JP2014060948A - Method for determining kinds of beer raw materials and products, and method for evaluating beer quality using the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a technique for determining kinds of barley, a hop, and beer yeast which are raw materials for beer used as a sample.SOLUTION: There are provided a method in which an extremely small amount of the remaining DNA in beer is highly purified by combining an enzyme method and a beads method, and kinds of barley, a hop, and beer yeast are determined by a nucleic acid amplification using primers characteristic to each of the barley, the hop, and the beer yeast, a method for comprehensively evaluating the beer quality, and a kit used for the method.

Description

  The present invention relates to a method for discriminating beer raw materials and products, and a method for evaluating beer quality using the method. More specifically, the present invention relates to a template DNA preparation method for amplifying DNA remaining in beer by PCR in the presence of appropriate primers, a beer raw material based on the results of PCR using the template DNA obtained by the preparation method, and The present invention relates to a product discrimination method and a beer quality evaluation method based on the discrimination result.

  Beer breaks down barley starch by the enzymatic activity of barley malt, ferment saccharides to be purified by yeast to produce alcohol and carbon dioxide, adjust the fermentation with added hops, and give a unique flavor It is a beverage that is widely enjoyed around the world.

  It is said that the quality and quality of beer are influenced by the type and characteristics of the raw barley, the type and the fermentation conditions of the barley yeast, and the type and quality of the hop as a secondary ingredient.

  For identification of varieties by PCR using barley seeds as a sample, Uchimura et al. (Non-patent Document 1: Yosuke Uchimura, Masahiko Furusho, Tomohiko Yoshida 2004. Variety identification by DNA markers in domestic Nijo barley. Nisakuki 73: 35- 41.) and Nagahama et al. (Non-patent document 2: Takashi Nagamine, Masayuki Amaya, Tatsuya Ikeda, Mika Ozeki, Naoto Haruyama, Tsuneo Kato, Toshinori Satsuki: Development and evaluation of major trait DNA markers for beer barley and their use in breeding Problem, Tochigi National Agricultural Research Report No 59: 45-54 (2007)).

  Regarding the microbial species identification of brewer's yeast, Sato and Tsuchiya (Patent Document 1: Japanese Patent Application Laid-Open No. 2002-233382 “Method for identifying brewer's yeast”) focused on the characteristics of the Lg-FLO1 gene, A strain identification technology for wild yeast has been developed, and Ogata and Tadami (Patent Document 2: Japanese Patent Laid-Open No. 2008-245636) describe S. cerevisiae of DNA derived from brewer's yeast. cerevisiae type nucleotide sequence and S. cerevisiae type nucleotide sequence. This paper reports on yeast identification technology using PCR primer pairs that specifically amplify the places where recombination of Bayanus type nucleotide sequences has occurred, and technology for estimating beer brewing aptitude such as aroma component generation.

  Regarding the identification of varieties of hops, which are secondary ingredients of beer, Tsuchiya et al. (Non-patent Document 3: Tsuchiya et al., Journal of fermentation and bioengineering, 84 ( 2), 103-107, 1997), and Murakami has reported a method for efficiently extracting DNA from hops and suitable primers for PCR when hop varieties are identified by the PCR method (patent) Document 3: JP-A-11-103895).

  The above barley, brewer's yeast, and hop identification technologies are all technologies that extract DNA from seeds, fungus bodies, or plant bodies, use them as templates, and perform PCR, not technologies that identify raw materials using beer as a sample. On the other hand, the present inventors have developed a technology for discriminating raw materials of brewed liquor such as wine, sake and beer by DNA discrimination using the PCR method, and whether corn or rice is added to beer. Can be identified by the PCR method (Patent Document 4: Japanese Patent No. 4953058 “Method for discriminating raw material plants in brewed liquor”).

  The above-mentioned technique reported by the present inventors is particularly suitable for discriminating rice varieties that are the main ingredients of sake and grape varieties that are the main ingredients of wine, but for beer, template DNA that can be extracted and purified. However, the degree of purification and amount of these are insufficient, and further improvement is necessary for the discrimination of a wide range of raw material plants.

JP 2002-233382 A JP-A-11-103895 JP 2008-245636 A Japanese Patent No. 4953058

Yosuke Uchimura, Masahiko Furusho, Tomohiko Yoshida 2004. Variety identification by DNA marker of domestic Nijo barley. Nisakuki 73: 35-41 Nagatake, et al., “Problems in Development and Evaluation of Breeding Barley Main Marker DNA Markers and Breeding Utilization”, Tochigi Agricultural Research Report No. 59: 45-54 (2007) Youichi Tsuchiya et al. , “Identification of hop varieties using specific primers derived from RAPD markers,” Journal of Fermentation and Bioengineering, 84 (2), 97-107.

  It is known that the quality of beer is affected by the type of raw barley, the type and fermentation conditions of brewer's yeast, and the quality of hops, which are secondary ingredients, and the quality of beer is determined by DNA discrimination using the PCR method. In order to estimate, it is necessary to develop DNA markers related to quality for each of the raw barley, brewer's yeast, and hops, and to develop and design suitable primers, and the high purity required for multiple PCRs Therefore, a large amount of template DNA must be extracted and purified from beer. To that end, the method for extracting and purifying DNA from brewed liquor reported in Japanese Patent No. 4953058 has been improved to efficiently extract highly pure template DNA. -It was necessary to newly develop technology for purification and technology for PCR primer selection.

The first object of the present invention is to provide a method for discriminating beer raw materials or products.
The second object of the present invention is to provide a beer quality evaluation method based on the discrimination result of beer raw materials.

As a result of intensive studies in order to solve the above problems, the present inventors have succeeded in using a DNA purification method using a DNA adsorption carrier (for example, a magnetic carrier and a magnet) and an alcohol partitioning method in combination. It has been found that pure DNA can be prepared easily and in large quantities.
In addition, in order to find a PCR primer suitable for discriminating beer raw materials or products and evaluating the quality of beer, the present inventors used this to identify barley, yeast and hops using DNA extracted and purified from beer as a template. Amplify a large number of DNA of specific components, extract and clone DNA from suitable DNA bands, determine the base sequence of each DNA, and redesign the most suitable primer pair group based on the results succeeded in.

  By using the DNA preparation method of the present invention, a large amount of high-purity template DNA can be prepared, so that PCR can be performed in the presence of various primer pairs for each of the barley, yeast, and hops per beer, In addition to making it possible to discriminate each raw material of the beer, the overall quality evaluation of the beer has become possible by taking into consideration the effects of barley, yeast, and hops on beer quality.

In summary, the present invention encompasses the following features.
(1) A method for discriminating beer raw materials or products, the method comprising: (a) DNA extracted and purified from a beer sample as a template, a plurality of barley discrimination primers, a plurality of hop discrimination primers, and a plurality of beers A step of amplifying the DNA separately or simultaneously using each primer group consisting of primers for yeast discrimination, (b) a step of detecting the DNA amplified in step (a), (c) a DNA in step (b) And determining the type of barley, brewer's yeast, and hops, which are the raw materials used in the production of beer, based on the detection result.

  (2) Barley polygalacturonase gene, barley hordein gene, barley starch synthase gene, barley protein Z gene, barley alpha amylase gene, barley beta amylase gene, barley lipoxygenase gene, barley yellow spot mosaic It is a primer for genes of all types from two or more selected from the group consisting of detection genes for viruses, barley powdery mildew-derived genes, barley trypsin inhibitor genes, barley cellulase genes, and barley β-glucanase genes. The method according to (1) above.

(3) The method according to (2) above, wherein the barley hordein gene is a hordein A gene, a hordein B gene, a hordein C gene, or a combination thereof.
(4) The method according to (2) or (3) above, wherein the barley protein Z gene is a protein Z7 gene, a protein Z4 gene, or a combination thereof.

(5) The method according to any one of (2) to (4) above, wherein the barley starch synthase gene is a barley starch granule-binding starch synthase gene.
(6) Any of the above (1) to (5), wherein the plurality of barley discrimination primers includes at least primers for detecting barley polygalacturonase gene, barley hordein gene, barley starch synthase gene and barley protein Z gene. The method of crab.

  (7) The barley polygalacturonase gene detection primer pair is composed of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 23, and the barley hordein gene detection primer pair is SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 3 And / or SEQ ID NO: 25 and / or SEQ ID NO: 4 and SEQ ID NO: 26, and the detection primer pair for the barley starch synthase gene consists of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 27, and the barley protein Z gene The detection primer pairs consisted of the base sequences of SEQ ID NO: 6 and SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 29, SEQ ID NO: 8 and SEQ ID NO: 30 and / or SEQ ID NO: 9 and SEQ ID NO: 31, The detection primer pair consists of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 32, and the detection primer pair of the barley lipoxygenase gene has the sequence A primer pair for detecting a barley yellow mosaic virus-derived gene consisting of the base sequences of No. 11 and SEQ ID NO: 33, and a primer pair for detecting a barley powdery mildew-derived gene consisting of the base sequences of SEQ ID NO: 12 and SEQ ID NO: 34 SEQ ID NO: 13 and SEQ ID NO: 35 and / or SEQ ID NO: 14 and SEQ ID NO: 36, and the barley trypsin inhibitor gene detection primer pair comprises the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 37, and barley cellulase The gene detection primer pair consists of the nucleotide sequences of SEQ ID NO: 16 and SEQ ID NO: 38, and the barley β-glucanase gene detection primer pair consists of the nucleotide sequences of SEQ ID NO: 17 and SEQ ID NO: 39 above (2 ) To (6).

(8) The plurality of hop discrimination primers are selected from a primer for detecting a hop gene belonging to an aroma, a primer for detecting a hop gene belonging to a fine aroma, and a primer for detecting a hop gene belonging to a bitter. The method according to any one of (1) to (7).
(9) A plurality of hop discrimination primers are a primer pair consisting of the base sequences of SEQ ID NO: 18 and SEQ ID NO: 40, a primer pair consisting of the base sequences of SEQ ID NO: 19 and SEQ ID NO: 41, and the bases of SEQ ID NO: 20 and SEQ ID NO: 42 The method according to (8) above, which is selected from a primer pair consisting of a sequence.

(10) Any one of the above (1) to (9), wherein the plurality of beer yeast discrimination primers are selected from a primer that makes it possible to distinguish between upper-fermenting yeast and lower-fermenting yeast and a primer for detecting a yeast thioredoxin gene The method described in 1.
(11) The method according to (10) above, wherein the plurality of beer yeast discrimination primers are selected from the primer pair of SEQ ID NO: 21 and SEQ ID NO: 43 and the primer pair of SEQ ID NO: 22 and SEQ ID NO: 44.

(12) The template DNA is (a1) a step in which starch degrading enzyme and proteolytic enzyme are allowed to act on a beer sample to decompose starch, starch degradation product and protein, (a2) DNA is recovered from the treatment liquid in step (a1) And (a3) adsorbing the DNA from step (a2) using a DNA-adsorbing solid phase carrier, and recovering the DNA from the solid phase carrier. ) To (11).
(13) The method according to (12) above, wherein the starch degrading enzyme is a thermostable amylase.

(14) The method according to (12) or (13) above, wherein the proteolytic enzyme is proteinase K.
(15) The method according to any one of (12) to (14) above, wherein the DNA-adsorbing solid phase carrier is glass beads or magnetic beads.

(16) Nucleic acid that is a method for evaluating the quality of beer and uses a barley discrimination primer, a hop discrimination primer, and a brewer's yeast discrimination primer using the method according to any one of (1) to (15) above. A step of discriminating the types of barley, brewer's yeast, and hops, which are raw materials used in the production of beer, by the amplification method, and using the discrimination result, comprehensive quality of beer taste and foam retention is obtained. Evaluating the method.
(17) The method according to (16) above, wherein the beer's taste is bitter, sour, astringent and umami.

(18) For the presence or absence of an amplified DNA band for deriving a discrimination result, binarize 1 and none 0, and estimate the quality of the beer by performing multivariate analysis with beer quality as an objective variable, The method according to (16) or (17) above.
(19) Barley discrimination primer derived from barley polygalacturonase gene, barley hordein gene, barley starch synthase gene, barley protein Z gene, barley α-amylase gene, barley β-amylase gene, barley lipoxygenase gene, barley yellow mosaic virus It is a primer for genes of all types from two or more selected from the group consisting of genes, barley powdery mildew-derived genes, barley trypsin inhibitor gene, barley cellulase gene, and barley β-glucanase gene detection primer. Primer for detecting a hop gene belonging to aroma, a primer for detecting a hop gene belonging to fine aroma, a primer for detecting a hop gene belonging to bitter, and a primer for discriminating brewer's yeast The method according to any one of the above (16) to (18), wherein is a primer that makes it possible to distinguish between upper surface fermenting yeast and lower surface fermenting yeast and a primer for detecting a yeast thioredoxin gene.

  (20) The barley polygalacturonase gene detection primer pair is composed of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 23, and the barley hordein gene detection primer pair is SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 3 And / or SEQ ID NO: 25 and / or SEQ ID NO: 4 and SEQ ID NO: 26, and the detection primer pair for the barley starch synthase gene consists of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 27, and the barley protein Z gene The detection primer pairs consisted of the base sequences of SEQ ID NO: 6 and SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 29, SEQ ID NO: 8 and SEQ ID NO: 30 and / or SEQ ID NO: 9 and SEQ ID NO: 31, The detection primer pair consists of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 32, and the detection primer pair for the barley lipoxygenase gene is arranged. A primer pair for detecting a barley yellow mosaic virus-derived gene comprising a nucleotide sequence of column No. 11 and SEQ ID NO: 33, comprising a nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 34, and a primer pair for detecting a barley powdery mildew-derived gene Is composed of the base sequences of SEQ ID NO: 13 and SEQ ID NO: 35 and / or SEQ ID NO: 14 and SEQ ID NO: 36, and the primer pair for detection of the barley trypsin inhibitor gene is composed of the base sequences of SEQ ID NO: 15 and SEQ ID NO: 37, The cellulase gene detection primer pair consists of the base sequence of SEQ ID NO: 16 and SEQ ID NO: 38, and the barley β-glucanase gene detection primer pair consists of the base sequence of SEQ ID NO: 17 and SEQ ID NO: 39, and hop discrimination Primer for primer consisting of the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 40, SEQ ID NO: 9 and a primer pair consisting of the base sequence of SEQ ID NO: 41, a primer pair consisting of the base sequence of SEQ ID NO: 20 and SEQ ID NO: 42, and the brewer's yeast discrimination primer is the base sequence of SEQ ID NO: 21 and SEQ ID NO: 43 The method according to (19) above, which is a primer pair consisting of and a primer pair consisting of the nucleotide sequences of SEQ ID NO: 22 and SEQ ID NO: 44.

  (21) A kit comprising a barley discrimination primer, a hop discrimination primer and a beer yeast discrimination primer for use in the method according to any one of (1) to (20) above, wherein the barley discrimination primer Barley polygalacturonase gene, barley hordein gene, barley starch synthase gene, barley protein Z gene, barley α-amylase gene, barley β-amylase gene, barley lipoxygenase gene, barley yellow mosaic virus-derived gene, barley powdery mildew A primer for two or more genes selected from the group consisting of detection primers for genes, barley trypsin inhibitor gene, barley cellulase gene, and barley β-glucanase gene, and hop discrimination primers belong to aroma For detection of hop genes A primer for detecting a hop gene belonging to lymer and fine aroma, and a primer for detecting a hop gene belonging to bitter, and a primer for distinguishing beer yeast from top fermenting yeast and bottom fermenting yeast And a kit for detecting a yeast thioredoxin gene.

  (22) The kit according to (21), wherein the barley discrimination primer includes at least primers for detecting a barley polygalacturonase gene, a barley hordein gene, a barley starch synthase gene, and a barley protein Z gene.

  (23) The barley polygalacturonase gene detection primer pair is composed of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 23, and the barley hordein gene detection primer pair is SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 3 And / or SEQ ID NO: 25 and / or SEQ ID NO: 4 and SEQ ID NO: 26, and the detection primer pair for the barley starch synthase gene consists of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 27, and the barley protein Z gene The detection primer pairs consisted of the base sequences of SEQ ID NO: 6 and SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 29, SEQ ID NO: 8 and SEQ ID NO: 30 and / or SEQ ID NO: 9 and SEQ ID NO: 31, The detection primer pair consists of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 32, and the detection primer pair for the barley lipoxygenase gene is arranged. A primer pair for detecting a barley yellow mosaic virus-derived gene comprising a nucleotide sequence of column No. 11 and SEQ ID NO: 33, comprising a nucleotide sequence of SEQ ID NO: 12 and SEQ ID NO: 34, and a primer pair for detecting a barley powdery mildew-derived gene Is composed of the base sequences of SEQ ID NO: 13 and SEQ ID NO: 35 and / or SEQ ID NO: 14 and SEQ ID NO: 36, and the primer pair for detection of the barley trypsin inhibitor gene is composed of the base sequences of SEQ ID NO: 15 and SEQ ID NO: 37, The cellulase gene detection primer pair consists of the base sequence of SEQ ID NO: 16 and SEQ ID NO: 38, and the barley β-glucanase gene detection primer pair consists of the base sequence of SEQ ID NO: 17 and SEQ ID NO: 39, and hop discrimination Primer for primer consisting of the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 40, SEQ ID NO: 9 and a primer pair consisting of the base sequence of SEQ ID NO: 41, a primer pair consisting of the base sequence of SEQ ID NO: 20 and SEQ ID NO: 42, and the brewer's yeast discrimination primer is the base sequence of SEQ ID NO: 21 and SEQ ID NO: 43 The kit according to (21) or (22) above, which is a primer pair comprising: and a primer pair comprising the nucleotide sequences of SEQ ID NO: 22 and SEQ ID NO: 44.

  According to the present invention, by using a nucleic acid amplification method such as PCR using DNA extracted and purified from beer as a template, it is possible to discriminate the variety of barley that is the main raw material of the beer, discriminate the brewer's yeast that is the fermenting microorganism of the beer, and It is possible to identify hops that are auxiliary materials.

According to the present invention, a beer product can be identified.
According to the present invention, it is possible to estimate the quality of the beer by a nucleic acid amplification method such as PCR using DNA extracted and purified from beer as a template, and in particular, binarize the DNA amplification results as explanatory variables, It is possible to estimate the quality of the beer from the result of amplification of the DNA of the beer product by performing multivariate analysis such as multiple regression analysis using the property and foam retention as the objective variables.

This figure shows the procedure of a method for preparing a template DNA for PCR from a beer sample. In the figure, TE represents Tris / EDTA buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). In the figure, PCI means phenol-chloroform-isoamyl alcohol (25: 24: 1). This figure shows the results of PCR using template DNAs with different extraction and purification methods. The PCR primer is a barley starch synthase primer, ie, the forward primer (F) is caa ttc atc cga tca ctc aat cat (SEQ ID NO: 45) and the reverse primer (R) is cag gcc gac aag gtg ctg (SEQ ID NO: 46). Annealing was performed at 58 ° C. In the figure, M is a marker, lanes 1 and 2 are isoplant I (Beer, DNA), lanes 3 and 4 are isoplant II (Beer, DNA), and lanes 5 and 6 are Dneasy plant. mini kit (QIAGEN) (Beer, DNA), lanes 7 and 8 are the method of the present invention (Beer, DNA), and lane 9 is barley starch synthase DNA. This figure shows the result of PCR using a barley gene-derived primer (SEQ ID NO: 1 to 15 in Table 1) as a template DNA from barley. In the figure, M is a marker, and lanes 1 to 16 represent template DNAs from the 16 barley varieties shown in Table 3. Band 1 represents polygalacturonase-related DNA, band 2 represents hordein A-related DNA, band 3 represents hordein B-related DNA, and band 4 represents hordein C-related DNA. Band 5 represents starch synthase related DNA, bands 6, 7 and 8 represent protein Z7 (serpin) related DNA, band 9 represents protein Z4 related DNA, and band 10 represents udon powder It represents DNA related to disease resistance gene (α-amylase), band 11 represents DNA related to lipoxygenase, band 12 represents DNA related to resistance to macular mosaic virus disease, and band 13 represents DNA related to powdery mildew disease resistance. Band 14 represents powdery mildew resistance (protein Z4) related DNA and band 15 represents trypsin. Representing an inhibitor-related DNA. This figure shows a barley lipoxygenase gene (SEQ ID NO: 11 in Table 1), β-glucanase gene (SEQ ID NO: 17), hordein A (SEQ ID NO: 2) and hordein C (SEQ ID NO: 4) using DNA from beer as a template. ) Shows discrimination of various beers by PCR using primers derived from. In the figure, M is a marker, and lanes 1 to 23 represent template DNA from 22 types of beer (1 to 22) and a comparative barley sample (23). Band 1 represents polygalacturonase-related DNA, band 2 represents hordein A-related DNA, band 4 represents hordein C-related DNA, and band 5 represents starch synthase-related DNA. Bands 6, 7 and 8 represent protein Z7 (serpin) related DNA, band 9 represents protein Z4 related DNA, and band 10 represents powdery mildew resistance gene (α-amylase) related DNA. , Band 11 represents lipoxygenase-related DNA and band 15 represents trypsin inhibitor-related DNA. This figure shows a comparison between the base sequence of a PCR amplification product from a beer sample (SEQ ID NO: 47; amplification product by Protein Z4 primer in Table 1) and the base sequence of Serpin (SEQ ID NO: 48). This figure shows the results of electrophoresis of DNA amplified by PCR of various beers using hop variety-identifying primers (SEQ ID NOs: 18 to 20 in Table 1). Lane M is a marker, lane a is hop variety Saatzer, lane b is hop variety Cascade, lane c is hop variety Galena, lane d is hop variety Liberty, lane 1 -22 are different types of beer samples. In the figure, w and y represent amplification products by the Hop-a primer and Hop-c primer in Table 1 described later, respectively. x represents an amplification product by the Hop-b primer in Table 1 described later. This figure shows the result of electrophoresis of the PCR amplification product (Za) of the yeast thioredoxin-2 gene in the beer sample (upper, lane M is a marker, and lanes 1 to 22 are numbers 22 shown in Table 3. PCR amplification product from seed beer, lane 23 represents the amplification product derived from the yeast thioredoxin-2 gene), the base sequence of the PCR amplification product (SEQ ID NO: 49) and the base sequence of standard thioredoxin-2 DNA (SEQ ID NO: 49) Comparison with () is shown. This figure shows a two-dimensional display of the taste measurement results of various beer samples by the taste recognition device. Numbers 1 to 22 represent the numbers of the 22 types of beer shown in Table 3. In the figure, (1) is the characteristic range of Lager beer, (2) is the characteristic range of Low-mal beer, (3) is the characteristic range of Ale beer, and (4) is the characteristic range of draft beer. It is. This figure shows the relationship between the taste by the taste recognition device and the PCR results in Table 3. Numbers 1 to 22 represent the numbers of 22 kinds of beers described in Table 3. R ² represents a coefficient of determination. This figure shows the test method of the foam retention property (stability) of beer. This figure is a graph showing estimation of beer foam retention (stability) by PCR. R ² represents a coefficient of determination. The numbers in the graph represent the numbers of the 22 beers shown in Table 3.

The present invention will be described in further detail.
1. The discriminating method of a beer raw material or a product The present invention, in the first aspect, is a discriminating method of a beer raw material or a product, the method comprising: (a) using a DNA extracted and purified from a beer sample as a template, Amplifying the DNA separately or simultaneously using a primer group comprising a discrimination primer, a plurality of hop discrimination primers and a plurality of brewer's yeast discrimination primers; (b) the DNA amplified in step (a) And (c) providing a method including the step of determining the types of barley, brewer's yeast, and hops, which are raw materials used in the production of beer, based on the detection results of DNA in step (b). .

  Whether the primers are used individually or in combination is based on the premise that the size positions of the ladders produced by the amplification products do not overlap under the same electrophoresis conditions. For this purpose, it is necessary to amplify the target gene using each primer individually in advance and confirm the size position of the amplified product.

  In this specification, “beer” refers to the decomposition of barley starch by the enzymatic activity of barley malt, fermenting the sugar to be purified by yeast to produce alcohol and carbon dioxide, and adjusting the fermentation by the added hops. In addition, it is a beverage that is enjoyed widely in the world with a unique flavor, brewed in countries such as Germany, Belgium, United Kingdom, Ireland, Austria, Czech, United States, China, Korea, Japan, Pilsner, Vienna, There are various styles such as Dunkel, Bock, White Ale, Lambic, Strong Ale, Saison, Pale Ale, Brown Ale, Stout, Kelsch, Weizen, Fruit Beer, Liquor East Beer, etc. There is beer.

  In this specification, “barley” means the scientific name Hordeum vulgare L. Examples of Nijo barley grown in Japan are as follows: Amagi Nijo, Asaka Gold, Miharu Gold, Hoshu, Kyushu Nijo 16, Nishino Gold, Daisen Gold, Kinuyutaka, Misato Golden , Sky Golden, Eggplant Nijo, Omi Yutaka, Tone Nijo, Myougi Nijo, Haruna Nijo, Kinuka Nijo, Mikamo Golden, Saitama Nijo, Takaho Golden, New Golden, Nishino Chika, Nishinohoshi, Harushizuku, Ryofu, Hoshi Examples of foreign-made Nijo barley such as Masari and Koharu Nijo include Harrington, Palas, Bowdin, Flagship, Marytime, Hamerin, Metcalfe, Stirling and Schooner.

  In this specification, “beer yeast” refers to yeast generally used for brewing beer, and in terms of biological classification, examples include Saccharomyces cerevisiae and Saccharomyces pastorianus. Historically, the distinction between “upper brewer's yeast” and “lower brewer's yeast” has been distinguished by the dynamics of brewer's yeast (floating near the surface or agglomerating and sinking) seen after fermentation in the brewing process. Now, among those skilled in the art, the lower brewer's yeast and the upper brewer's yeast are different species in biological classification, the upper brewer's yeast is mainly classified as S. cerevisiae, and the lower brewer's yeast is Saccharomyces.・It is a common view that Leviciae is a hybrid of S. bayanus and is classified as S. pastorianus. In this specification, “genomic DNA of beer yeast” In addition to Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Saccharomyces pastorianus (Saccharomyces pastorianus), it also includes the genomic DNA of Saccharomyces bayanus.

  Regarding the “hop” in the present specification, the main cultivated hop varieties in the world to be identified in the present invention include German fine aroma, Hersbrucker, Hallertauer Tradition, Hallertauer, Sparter Select, Perle, Tettanger, bitter belonging to aroma. North Brewer, Brewers Gold, Magnum, Sazer belonging to Czech fine aroma, Bor belonging to bitter, Strissel Spalt belonging to French aroma, Lublin belonging to Polish aroma, Savinjsk belonging to Slovenia aroma. Golding, Super Styrian belonging to Bitter, Fugle, American Tettnanger, Willamette, Crystal, Liberty belonging to Bitter, Nugget, Cluster, chinook, Galena belonging to Bitter, P. aroma of New Zealand. Hallertau, New Zealand Hallertau, Green. You can list Bullet, Stickle Brat, Australian Pride of Ringwood, Kirin No. 2 belonging to Japanese bitter, Tomidori, Kitamidori and Eastern Gold. From these hop leaves, spikelets, or pellets and veils of spikelets, the genomic DNA that serves as a template for PCR is extracted and purified, but most of the hops currently used are pellets. It is processed (compressed after crushing).

  The method of the present invention makes it possible not only to discriminate beer materials but also to identify beer products by discriminating the types of barley, brewer's yeast, and hops that are raw materials used in the production of beer. This is based on the improvement of the technology for purifying template DNA from beer samples and the selection of specific gene groups advantageous for discrimination from the gene groups related to barley, brewer's yeast and hops.

1.1. Preparation of template DNA Purification of template DNA from a beer sample includes the following steps (a1) to (a3): (a1) Starch, starch degradation product and protein are allowed to act on a beer sample by causing amylolytic enzyme and proteolytic enzyme to act. Disassembling step,
(A2) a step of recovering DNA from the treatment liquid of step (a1), and (a3) adsorbing DNA from step (a2) using a DNA-adsorbing solid phase carrier and recovering the DNA from the solid phase carrier Step to do,
Can be carried out by a method comprising:

  Compared with the method described in the CTAB method, the alkali phenol method, the ISOPLANT kit method, the Qiagen kit method, the Takara kit method, and Japanese Patent No. 4953058, a high molecular weight template DNA can be prepared. There is also the advantage that it is possible to amplify and discriminate DNA of raw barley, yeast that is a fermenting microorganism, and hops that are secondary raw materials by PCR, which has a high degree of DNA purification and remains in beer in beer. .

  In step (a1), starch, starch degradation products, and proteins, which are main components other than nucleic acids, are removed. The beer sample is lyophilized to recover components other than water. Specifically, by lowering the ambient pressure of frozen beer using a vacuum pump, water is sublimated and dried at a low temperature at which the beer does not melt to obtain a powder. Thereafter, the powder is dissolved in a buffer solution for dissolving DNA, and amylolytic enzyme and proteolytic enzyme are allowed to act.

  Various buffer solutions such as glycine / hydrochloric acid, citric acid / sodium phosphate, sodium phosphate, tris / hydrochloric acid, boric acid, glycine / sodium hydroxide, sodium bicarbonate / sodium hydroxide, etc. Britton and Robinson's broad buffer solution, Good's buffer solution, for example, chaotropic ions such as guanidine hydrochloride, guanidine isothiocyanate, guanidine thiocyanate and / or sodium lauryl sulfate, Triton X-100, sodium cholate, Tween 20, etc. The buffer solution which added 0.05 to 10weight% of surfactant can be mentioned. By adding chaotropic ions or surfactants, proteolysis can be performed while suppressing the reaction of the DNA degrading enzyme.

  For the starch decomposition in the present invention, a starch-degrading enzyme such as amylase (for example, α-amylase, β-amylase, glucoamylase), for example, a thermostable amylase such as Bacillus licheniformis can be used. As the thermostable amylase, a commercially available product such as α-amylase (manufactured by Sigma Aldrich, St. Louis, USA) can be used. As the proteolytic enzyme, various proteolytic enzymes such as pepsin, trypsin, chymotrypsin, subtilisin, papain, ficin, and bromelain can be used, and proteinase K that can degrade proteins in the presence of SDS is preferable. Proteinase K is a serine protease derived from the fungus Triturachium album and is sold by Takara Bio Inc., Invitrogen Corp., and the like. In the treatment with these enzymes, for example, 250 mg of beer powder is dissolved in 2.5 mL of DNA elution buffer, and amylolytic enzyme and proteolytic enzyme are added in appropriate amounts to express enzymatic activity. The former is at a temperature of 80 ° C. The latter can be carried out by reacting each at 60 ° C. for 60 minutes.

In step (a2), DNA is recovered from the treatment liquid in step (a1).
Specifically, after enzyme treatment, DNA is precipitated from the treatment solution with alcohol (preferably about 99% ethanol), recovered as a precipitate by centrifugation, and DNA is eluted (or extracted) with 70% ethanol on ice. And then phenol treatment and DNA purification by chloroform / isoamyl alcohol treatment. Alternatively, in this step, extraction can be carried out using a phenol / chloroform (usually about 1: 1 volume ratio) solution instead of phenol / chloroform / isoamyl alcohol. By using isoamyl alcohol, the compatibility with water is improved as compared with the case of chloroform alone, so that the organic solvent phase and the aqueous phase are easily separated. The volume ratio of isoamyl alcohol: chloroform can usually be about 1:25. Furthermore, ethanol or (iso) propanol is usually used as the alcohol for precipitating DNA. The elution of DNA with 70% ethanol in this step is important for the recovery of DNA from beer, together with the following step (a3) (especially the use of a DNA-adsorbing solid phase carrier). In elution of DNA with 70% ethanol, treatment within 3 minutes is desirable to avoid elution of the dye. The ethanol concentration at this time may be 68% to 72%, but the optimum concentration is 70%, and the balance is water.

  After the above steps, RNA may be decomposed and removed by RNase if necessary.

  In step (a3), the DNA from step (a2) is adsorbed using a DNA-adsorbing solid phase carrier, and the DNA is recovered from the solid phase carrier. This step is also important and essential for efficient recovery of DNA from beer.

  Examples of the DNA-adsorbing solid phase carrier include any shape made of glass or metal (for example, magnetic metal). The shape body is preferably a microsphere (that is, a bead) in terms of operability and ease of collection.

  Specifically, after adsorbing DNA to magnetic beads, separating the beads from the solution portion with a magnet, washing with ethanol or (iso) propanol solution containing salt, then desorbing DNA from the magnetic beads with DNA eluate And purify. The obtained DNA is dissolved in TE (Tris / EDTA) buffer to form template DNA. Examples of the DNA eluate include pure water, TE, and a buffer solution containing 0.5 M or less salt (such as NaCl).

1.2. Amplification and detection of DNA In the above step (a), each primer comprising a plurality of barley discrimination primers, a plurality of hop discrimination primers, and a plurality of beer yeast discrimination primers using DNA obtained from a beer sample as a template The DNA is amplified using groups separately or simultaneously.

  In the method of the present invention, DNA extracted and purified from beer is used as a template, and nucleic acid amplification methods such as PCR using a barley discrimination primer, a hop discrimination primer, and a brewer's yeast discrimination primer, the raw material barley, hop and It is characterized by simultaneously determining three types of brewer's yeast.

  The nucleic acid amplification method is an arbitrary method capable of amplifying DNA, for example, PCR (Polymerase Chain reaction) method, LAMP (Loop-Mediated Isolation Amplification) method (T. Notomi et al. Nucleic Acid Res. 2000, 28 (28)). : E63), ICAN method (isothermal gene amplification method) and the like.

  The PCR method is a general nucleic acid amplification method. In the presence of one or more primers in the primer group, the PCR is performed on the primer binding portion by repeating a cycle consisting of three reactions of denaturation, annealing, and extension. This refers to a reaction for amplifying the sandwiched DNA, specifically, a denaturation step at about 94 ° C. to make double-stranded DNA into a single strand, and an annealing step at about 35 to 68 ° C. to bind the primer to the template DNA And repeating the extension step at about 72 ° C. for extending the primer along the template using a thermostable DNA polymerase (eg, Taq DNA polymerase, Pfu DNA polymerase, etc.) as a cycle for about 20 to 40 cycles.

  The LAMP method does not require a denaturation reaction and can carry out an amplification reaction at a constant temperature of 60 to 65 ° C. In this method, the primer is designed to produce a loop structure at the primer binding site of the initial amplification product.

  The ICAN method is a method of amplifying DNA isothermally (about 50 to 60 ° C.) using an RNA / DNA chimera primer, a DNA polymerase having strand displacement activity and template exchange activity, and RNaseH.

The number of bases of the primer is usually 19 to 30 bases, preferably 21 to 24 bases.
The primers used in the amplification reaction are a plurality of barley discrimination primers, a plurality of hop discrimination primers, and a plurality of brewer's yeast discrimination primers. Specific examples of each primer are as follows.

<Barley discrimination primer>
Barley polygalacturonase gene, barley hordein gene, barley starch synthase gene, barley protein Z gene, barley α-amylase gene, barley β-amylase gene, barley lipoxygenase gene, barley macular mosaic virus-derived gene A barley powdery mildew-derived gene, a barley trypsin inhibitor gene, a barley cellulase gene, and a barley β-glucanase gene and a barley β-amylase gene detection primer. , A gene primer.

  Here, the barley hordein gene is a hordein A gene, a hordein B gene, a hordein C gene, or a combination thereof, and preferably a combination of three kinds of genes.

The barley protein Z gene is protein Z7, protein Z4 gene, or a combination thereof, preferably a combination of two genes.
The barley starch synthase gene is a barley starch granule-binding starch synthase gene.

  As a plurality of barley discrimination primers, for example, at least barley polygalacturonase gene, barley hordein gene (hordein A gene and hordein B gene, hordein C gene), barley starch synthase gene and barley protein Z gene (protein Z7 gene) And protein Z4 gene) detection primers, all of the tested barley varieties shown in the table can be identified, as is apparent from the results in Table 2 below.

  The plummer sequence for detecting the above gene is not particularly limited as long as it is a sequence characteristic of each gene. Examples of the primer pair (forward primer (F) and reverse primer (R)) for detecting the gene will be described below, but the gene detection primer is not limited to the specific primer illustrated.

  The barley polygalacturonase gene detection primer pair consists of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 23, and the barley hordein gene detection primer pair consists of SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 3, and SEQ ID NO: 25 and / or consisting of the base sequences of SEQ ID NO: 4 and SEQ ID NO: 26, and the primer pair for detecting the barley starch synthase gene consists of the base sequences of SEQ ID NO: 5 and SEQ ID NO: 27, and the primer for detecting the barley protein Z gene Primers for detection of barley α-amylase gene consisting of the base sequences of SEQ ID NO: 6 and SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 29, SEQ ID NO: 8 and SEQ ID NO: 30 and / or SEQ ID NO: 9 and SEQ ID NO: 31 The pair consists of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 32, and the primer pair for detection of the barley lipoxygenase gene is SEQ ID NO: 1. And the detection primer pair of the barley yellow mosaic virus-derived gene consists of the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 34, and the detection primer pair of the barley powdery mildew-derived gene has the sequence No. 13 and SEQ ID NO: 35 and / or SEQ ID NO: 14 and SEQ ID NO: 36, the barley trypsin inhibitor gene detection primer pair consists of SEQ ID NO: 15 and SEQ ID NO: 37, and the barley cellulase gene The detection primer pair consists of the nucleotide sequences of SEQ ID NO: 16 and SEQ ID NO: 38, and the detection primer pair of the barley β-glucanase gene consists of the nucleotide sequences of SEQ ID NO: 17 and SEQ ID NO: 39 (Table 1).

The gene sequences that are the basis of these specific primer sequences are available from the NCBI DNA Data Bank (GenBank), DDBJ (Japan DNA Data Bank), etc., where the DDBJ gene and registration number (base The relationship with (array) is as follows.
Barley polygalacturonase gene, EF427919
Barley hordein A gene, AF474373
Barley hordein B gene, Q40020
Barley hordein C gene, Q40020
Barley starch synthase gene, X07931
Barley protein Z7 gene, X95277
Barley protein Z4 gene, P06293
Barley α-amylase gene, AF427791
Barley lipoxygenase gene, L35931
Barley powdery mildew-derived gene, Q43492
Barley trypsin inhibitor gene, X65875
Barley β-glucanase gene, EU267181

  The primer for detecting the gene is preferably selected from sequences derived from the coding region of the gene (base sequence of the registration number) based on the registered base sequence, in addition to the specific primer sequence described above. Alternatively, it may be selected from a sequence derived from a non-coding region if the gene can be specifically detected. Examples of non-coding regions include intron regions, 3′-untranslated regions or 5′-untranslated regions.

<Primer for hop discrimination>
The primer for hop discrimination can be selected from, for example, a primer for detecting a hop gene belonging to an aroma, a primer for detecting a hop gene belonging to a fine aroma, and a primer for detecting a hop gene belonging to a bitter.

  Specific examples of the hop discrimination primer pair are selected from the primer pair of SEQ ID NO: 18 and SEQ ID NO: 40, the primer pair of SEQ ID NO: 19 and SEQ ID NO: 41, and the primer pair of SEQ ID NO: 20 and SEQ ID NO: 42. It is not limited to primers (Table 1).

  The hop gene used as the basis of the primer sequence exemplified above is described in, for example, Japanese Patent No. 4097288. In this patent document, for example, SEQ ID NO: 1 (CTATTTCGGCTAGTTCTGC), SEQ ID NO: 2 (CTATTTTGGCCAGTTTTGT), SEQ ID NO: 3 A number such as (AGCTGAGCAAGCTTCTTTG) is assigned. Therefore, in addition to the specific primer sequence described above, it is also possible to design a primer based on a registered base sequence, perform PCR, and select a primer sequence from the coding sequence or non-coding sequence of the DNA to be amplified.

<Primer for distinguishing beer yeast>
The primer for discriminating beer yeast can be selected from, for example, a primer that makes it possible to distinguish between upper fermenting yeast and lower fermenting yeast and a primer for detecting a yeast thioredoxin gene.

  Specific examples of the primer pair for discriminating beer yeast are selected from a primer pair consisting of the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 43 and a primer pair consisting of the nucleotide sequences of SEQ ID NO: 22 and SEQ ID NO: 44. It is not limited to primers (Table 1).

The brewer's yeast genes that serve as the basis for the primer sequences exemplified above are registered in DDBJ and GenBank, and given registration numbers such as AB300409 and U40843, for example. Therefore, in addition to the specific primer sequence described above, it is also possible to select a primer sequence from the coding sequence or non-coding sequence of the gene based on the registered base sequence.
In step (b) above, the DNA amplified in step (a) is detected.

  In general, DNA can be detected by electrophoresis. Electrophoresis may be performed under any electrophoresis conditions as long as the target DNA can be detected. In general, polyacrylamide gel, agarose gel, or the like can be used as the type of gel.

  As a specific example of the electrophoresis conditions, a 2% agarose gel was used, and the electrophoresis apparatus used was a mupid manufactured by Cosmo Bio Co., Ltd., and the electrophoresis was performed at a DC voltage of 100 V for 30 minutes.

  The amplification product of the gene can be detected as a band at a specific molecular weight size by subjecting it to electrophoresis using the predetermined electrophoresis conditions as described above. At this time, usually, electrophoresis is performed using an electrophoresis buffer containing ethidium bromide (EtBr), and then the gel is taken out and irradiated with ultraviolet rays, the band can be visualized. EtBr allows DNA to be detected because it has the property of entering between the double strands of DNA.

  In the step (c), barley and hops, which are raw materials used for the production of beer, based on the detection results of the barley, hop and a plurality of genes derived from brewer's yeast presumed to be present in the beer sample And the type of brewer's yeast.

  If necessary, the base sequence of the amplification product can be determined and aligned with the known base sequence of the target gene to confirm that the amplification product is the target gene.

  Table 3 described below shows the results of determining the presence or absence (+, −) of the target gene when PCR was performed using the above-described primers (Table 1) for 22 types of beer commercially available. According to this, all 22 types of beer tested, together with the beer raw materials used (barley variety (Table 2), types of brewer's yeast used for fermentation, types of hops as an auxiliary material), the present invention The results in Table 3 show that the determination can be made by the method. In particular, it is possible to distinguish barley varieties (16 types) as the main raw material. In addition, since the beer raw material and the beer product can be distinguished, the method of the present invention makes it possible to estimate the production conditions of beer from the raw material side.

2. In the second aspect, the present invention also provides a method for evaluating beer quality. In this method, the barley, brewer's yeast, and hops, which are the raw materials used in the production of beer, determined by the nucleic acid amplification method using the barley discriminating primer, the hop discriminating primer, and the brewer's yeast discriminating primer in the above method. A step of discriminating the type, and a step of comprehensively evaluating the quality of the beer's taste and foaming retention properties (further, such as throat squeezing) using the discrimination result.

  Barley discrimination primers are: barley polygalacturonase gene, barley hordein gene, barley starch synthase gene, barley protein Z gene, barley α-amylase gene, barley β-amylase gene, barley lipoxygenase gene, barley macular mosaic virus-derived gene, barley Primers for all kinds of genes selected from the group consisting of detection genes for powdery mildew-derived genes, barley trypsin inhibitor gene, barley cellulase gene, and barley β-glucanase gene. A primer for detecting a hop gene belonging to an aroma, a primer for detecting a hop gene belonging to a fine aroma, and a primer for detecting a hop gene belonging to a bitter, and a primer for discriminating brewer's yeast. A 酵酵 mother and bottom fermenting yeast is a detection primer of the primer and yeast thioredoxin gene to allow identification.

  Specifically, the barley polygalacturonase gene detection primer pair is composed of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 23, and the barley hordein gene detection primer pair is SEQ ID NO: 2, SEQ ID NO: 24, sequence Bare protein Z consists of the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 25 and / or SEQ ID NO: 4 and SEQ ID NO: 26, and the primer pair for detection of barley starch synthase gene consists of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 27 The gene detection primer pair comprises the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 29, SEQ ID NO: 8 and SEQ ID NO: 30 and / or SEQ ID NO: 9 and SEQ ID NO: 31, and barley α-amylase The gene detection primer pair is composed of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 32, and the barley lipoxygenase gene detection primer pair is A primer pair for detecting a barley yellow mosaic virus-derived gene consisting of the base sequences of SEQ ID NO: 11 and SEQ ID NO: 33, comprising a base sequence of SEQ ID NO: 12 and SEQ ID NO: 34, and a primer pair for detecting a barley powdery mildew-derived gene Is composed of the base sequences of SEQ ID NO: 13 and SEQ ID NO: 35 and / or SEQ ID NO: 14 and SEQ ID NO: 36, and the primer pair for detection of the barley trypsin inhibitor gene is composed of the base sequences of SEQ ID NO: 15 and SEQ ID NO: 37, The cellulase gene detection primer pair consists of the base sequence of SEQ ID NO: 16 and SEQ ID NO: 38, and the barley β-glucanase gene detection primer pair consists of the base sequence of SEQ ID NO: 17 and SEQ ID NO: 39, and hop discrimination Primer for primer consisting of the base sequence of SEQ ID NO: 18 and SEQ ID NO: 40, SEQ ID NO: 19 and a primer pair consisting of the base sequence of SEQ ID NO: 41, a primer pair consisting of the base sequence of SEQ ID NO: 20 and SEQ ID NO: 42, and the primer for discriminating beer yeast is the base sequence of SEQ ID NO: 21 and SEQ ID NO: 43 And a primer pair consisting of the nucleotide sequences of SEQ ID NO: 22 and SEQ ID NO: 44.

  Primers for discriminating varieties or brewing aptitudes of barley are, for example, two or more, preferably 7 or more to all types of primers of SEQ ID NOs: 1 to 17 in Table 1, hop cultivar discrimination or brewing aptitude The primer for discrimination is, for example, two or more kinds of primers of SEQ ID NOS: 18, 19, and 20 in Table 1, and the primer for discriminating the strain of beer yeast or beer brewing suitability is, for example, SEQ ID NO: 21, Two of 22 primers. Preferably, all the primers listed in Table 1 for barley, hops and brewer's yeast are used.

  The results of the presence or absence of amplified DNA for the presence of a plurality of genes derived from barley, hops and brewer's yeast used to discriminate beer ingredients and products, bitterness, sourness, astringency, umami, foam in 22 types of beer For example, the results shown in FIGS. 8, 9 and 11 are obtained when graphed in combination with the degree of characteristics such as the holding force. This means that the quality of beer's taste and foam retention (further, etc.) can be estimated and evaluated comprehensively using the discrimination results obtained by the method according to 1 of the present invention. It shows what you can do.

  That is, as a specific example of the quality evaluation of beer in the present invention, in addition to the sensory test by humans, the change in membrane potential accompanying the adsorption of the taste component to the artificial lipid membrane is electrically detected and evaluated as taste recognition data Quality evaluation using bitterness, sourness, astringency, umami and the like as objective variables by a taste recognition device (for example, manufactured by Intelligent Sensor Technology).

  Furthermore, as an example of the quality evaluation of beer in the present invention, there can be mentioned a quality evaluation using as an index the foam holding power when beer is poured into a container.

  Specifically, as an example of the quality evaluation of beer in the present invention, the presence or absence of DNA amplification by PCR using template DNA prepared from beer with beer taste and beer foam retention by a taste recognition device as target variables Is binarized (for example, 1 for amplified and 0 for non-amplified) as an explanatory variable, and beer taste and foam retention by multivariate analysis such as multiple regression analysis and principal component analysis It can be mentioned that the quality of beer such as is evaluated (see FIGS. 8 to 11).

  FIG. 8 shows a two-dimensional display of the taste measurement results of various beer samples by the taste recognition device. The number on the vertical axis shows the degree of bitterness, and the horizontal axis shows the degree of sourness. As the positive (+) number increases, the bitterness or sourness increases, and conversely, the negative (-) number increases. Bitterness or sourness decreases. Numbers 1 to 22 represent the numbers of the 22 types of beer described in Table 3. For each beer, the bitterness and sourness were measured using a taste recognition device, and the respective values were plotted. In the figure, (1) is the characteristic range of Lager beer, (2) is the characteristic range of Low-mal beer, (3) is the characteristic range of Ale beer, and (4) is the characteristic range of draft beer. It is. From FIG. 8, it can be seen that the beers of (1), (2), (3) and (4) each have a characteristic taste in sourness and bitterness, as has been said conventionally.

FIG. 9 shows the relationship between the taste (sour taste, bitterness and astringency) by the taste recognition apparatus and the PCR results (amplification results (ie, + or −) in Table 3). About three graphs, a vertical axis | shaft shows the degree of the actual value (measurement value by a taste recognition apparatus) of sourness, bitterness, or astringency, and a horizontal axis shows the degree of the estimated value of sourness, bitterness, or astringency. As the positive (+) number increases, sourness, bitterness or astringency increases. Conversely, as the negative (−) number increases, sourness, bitterness or astringency decreases. Numbers 1 to 22 represent the numbers of the 22 types of beer described in Table 3. R ² represents a coefficient of determination. The estimated values in these graphs are values calculated by substituting the amplification results (+ or −) by PCR primers in Table 3 into the following estimation formula.

  The sourness estimation formula is 0.54 (6) +9.22 (7) +3.19 (21) +0.61 (15) -4.84 (9) -4.46 (18) -3.53 (22 ) -0.49 (8) -0.11 (20) -0.58 (19) -1.98 (1 (or 1a or 1F))-0.55 (10) -0.17 (11)- 2.68 (17) -2.05. Here, the numbers in parentheses are the DNA marker numbers or primer numbers in Table 1, where 1 is when the DNA is amplified, 0 when it is not amplified, and the PCR amplification result in Table 3 is positive (+). In this case, “1” is assigned in parentheses, and in the case of minus (−), “0” is assigned in parentheses.

  The estimation formula of bitterness is 2.61 (18) +1.59 (22) +0.45 (20) +1.91 (1 (or 1a or 1F)) + 0.05 (15) +3.72 (17) -1 .41 (6) -1.83 (7) -0.36 (9) -1.85 (21) -0.67 (8) -2.78 (19) -2.12 (10) -1. 47 (11) +0.10. Here, the numbers in parentheses are the DNA marker numbers or primer numbers in Table 1, where 1 is when the DNA is amplified, 0 when it is not amplified, and the PCR amplification result in Table 3 is positive (+). In this case, “1” is assigned in parentheses, and in the case of minus (−), “0” is assigned in parentheses.

  The astringent estimation formula is 0.06 (9) +0.91 (18) +0.41 (22) +0.13 (8) +0.06 (20) +0.52 (1 (or 1a or 1F)) + 0. 13 (15) +0.93 (17) −0.37 (6) −0.39 (7) −0.35 (21) −0.78 (19) −0.06 (10) −0.16 ( 11) -0.82. Here, the numbers in parentheses are the DNA marker numbers or primer numbers in Table 1, where 1 is when the DNA is amplified, 0 when it is not amplified, and the PCR amplification result in Table 3 is positive (+). In this case, “1” is assigned in parentheses, and in the case of minus (−), “0” is assigned in parentheses.

  From FIG. 9, the sourness, bitterness or astringency of various beer samples is estimated by multiple regression analysis based on the results of PCR using the DNA prepared from the beer sample by the method according to the present invention as a template and using various primers shown in the present invention. It turns out that it is possible.

FIG. 11 is a graph showing estimation of beer foam retention by PCR. The vertical axis is an actual measurement value, and the horizontal axis is an estimated value calculated from the following estimation formula. R ² represents a coefficient of determination. The numbers in the graph represent the numbers of 22 types of beer shown in Table 3. The formula for estimating the stability of the foam is 140.65 (7) +68.18 (11) +26.26 (9) +12.84 (21) +111.22 (22) +11.00 (8) +1.37 (15 ) -1.98 (6) -7.31 (18) -22.75 (20) -20.95 (19) -13.70 (1 (or 1a or 1F))-8.81 (10)- 40.36 (17) +122.36, where the number in parentheses represents the DNA marker number or primer number in Table 1, with 1 when the DNA is amplified and 0 when the DNA is not amplified. If the PCR amplification result of 3 is plus (+), “1” is substituted in parentheses, and if it is minus (−), “0” is substituted in parentheses.

  From FIG. 11, it is possible to estimate the bubble stability of beer by multiple regression analysis based on PCR results using the primers shown in the present invention using DNA prepared from various beer samples by the method of the present invention as a template. I know that there is.

  So far, protein Z (serpin) in barley has been reported to affect the foaming properties of beer (T. Imure et al., Theor. Appl. Genetics, 2011, 122: 199-210) and lipoxygenase. Is related to lipid oxidation and to beer flavor deterioration. On the other hand, according to the method of the present invention, since many genes of barley, brewer's yeast and hop are targeted, the quality of beer can be evaluated comprehensively.

3. Kit In the third aspect, the present invention also provides a barley discrimination primer for use in the method described in 1 (beer raw material, production conditions or product discrimination) and 2 (beer quality evaluation), A kit comprising a hop discrimination primer and a beer yeast discrimination primer, wherein the barley discrimination primer comprises a barley polygalacturonase gene, a barley hordein gene, a barley starch synthase gene, a barley protein Z gene, a barley α-amylase gene, 2 selected from the group consisting of primers for detecting barley β-amylase gene, barley lipoxygenase gene, barley yellow mosaic virus-derived gene, barley powdery mildew-derived gene, barley trypsin inhibitor gene, barley cellulase gene, and barley β-glucanase gene Genes from all species to all species A primer for detecting a hop gene belonging to an aroma, a primer for detecting a hop gene belonging to a fine aroma, and a primer for detecting a hop gene belonging to a bitter, and a brewer's yeast Provided is a kit in which the discrimination primer is a primer that makes it possible to distinguish between top fermenting yeast and bottom fermenting yeast and a primer for detecting a yeast thioredoxin gene.

  The barley discrimination primer preferably contains at least a detection primer for barley polygalacturonase gene, barley hordein gene, barley starch synthase gene and barley protein Z gene.

  Specifically, the primer pairs for detection of barley polygalacturonase gene are SEQ ID NO: 1 and SEQ ID NO: 23, and the primer pair for detection of barley hordein gene is SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 3 and SEQ ID NO: 25 and / or SEQ ID NO: 4 and SEQ ID NO: 26, and the primer pair for detection of barley starch synthase gene is SEQ ID NO: 5 and SEQ ID NO: 27, and the primer pair for detection of barley protein Z gene Are SEQ ID NO: 6 and SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 29, SEQ ID NO: 8 and SEQ ID NO: 30 and / or SEQ ID NO: 9 and SEQ ID NO: 31, and the primer pair for detection of barley α-amylase gene is a sequence SEQ ID NO: 10 and SEQ ID NO: 32, and the detection primer pair for the barley lipoxygenase gene is SEQ ID NO: 11 and SEQ ID NO: 33. A primer pair for detecting a gene derived from zygovirus is SEQ ID NO: 12 and SEQ ID NO: 34, and a primer pair for detecting a gene derived from barley powdery mildew is SEQ ID NO: 13 and SEQ ID NO: 35 and / or SEQ ID NO: 14 and SEQ ID NO: 36, the barley trypsin inhibitor gene detection primer pair is SEQ ID NO: 15 and SEQ ID NO: 37, the barley cellulase gene detection primer pair is SEQ ID NO: 16 and SEQ ID NO: 38, and barley β-glucanase The gene detection primer pair is SEQ ID NO: 17 and SEQ ID NO: 39, and the hop discrimination primer belongs to a hop gene detection primer belonging to an aroma, a hop gene detection primer belonging to a fine aroma, and a bitter It is a primer for detection of hop genes, and beer yeast size Use primer, a primer pair of primers and SEQ ID NO: 22 and SEQ ID NO: 44 SEQ ID NO: 21 and SEQ ID NO: 43.

  The primer generally has a length of 19 to 30 bases, preferably 21 to 24 bases, and can be synthesized using, for example, an automatic nucleic acid synthesizer utilizing the phosphoramidite method.

  The kit includes the above-mentioned primers individually or appropriately combined and packaged. In addition, if necessary, a DNA purification reagent (eg, extraction buffer such as TE, phenol, chloroform, isoamyl alcohol from beer samples). , Ethanol, (iso) propanol and other extraction / precipitation solvents, magnetic beads and other DNA adsorbent solid phase carriers, elution buffers, etc., and nucleic acid amplification reagents such as PCR (buffers, heat-resistant DNA polymerases, dNTPs (N = A, T, C, G), etc.), and electrophoresis reagents (gel preparation reagents, electrophoresis buffers, detection reagents such as ethidium bromide, etc.) can be included as appropriate.

  Embodiments of the present invention will be described below by way of examples. However, the present invention is not limited to these examples.

[Example 1]
<Advantage of preparation method of template DNA for PCR from beer>
Conventional methods for preparing template DNA for PCR: (1) CTAB method, (2) alkali phenol method, (3) ISOPLANT kit method, (4) Qiagen kit method, (5) Takara kit method, (6) Japan Compared with the method described in Japanese Patent No. 4953058, the “enzymatic method / bead method combined method”, which is a template DNA preparation method of the present invention, can prepare a high molecular weight template DNA. As shown, it was deposited in the form of lint. Moreover, it was possible to amplify and discriminate the raw barley, the yeast that is a fermenting microorganism, and the hop DNA that is a secondary raw material by PCR, which have a high degree of DNA purification and remain in beer in trace amounts.

  The specific template DNA preparation method is as follows. Dissolve the powder obtained by freeze-drying various sample beers in a DNA dissolution buffer, act on heat-resistant amylase and proteolytic enzyme to decompose starch, starch degradation products and protein, then centrifuge and remove the supernatant. Was purified by phenol treatment and chloroform / isoamyl alcohol mixed solvent (25: 1) treatment, followed by alcohol (ethanol) precipitation, and then DNA dissolved in 70% ethanol was alcohol precipitated again while cooling with ice water. After dissolving in TE, adsorbing DNA to magnetic beads, separating the beads from the solution portion with a magnet, washing the beads with a buffer containing salt, and then washing the glass beads or the magnetic beads with a DNA eluate DNA is desorbed from the template to make template DNA, and a barley discrimination primer ( Tachi synthase, results PCR was performed in SEQ ID NO: 5) the presence of Table 1 shown in FIGS.

  As an example of comparison of the template DNA extraction and purification method with the conventional method, FIG. 2 shows a template extracted from beer by commercially available DNA extraction kits ISOPLANT I, ISOPLANT II (Nippon Gene) and DNEASY (Qiagen). The results of PCR using DNA and template DNA extracted and purified from beer by the method of the present invention using a starch synthase primer are shown. In the case of template DNA prepared using a commercially available DNA extraction kit, amplified DNA does not appear, whereas in the case of template DNA extracted and purified by the method of the present invention, it is the same as in the case of template DNA directly extracted and purified from barley. Amplified DNA (arrow in FIG. 2) appeared.

[Example 2]
<Detection of various barley protein serpin DNA>
Using DNA prepared from various barleys (16 barley varieties shown in Table 3 below) as a template, as a barley discrimination primer, a primer derived from the serpin gene, which is a major protein of barley (SEQ ID NOs: 6, 7, 8, 9) PCR was performed in the presence of coexistence. As a result, as shown in FIG. 3, a serpin amplification band was detected in most barley. From this agarose gel, a DNA band corresponding to the barley serpin gene was cut out and cloned, and the nucleotide sequence was examined. As a result, as shown in FIG. 5, it coincided with the nucleotide sequence of barley serpin.

[Example 3]
<Detection of various barley DNA in beer>
In the same manner as in Example 1, DNA prepared from sample beer was used as a template, and as components affecting beer quality, the barley lipoxygenase gene (SEQ ID NO: 11 in Table 1), β-glucanase gene (SEQ ID NO: 17), hordein A PCR was performed in the presence of primers derived from (SEQ ID NO: 2) and hordein C (SEQ ID NO: 4). As a result, as shown in FIG. 4, amplification bands of the respective genes were detected in several types of beer.

  Similarly, as shown in Table 2, by using 18 kinds of DNA markers, it was possible to distinguish all 16 kinds of barley from each other. Here, the primers 1a and 1b in Table 2 are composed of the sequences of SEQ ID NOS: 1 and 23 in Table 1, respectively.

[Example 4]
<Amplification of hop DNA in beer>
PCR was performed in the same manner as in Example 1 using DNA prepared from sample beer as a template, and coexisting with a hop gene-derived primer (SEQ ID NOs: 18, 19, and 20 in Table 1) as a hop detection primer. As a result, as shown in Table 3 and FIG. 6, hop amplification bands were detected in several types of beer.

[Example 5]
<Amplification of DNA of brewer's yeast in beer>
In the same manner as in Example 1, DNA prepared from sample beer was used as a template, and PCR was performed in the presence of primers derived from a brewer's yeast gene (SEQ ID NOs: 21 and 22 in Table 1) as primers for distinguishing brewer's yeast. As a result, as shown in Table 3, amplified DNA bands derived from yeast genes were detected in several types of beer. As shown in FIG. 7, DNA extracted and purified from beer was used as a template, and coexisting with yeast gene thioredoxin-related primers. The base sequence of the DNA amplified by PCR matched the sequence described in the literature of the base sequence of yeast thioredoxin.

[Example 6]
<Distinguishing examples of various beers around the world>
Commercially available foreign beer, Czech budova, US Brooklyn lager, Belgian Stella Artois, Tahiti hinano, Dutch cloche, Thai shinha, Danish Carlsberg, British frers London pride, German Paulaner hfe Weiss , Australian VB Victoria Bitter, Mexican Sol, American Primo, German Erdinger Weiss Beer, British Dutch Original Organic Dolby Ale, Peruvian Crystal, 2 Domestic Sapporo Beers, 1 Suntory Beer and 1 Kirin Beer A seed was used. As shown in FIG. 4 and Table 3, each beer shows characteristic results in barley type, yeast type, barley enzyme, hop type, etc., and identifies all 22 types of beer tested. Was possible.

[Example 7]
<Evaluation of taste by taste recognition apparatus and estimation of taste by PCR results in Table 3>
Twenty-two types of beer shown in Table 3 were used as samples, and the taste (bitterness, sourness, astringency) was evaluated using the taste sensor SA202 of Intelligent Sensor Technology. As an example, FIG. 8 shows a two-dimensional display of the evaluation results obtained by the taste recognition device for sourness and bitterness. In addition, multiple regression analysis (Evaluation formulas for sourness, bitterness, and astringency) with the taste evaluation results of acidity, bitterness, and astringency measured by the taste recognition device as objective variables and the PCR results shown in Table 3 as explanatory variables (Multiple regression equation) (above) was used. The coefficient of determination for sourness is 0.75, the coefficient of determination for bitterness is 0.68, and the coefficient of determination for astringency is 0.76, which are high values. By multivariate analysis of PCR results using template DNA extracted and purified from beer It was shown that the taste of beer, such as bitterness, acidity and astringency can be estimated.

[Example 8]
<Estimation by beer foam retention test and PCR results>
Using 22 types of beer shown in Table 3 as samples, the foaming property of beer was quantitatively evaluated by the method shown in FIG. That is, with a beer temperature constant at room temperature of 25 ° C., a fixed amount (250 mL) of beer is placed in the upper beaker, 100 mL of beer is poured into the lower graduated cylinder with a constant pressure difference by siphon, and the volume of bubbles after a fixed time Was measured as the foam retention of the sample beer.

  Using this beer foaming property as an objective variable and the PCR results in Table 3 as explanatory variables, a multiple regression analysis (using the foam stability estimation formula (multiple regression equation) (above)) was performed, and the dispersion shown in FIG. Got the figure. The coefficient of determination of this multiple regression analysis was 0.87, and it became possible to estimate the foam retention of the beer by performing multiple regression analysis on the PCR results obtained from the DNA template extracted and purified from beer.

[Example 9]
<Relationship between sensory test results of beer and quality estimation by PCR>
Using the various beers shown in Table 3 as samples, the comprehensive evaluation results of sensory tests conducted by 8 expert researchers were used as objective variables, and the relationship with the PCR results shown in Table 3 was subjected to multiple regression analysis in the same manner as described above. As a result, a determination coefficient of 0.82 was obtained. As a result, it was shown that the quality estimation of beer by the PCR method in the present invention can also be applied to sensory test results.

  The present invention prepares high-purity template DNA for PCR using beer as a sample with high efficiency, and comprehensively estimates the quality of the beer by discriminating each of barley, yeast, and hops. This technology has high utility value for the beer brewing industry, inspection institutions, and the barley, yeast and hop breeding, production and sales industries.

SEQ ID NOs: 1-46: Primers

Claims (23)

A method for discriminating beer raw materials or products, the method comprising: (a) using a DNA extracted and purified from a beer sample as a template, a plurality of barley discrimination primers, a plurality of hop discrimination primers and a plurality of beer yeast discrimination A step of amplifying the DNA using each primer group consisting of primers separately or simultaneously, a step of detecting the DNA amplified in step (a), (c) a result of detecting the DNA in step (b) And determining the type of barley, brewer's yeast, and hops, which are raw materials used in the production of beer, based on the method.   Barley polygalacturonase gene, barley hordein gene, barley starch synthase gene, barley protein Z gene, barley α-amylase gene, barley β-amylase gene, barley lipoxygenase gene, barley macular mosaic virus-derived gene 2. A primer for genes of all kinds from two or more selected from the group consisting of primers for detecting barley powdery mildew-derived genes, barley trypsin inhibitor genes, barley cellulase genes, and barley β-glucanase genes. The method described in 1.   The method according to claim 2, wherein the barley hordein gene is a hordein A gene, a hordein B gene, a hordein C gene, or a combination thereof.   The method according to claim 2 or 3, wherein the barley protein Z gene is a protein Z7 gene, a protein Z4 gene, or a combination thereof.   The method according to any one of claims 2 to 4, wherein the barley starch synthase gene is a barley starch granule-binding starch synthase gene.   The plurality of barley discrimination primers include at least primers for detecting barley polygalacturonase gene, barley hordein gene, barley starch synthase gene, and barley protein Z gene. Method.   The barley polygalacturonase gene detection primer pair consists of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 23, and the barley hordein gene detection primer pair includes SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 3 and SEQ ID NO: 25 and / or consisting of the base sequences of SEQ ID NO: 4 and SEQ ID NO: 26, and the primer pair for detecting the barley starch synthase gene consists of the base sequences of SEQ ID NO: 5 and SEQ ID NO: 27, and the primer for detecting the barley protein Z gene Primers for detection of barley α-amylase gene consisting of the base sequences of SEQ ID NO: 6 and SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 29, SEQ ID NO: 8 and SEQ ID NO: 30 and / or SEQ ID NO: 9 and SEQ ID NO: 31 The pair consists of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 32, and the primer pair for detection of the barley lipoxygenase gene is SEQ ID NO: 1. And the detection primer pair of the barley yellow mosaic virus-derived gene consists of the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 34, and the detection primer pair of the barley powdery mildew-derived gene has the sequence No. 13 and SEQ ID NO: 35 and / or SEQ ID NO: 14 and SEQ ID NO: 36, the barley trypsin inhibitor gene detection primer pair consists of SEQ ID NO: 15 and SEQ ID NO: 37, and the barley cellulase gene The detection primer pair consists of the nucleotide sequences of SEQ ID NO: 16 and SEQ ID NO: 38, and the detection primer pair of the barley β-glucanase gene consists of the nucleotide sequences of SEQ ID NO: 17 and SEQ ID NO: 39. The method of any one of these.   The plurality of hop discrimination primers are selected from a primer for detecting a hop gene belonging to an aroma, a primer for detecting a hop gene belonging to a fine aroma, and a primer for detecting a hop gene belonging to a bitter. 8. The method according to any one of items 7.   A plurality of hop discrimination primers are composed of a primer pair consisting of the base sequences of SEQ ID NO: 18 and SEQ ID NO: 40, a primer pair consisting of the base sequences of SEQ ID NO: 19 and SEQ ID NO: 41, and the base sequences of SEQ ID NO: 20 and SEQ ID NO: 42 9. The method of claim 8, wherein the method is selected from a primer pair.   The method according to any one of claims 1 to 9, wherein the plurality of beer yeast discrimination primers are selected from a primer that makes it possible to distinguish between upper fermentation yeast and lower fermentation yeast and a primer for detection of a yeast thioredoxin gene. .   The method according to claim 10, wherein the plurality of brewer's yeast discrimination primers are selected from a primer pair consisting of the base sequences of SEQ ID NO: 21 and SEQ ID NO: 43 and a primer pair consisting of the base sequences of SEQ ID NO: 22 and SEQ ID NO: 44. .   Template DNA, (a1) a step of causing starch-degrading enzyme and proteolytic enzyme to act on a beer sample to decompose starch, starch degradation product and protein, (a2) a step of recovering DNA from the treatment liquid of step (a1), And (a3) using a DNA-adsorbing solid phase carrier to adsorb the DNA from step (a2) and recovering the DNA from the solid phase carrier. The method according to any one of the above.   The method according to claim 12, wherein the amylolytic enzyme is a thermostable amylase.   The method according to claim 12 or 13, wherein the proteolytic enzyme is proteinase K.   The method according to any one of claims 12 to 14, wherein the DNA-adsorbing solid phase carrier is a glass bead or a magnetic bead.   A method for evaluating the quality of beer, wherein a beer is obtained by a nucleic acid amplification method using a barley discrimination primer, a hop discrimination primer and a brewer's yeast discrimination primer using the method according to any one of claims 1 to 15. Discriminating the types of barley, brewer's yeast and hops, which are the raw materials used in the manufacture of the product, and comprehensively evaluating the quality of beer's taste and foam retention using the discrimination results Including said method.   The method according to claim 16, wherein the taste of beer is bitter, sour, astringent and umami. The presence or absence of an amplified DNA band for deriving a discrimination result is binarized to be 1 and 0 to 0, and the quality of the beer is estimated by performing multivariate analysis using beer quality as an objective variable. Or the method according to 17;   Barley discrimination primers are: barley polygalacturonase gene, barley hordein gene, barley starch synthase gene, barley protein Z gene, barley α-amylase gene, barley β-amylase gene, barley lipoxygenase gene, barley macular mosaic virus-derived gene, barley Primers for all kinds of genes selected from the group consisting of detection genes for powdery mildew-derived genes, barley trypsin inhibitor gene, barley cellulase gene, and barley β-glucanase gene. A primer for detecting a hop gene belonging to an aroma, a primer for detecting a hop gene belonging to a fine aroma, and a primer for detecting a hop gene belonging to a bitter, and a primer for discriminating brewer's yeast. A detection primer of the primer and yeast thioredoxin genes that allow identifying and 酵酵 mother and bottom fermenting yeast, the method according to any one of claims 16 to 18.   The barley polygalacturonase gene detection primer pair consists of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 23, and the barley hordein gene detection primer pair includes SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 3 and SEQ ID NO: 25 and / or consisting of the base sequences of SEQ ID NO: 4 and SEQ ID NO: 26, and the primer pair for detecting the barley starch synthase gene consists of the base sequences of SEQ ID NO: 5 and SEQ ID NO: 27, and the primer for detecting the barley protein Z gene Primers for detection of barley α-amylase gene consisting of the base sequences of SEQ ID NO: 6 and SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 29, SEQ ID NO: 8 and SEQ ID NO: 30 and / or SEQ ID NO: 9 and SEQ ID NO: 31 The pair consists of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 32, and the primer pair for detection of the barley lipoxygenase gene is SEQ ID NO: 1. And the detection primer pair of the barley yellow mosaic virus-derived gene consists of the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 34, and the detection primer pair of the barley powdery mildew-derived gene has the sequence No. 13 and SEQ ID NO: 35 and / or SEQ ID NO: 14 and SEQ ID NO: 36, the barley trypsin inhibitor gene detection primer pair consists of SEQ ID NO: 15 and SEQ ID NO: 37, and the barley cellulase gene The detection primer pair consists of the base sequence of SEQ ID NO: 16 and SEQ ID NO: 38, and the detection primer pair of the barley β-glucanase gene consists of the base sequence of SEQ ID NO: 17 and SEQ ID NO: 39, and the hop discrimination primer , A primer pair consisting of the base sequences of SEQ ID NO: 18 and SEQ ID NO: 40, SEQ ID NO: 19 and sequence A primer pair consisting of the base sequence of No. 41, a primer pair consisting of the base sequences of SEQ ID NO: 20 and SEQ ID NO: 42, and a primer for discriminating beer yeasts consisting of the base sequences of SEQ ID NO: 21 and SEQ ID NO: 43 The method according to claim 19, wherein the pair is a primer pair consisting of the nucleotide sequences of SEQ ID NO: 22 and SEQ ID NO: 44.   A kit comprising a barley discrimination primer, a hop discrimination primer and a beer yeast discrimination primer for use in the method according to any one of claims 1 to 20, wherein the barley discrimination primer is a barley poly Galacturonase gene, barley hordein gene, barley starch synthase gene, barley protein Z gene, barley α-amylase gene, barley β-amylase gene, barley lipoxygenase gene, barley yellow mosaic virus-derived gene, barley powdery mildew-derived gene, barley trypsin A primer for two or more genes selected from the group consisting of a primer for detecting an inhibitor gene, a barley cellulase gene, and a barley β-glucanase gene, wherein the hop discrimination primer is a hop gene belonging to aroma. Detection primer, A primer for detecting a hop gene belonging to key aroma and a primer for detecting a hop gene belonging to bitter, and a primer for distinguishing beer yeast from bottom fermented yeast and bottom fermented yeast and yeast thioredoxin The kit, which is a primer for detecting a gene.   The kit according to claim 21, wherein the barley discrimination primer includes at least primers for detecting a barley polygalacturonase gene, a barley hordein gene, a barley starch synthase gene, and a barley protein Z gene.   The barley polygalacturonase gene detection primer pair consists of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 23, and the barley hordein gene detection primer pair includes SEQ ID NO: 2, SEQ ID NO: 24, SEQ ID NO: 3 and SEQ ID NO: 25 and / or consisting of the base sequences of SEQ ID NO: 4 and SEQ ID NO: 26, and the primer pair for detecting the barley starch synthase gene consists of the base sequences of SEQ ID NO: 5 and SEQ ID NO: 27, and the primer for detecting the barley protein Z gene Primers for detection of barley α-amylase gene consisting of the base sequences of SEQ ID NO: 6 and SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 29, SEQ ID NO: 8 and SEQ ID NO: 30 and / or SEQ ID NO: 9 and SEQ ID NO: 31 The pair consists of the nucleotide sequences of SEQ ID NO: 10 and SEQ ID NO: 32, and the primer pair for detection of the barley lipoxygenase gene is SEQ ID NO: 1. And the detection primer pair of the barley yellow mosaic virus-derived gene consists of the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 34, and the detection primer pair of the barley powdery mildew-derived gene has the sequence No. 13 and SEQ ID NO: 35 and / or SEQ ID NO: 14 and SEQ ID NO: 36, the barley trypsin inhibitor gene detection primer pair consists of SEQ ID NO: 15 and SEQ ID NO: 37, and the barley cellulase gene The detection primer pair consists of the base sequence of SEQ ID NO: 16 and SEQ ID NO: 38, and the detection primer pair of the barley β-glucanase gene consists of the base sequence of SEQ ID NO: 17 and SEQ ID NO: 39, and the hop discrimination primer , A primer pair consisting of the base sequences of SEQ ID NO: 18 and SEQ ID NO: 40, SEQ ID NO: 19 and sequence A primer pair consisting of the base sequence of No. 41, a primer pair consisting of the base sequences of SEQ ID NO: 20 and SEQ ID NO: 42, and a primer for discriminating beer yeasts consisting of the base sequences of SEQ ID NO: 21 and SEQ ID NO: 43 The kit according to claim 21 or 22, wherein the kit is a pair and a primer pair comprising the nucleotide sequences of SEQ ID NO: 22 and SEQ ID NO: 44.

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