SEQ ID NO: 2) of shochu yeast (A) and sake yeast (B).

  In the figure, (A) Lane 1 is a molecular weight marker (Molecular weight marker: length is 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 13 are, in order, KF1, KF3, CAN1, MK021, I-33, C14, C4, H5, K2, Aw101, S-2, S-3, and above, shochu yeast. (B) Lanes 1 and 13 are molecular weight markers (lengths are 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 12 are, in order, K-6, K-7, K-9, K-10, K-11, K-13, K-14, K-1501, K-1601, K-1701, and above, sake yeast. . Lane 12 is a laboratory yeast at S288C. Lanes 14 to 18 are K-601, K-701, K-901, K1001, K-1401, and above, sake yeast. Lane 19 is a laboratory yeast at S288C.

  In sake yeast and S288C, an amplified DNA fragment of 3.2 kb is observed, but no amplified DNA fragment is observed in shochu yeast excluding H5. The amplified DNA fragment of H5 is about twice as long (6.2 kbp) as that of sake yeast or S288C.

<Result 2>
FIG. 2 shows the results of electrophoresis of the amplified DNA fragments using the primer C (SEQ ID NO: 3) and the primer D (SEQ ID NO: 4) of wine yeast (A), shochu yeast (B) and sake yeast (C).

In the figure, (A) lanes 1 and 14 are molecular weight markers (length: 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 12 are association wine yeast No. 1, association wine yeast No. 3, association wine yeast No. 4, L2323, BM45, 71B, K1V1116, CS2, CEG, SIHA7, W-3, and more. . Lane 13 is a laboratory yeast at S288C. Lanes 15 to 21 are L27, EC1118, T73, 43, BC, SIHA4, BDX, wine yeast. Lane 22 is a laboratory yeast at S288C. (B) Lane 1 is Molecular weight marker (length is 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 13 are, in order, KF1, KF3, CAN1, MK021, I-33, C14, C4, H5, K2, Aw101, S-2, S-3, and above, shochu yeast.
(C) Lanes 1 and 13 are molecular weight markers (lengths are 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 12 are, in order, K-6, K-7, K-9, K-10, K-11, K-13, K-14, K-1501, K-1601, K-1701, and above, sake yeast. . Lane 12 is a laboratory yeast at S288C. Lanes 14 to 18 are K-601, K-701, K-901, K1001, K-1401, and above, sake yeast. Lane 19 is a laboratory yeast at S288C.

  An amplified DNA fragment of about 400 bp is found in wine yeast, but in other yeasts it is only found in shochu yeast KF1 and laboratory yeast S288C.

<Result 3>
FIG. 3 shows the results of electrophoresis of the amplified DNA fragments using the primer E (SEQ ID NO: 5) and the primer F (SEQ ID NO: 6) of wine yeast (A), shochu yeast (B) and sake yeast (C).

  In the figure, (A) Lanes 1 and 14 are molecular weight markers (Molecular weight marker: length is 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 13 are association wine yeast No. 1, association wine yeast No. 3, association wine yeast No. 4, L2323, BM45, 71B, K1V1116, CS2, CEG, SIHA7, W-3, and more, wine yeast . Lane 13 is a laboratory yeast at S288C. Lanes 15 to 21 are L27, EC1118, T73, 43, BC, SIHA4, BDX, wine yeast. Lane 22 is a laboratory yeast at S288C. (B) Lane 1 is a Molecular weight marker (length is 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top), and Lanes 2 to 13 are KF1, KF3, CAN1, MK021, I-33, C14, C4 in order H5, K2, Aw101, S-2, S-3, and above, shochu yeast. (C) Lanes 1 and 13 are molecular weight markers (lengths are 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 11 are, in order, K-6, K-7, K-9, K-10, K-11, K-13, K-14, K-1501, K-1601, K-1701, and above, sake yeast. . Lane 12 is a laboratory yeast at S288C. Lanes 14 to 18 are K-601, K-701, K-901, K1001, K-1401, and above, sake yeast. Lane 19 is a laboratory yeast at S288C.

  As is clear from the above results, a 640 bp amplified DNA fragment is commonly found in wine yeast, a 1200 bp amplified DNA fragment is commonly found in sake yeast, and 930 bp amplified except for KF1, I-33 and C14 in shochu yeast. DNA fragments are commonly found. The 930 bp amplified DNA fragment found in shochu yeast is not found in wine yeast, and the 1200 bp amplified DNA fragment is found only in L27 in wine yeast. The pattern of the amplified DNA fragment is the same as that for sake yeast KF1 represented by grape yeast 1; shochu yeast I-33 for wine yeast SIHA7; shochu yeast C14 for wine yeast K1V1116 and EC1118. However, no match can be found between sake yeast and wine yeast.

<Result 4>
FIG. 4 shows the results of electrophoresis of amplified DNA fragments obtained from the primers G (SEQ ID NO: 7) and H (SEQ ID NO: 8) of wine yeast (A), shochu yeast (B), and sake yeast (C).

  In the figure, (A) Lanes 1 and 14 are molecular weight markers (Molecular weight marker: length is 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 12 are association wine yeast No. 1, association wine yeast No. 3, association wine yeast No. 4, L2323, BM45, 71B, K1V1116, CS2, CEG, SIHA7, W-3, and more. . Lane 13 is a laboratory yeast at S288C. Lanes 15 to 21 are L27, EC1118, T73, 43, BC, SIHA4, BDX, wine yeast. Lane 22 is a laboratory yeast at S288C. (B) Lane 1 is a molecular weight marker (length is 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 13 are, in order, KF1, KF3, CAN1, MK021, I-33, C14, C4, H5, K2, Aw101, S-2, S-3, and above, shochu yeast. (C) Lanes 1 and 13 are molecular weight markers (lengths are 7 kbp, 5 kbp, 3 kbp, 2 kbp, 1.5 kbp in order from the top). Lanes 2 to 11 are, in order, K-6, K-7, K-9, K-10, K-11, K-13, K-14, K-1501, K-1601, K-1701, and above, sake yeast. . Lane 12 is a laboratory yeast at S288C. Lanes 14 to 18 are K-601, K-701, K-901, K1001, K-1401, and above, sake yeast. Lane 19 is a laboratory yeast at S288C.

  A 430 bp amplified DNA fragment is commonly observed in wine yeast, and a 430 bp amplified DNA fragment is observed only in KF1 and H5 in shochu yeast. Amplified DNA fragments are not observed in sake yeast.

  As is clear from the above, each yeast can be classified and discriminated by selecting various combinations of primers and examining the presence or absence of DNA fragments to be amplified and / or the length of DNA to be amplified. .

  According to the present invention, as compared with the conventional method of determining by changing the small preparation test, the drug and the culture condition, the effect of being able to be determined in a short time is exhibited, and the determination can be made clearly and stably. The results are excellent, reproducible, and easy to operate.

  Furthermore, according to the present invention, it is possible to distinguish between brewer's yeasts of different uses as well as contamination of wild yeasts other than brewer's yeasts. The yeast can be easily and accurately classified and discriminated between them, and it is possible to clearly discriminate the yeast in the mash and the wild yeast.

It is a drawing substitute photograph which shows the pattern of electrophoresis (Result 1). It is a drawing substitute photograph which shows the pattern of electrophoresis (Result 2). It is a drawing substitute photograph which shows the pattern of electrophoresis (Result 3). It is a drawing substitute photograph which shows the pattern of electrophoresis (Result 4).

Claims (2)

  Genomic DNA of wine yeast, shochu yeast, and sake yeast is amplified by PCR using a DNA fragment consisting of the nucleotide sequence represented by SEQ ID NO: 5 (primer E) and SEQ ID NO: 6 (primer F) as a primer. Characterized in that, by directly electrophoresis the amplified gene fragment, the yeast is discriminated based on the presence or absence of the amplified gene fragment, the difference in the number of the gene fragments, or the difference in the size of the gene fragments. A method for distinguishing sake yeast, shochu yeast and wine yeast.   Genomic DNA of sake yeast, shochu yeast, and wine yeast is amplified by PCR using a DNA fragment consisting of the base sequence represented by SEQ ID NO: 7 (primer G) and SEQ ID NO: 8 (primer H) as a primer. Characterized in that, by directly electrophoresis the amplified gene fragment, the yeast is discriminated based on the presence or absence of the amplified gene fragment, the difference in the number of the gene fragments, or the difference in the size of the gene fragments. Method for distinguishing sake yeast, shochu yeast and wine yeast.

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