JP4953058B2 - Method of distinguishing raw material plants in brewed sake - Google Patents

Description

  The present invention relates to a method for discriminating DNA of a plant plant in brewed sake.

  DNA discrimination technology for raw material varieties such as cooked rice and rice cake is being developed (Patent Document 1, Patent Document 2, Non-Patent Document 3). In the case of cooked rice, amylase and protease are allowed to act (hereinafter referred to as “enzymatic method”), DNA is extracted and purified by cetyltrimethylammonium bromide (CTAB) treatment and phenol treatment, and this is used as a template DNA as a primer. Is amplified by PCR, electrophoresed, and the rice varieties are identified based on the DNA polymorphism (Patent Document 1). In the case of cocoons, lyophilize the cocoons into powder, extract and purify the DNA by the enzyme method, CTAB treatment, and phenol treatment, and amplify them by PCR using primers as template DNA and perform electrophoresis. Thus, the raw material varieties of salmon are identified based on DNA polymorphism (Patent Document 2).

  In the case of brewed sake such as sake and wine, the manufacturer's technology, the type of microorganism used for fermentation, or the production conditions have a great influence on the quality. It is known to affect. In the case of sake, sake brewing suitable rice such as Yamada Nishiki and Hyakumangoku is known, and many high-grade sakes are produced using these brewing suitable rice as raw materials, and the varieties of raw rice are listed on the packaging label There are many cases. If it is possible to confirm whether the varieties listed on the packaging label and the varieties actually used are the same, it is possible to prevent / suppress the counterfeiting of the labeling of the premium liquor. Furthermore, if the relationship between the quality of liquor and the raw material plant or the raw material varieties can be examined, it can be used for future development and quality improvement of brewed sake.

  Therefore, there is a need for a technique that can identify the type or variety of the raw material plant used in brewed sake as a sample.

  However, fermented products such as sake and wine are originally liquid, have a high water content, have little solids, and the rice DNA is also degraded by microorganisms during the production process (fermentation stage). It has been considered impossible to apply the plant DNA variety discrimination technology.

  In addition, in the detection of genetically modified plants (GMO) and their processed products, the transgene is detected by PCR using primers designed based on the sequence of the transgene, but in the case of fermented processed products such as soy sauce. It has been impossible to detect the presence or absence of genetic recombination due to the degradation of DNA in the fermentation process and the presence of DNA from fermenting microorganisms.

  In other words, in order to determine the type or variety of the raw material plant by DNA analysis using a brewed liquor product available at the distribution and consumption stage as a sample, a solid containing a large amount of the DNA of the raw material plant does not pass through the fermentation process. Unlike rice and rice bran, conditions that do not cause degradation, denaturation, polymerization, etc. due to heating of trace amounts of raw plant-derived DNA or fragments remaining in the liquid while being degraded by microorganisms during fermentation To the concentration required for primer binding in PCR (annealing).

  Unlike rice and koji, in the case of brewed sake, in addition to the target DNA, koji molds and yeast cells or their residues coexist in addition to the target DNA. Certain starches, proteins, lipids, cell walls, etc. are also decomposed during the fermentation process, reaction between components proceeds, etc., and it has a very complex composition, separating sample DNA from contaminants such as carbohydrates, proteins, and lipids. In order to obtain high-quality DNA by purification, it is necessary to further devise the enzyme method.

  In the case of brewed liquor, unlike the case of cooked rice and koji in the previous reports, the coexistence of koji mold, yeast, hops, grape skin, etc. contains a large amount of pigment components such as polyphenols. Furthermore, reaction between components including Maillard reaction occurs by heat sterilization of raw materials, fermentation and fermentation stop treatment such as “burning”, and yellow and reddish brown pigment components increase. Many of pigment components such as polyphenol and melanoidin have enzyme inhibitory action and inhibit PCR.

  In addition, in order to amplify the isolated plant DNA or fragment thereof by PCR and distinguish the source plant or cultivar by its polymorphism, do not amplify mixed DNA such as Neisseria gonorrhoeae or yeast. Primers that only amplify DNA are required. Even when a partially degraded DNA fragment is used as a template, it is necessary to select a particularly suitable primer from among plant gene-specific primers so that amplified DNA that can distinguish between plant species can be obtained. .

  Furthermore, if the DNA derived from the plant plant or a fragment thereof can be used as a template for PCR and an identifiable DNA polymorphism appears in the presence of primers suitable for DNA amplification, PCR will be completed successfully. Appropriate reaction conditions must be set.

  In addition to many technical difficulties as described above, since the DNA discrimination technology of the raw rice and koji raw material varieties cannot be applied to brewed liquor as it is, the type or cultivar of the raw material plant is discriminated using only the brewed liquor as a sample. To do was something that those skilled in the art did not recall, and had many problems to be solved.

Japanese Patent No. 3048149 JP 2002-306173 A Nakamura Sumiko et al., Journal of Japanese Society for Agricultural Chemistry, 2004, Vol. 78, No. 10, p.984-993

  An object of the present invention is to provide a method for discriminating the type or variety of a raw material plant by DNA analysis using a brewed liquor product available at a distribution and consumption stage as a sample.

  As a result of intensive studies on the above problems, the present inventors have obtained a high concentration of the raw material plant-derived DNA or a fragment thereof that remains slightly in the brewed liquor, and further DNA degradation and denaturation occur in the process of increasing the concentration. As a result, a method has been found for making brewed liquor into a powder or thin film by non-heat concentration.

  In addition, in order to extract good-quality DNA from a sample, it was found that residual proteins can be thoroughly removed by repeating phenol treatment and the like immediately after heat-resistant amylase and protease K treatment.

  Furthermore, it has been found that high-quality PCR template DNA can be extracted and purified in high yields using brewed liquor as a raw material when combined with extraction and purification methods such as the CTAB method after degrading and removing starch and proteins by enzymatic methods. It was.

  It was found that the pigment component contained in the brewed liquor can be removed using an organic solvent when extracting and purifying the template DNA from the brewed liquor.

  Furthermore, in PCR for amplifying template DNA, it discovered that the primer derived from a plant gene was used as a primer used for PCR so that microorganism-derived DNA might not be amplified.

  That is, the present invention is summarized as follows.

  In the first aspect of the present invention, in the first embodiment, DNA is extracted and purified from brewed liquor, used as a template, PCR is performed in the presence of a plant gene-derived primer, and the type of the starting plant is discriminated based on the polymorphism of the amplified DNA. .

  In the second embodiment, DNA is extracted and purified from brewed liquor and used as a template, PCR is performed in the presence of a primer derived from a plant gene, and the source plant variety is discriminated based on the polymorphism of the amplified DNA.

  In another embodiment, the brew is sake, beer or wine.

  In another embodiment, the method further includes the step of pulverizing the brewed liquor by freeze drying, air flow drying, or spray drying.

  In another embodiment, the method further comprises the step of concentrating the brewed liquor into a thin film by cold air drying, cold air vacuum drying or vacuum centrifugal drying.

  In another embodiment, the DNA extraction / purification comprises methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, isoamyl alcohol, benzyl alcohol, acetone, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, chloroform and phenol. The pigment component is removed using one or more organic solvents selected from the group.

  In another embodiment, a thermostable amylolytic enzyme and / or proteolytic enzyme is allowed to act on the brewed liquor to degrade starch and / or protein, and the template DNA is extracted and purified.

  In another embodiment, when the brew is sake, the primers are A6 (SEQ ID NOs: 1 and 2), A7 (SEQ ID NOs: 3 and 4), A65 (SEQ ID NOs: 5 and 6), B1 (SEQ ID NO: 7 and 8), G28 (SEQ ID NO: 9 and 10), B43 (SEQ ID NO: 11 and 12), E30 (SEQ ID NO: 13 and 14), F6 (SEQ ID NO: 15 and 16), F30 (SEQ ID NO: 17 and 18), G4 (SEQ ID NO: 19 and 20), J6 (SEQ ID NO: 21 and 22), BL3 (SEQ ID NO: 23 and 24), M11 (SEQ ID NO: 25 and 26), Zein (SEQ ID NO: 27 and 28), P5 (SEQ ID NO: 29) And 30), S13 (SEQ ID NO: 31 and 32), M2CG (SEQ ID NO: 33 and 34), T16 (SEQ ID NO: 35 and 36), WK9 (SEQ ID NO: 37 and 38), P3 (SEQ ID NO: 39 and 40), SS1 (SEQ ID NO: 41 and 42), SS2 (SEQ ID NO: 43 and 44), SS2-2 (SEQ ID NO: 45 and 46), GBSS (SEQ ID NO: 47 and 48), DB (SEQ ID NO: 49 and 50), Pro10 (SEQ ID NO: 51 and 52), Pro13 (SEQ ID NO: 53) And 54), Glu (SEQ ID NO: 55 and 56), Mochi (SEQ ID NO: 57 and 58), BL3-3 (SEQ ID NO: 59 and 60), BL1 (SEQ ID NO: 61 and 62), BL2 (SEQ ID NO: 63 and 64) , BL4 (SEQ ID NO: 65 and 66), BL4-2 (SEQ ID NO: 67 and 68), BL65 (SEQ ID NO: 69 and 70), BL81 (SEQ ID NO: 71 and 72), BL6 (SEQ ID NO: 73 and 74), Mitol ( SEQ ID NO: 75 and 76), Chlo1 (SEQ ID NO: 77 and 78), GluSNP (SEQ ID NO: 79 and 80), BARGBSS (SEQ ID NO: 83 and 84), Ipoamy (SEQ ID NO: 87 and 88), Sugacss (SEQ ID NO: 89 and 90) ), B7 (SEQ ID NOs: 91 and 92), G22 (SEQ ID NOs: 93 and 94) and SBE (SEQ ID NOs: 100 and 101).

  In another embodiment, when the brew is beer, the primers are A6 (SEQ ID NO: 1 and 2), A7 (SEQ ID NO: 3 and 4), A65 (SEQ ID NO: 5 and 6), B1 (SEQ ID NO: 7 and 8), G28 (SEQ ID NO: 9 and 10), B43 (SEQ ID NO: 11 and 12), E30 (SEQ ID NO: 13 and 14), F6 (SEQ ID NO: 15 and 16), F30 (SEQ ID NO: 17 and 18), G4 (SEQ ID NO: 19 and 20), J6 (SEQ ID NO: 21 and 22), BL3 (SEQ ID NO: 23 and 24), M11 (SEQ ID NO: 25 and 26), Zein (SEQ ID NO: 27 and 28), P5 (SEQ ID NO: 29) And 30), S13 (SEQ ID NO: 31 and 32), M2CG (SEQ ID NO: 33 and 34), T16 (SEQ ID NO: 35 and 36), WK9 (SEQ ID NO: 37 and 38), P3 (SEQ ID NO: 39 and 40), SS1 (SEQ ID NO: 41 and 42), SS2 (SEQ ID NO: 43 and 44), SS2-2 (SEQ ID NO: 45 and 46), GBSS (SEQ ID NO: 47 and 48), DB (SEQ ID NO: 49 and 50), Pro10 (SEQ ID NO: 51 and 52), Pro13 (SEQ ID NO: 53) And 54), Glu (SEQ ID NO: 55 and 56), Mochi (SEQ ID NO: 57 and 58), BL3-3 (SEQ ID NO: 59 and 60), BL1 (SEQ ID NO: 61 and 62), BL2 (SEQ ID NO: 63 and 64) , BL4 (SEQ ID NO: 65 and 66), BL4-2 (SEQ ID NO: 67 and 68), BL65 (SEQ ID NO: 69 and 70), BL81 (SEQ ID NO: 71 and 72), BL6 (SEQ ID NO: 73 and 74), BARGBSS ( SEQ ID NO: 83 and 84), MAIZGBSS (SEQ ID NO: 85 and 86), B7 (SEQ ID NO: 91 and 92), G22 (SEQ ID NO: 93 and 94), SBE (SEQ ID NO: 100 and 101) and primers derived from soybean protein glycinin ( One or two or more primers selected from the group consisting of SEQ ID NOs: 106 and 107).

  In another embodiment, when the brew is wine, the primers are A6 (SEQ ID NOs: 1 and 2), A7 (SEQ ID NOs: 3 and 4), A65 (SEQ ID NOs: 5 and 6), B1 (SEQ ID NO: 7 and 8), G28 (SEQ ID NO: 9 and 10), B43 (SEQ ID NO: 11 and 12), E30 (SEQ ID NO: 13 and 14), F6 (SEQ ID NO: 15 and 16), F30 (SEQ ID NO: 17 and 18), G4 (SEQ ID NO: 19 and 20), J6 (SEQ ID NO: 21 and 22), BL3 (SEQ ID NO: 23 and 24), M11 (SEQ ID NO: 25 and 26), Zein (SEQ ID NO: 27 and 28), P5 (SEQ ID NO: 29) And 30), S13 (SEQ ID NO: 31 and 32), M2CG (SEQ ID NO: 33 and 34), T16 (SEQ ID NO: 35 and 36), WK9 (SEQ ID NO: 37 and 38), P3 (SEQ ID NO: 39 and 40), SS1 (SEQ ID NO: 41 and 42), SS2 (SEQ ID NO: 43 and 44), SS2-2 (SEQ ID NO: 45 and 46), GBSS (SEQ ID NO: 47 and 48), DB (SEQ ID NO: 49 and 50), Pro10 (SEQ ID NO: 51 and 52), Pro13 (SEQ ID NO: 53) And 54), Glu (SEQ ID NO: 55 and 56), Mochi (SEQ ID NO: 57 and 58), BL3-3 (SEQ ID NO: 59 and 60), BL1 (SEQ ID NO: 61 and 62), BL2 (SEQ ID NO: 63 and 64) , BL4 (SEQ ID NO: 65 and 66), BL4-2 (SEQ ID NO: 67 and 68), BL65 (SEQ ID NO: 69 and 70), BL81 (SEQ ID NO: 71 and 72), BL6 (SEQ ID NO: 73 and 74), VITGT ( SEQ ID NOs: 81 and 82), B7 (SEQ ID NOs: 91 and 92), G22 (SEQ ID NOs: 93 and 94), SBE (SEQ ID NOs: 100 and 101), primers derived from the grape mitochondrial cytochrome gene (SEQ ID NOs: 102 and 103) and grape shovel One or two or more types of primers selected from the group consisting of saccharide-binding protein gene-derived primers (SEQ ID NOs: 104 and 105).

  In another embodiment, the primer is part of a plant-derived repetitive sequence.

  In another embodiment, the polymorphism is discriminated by SNP discrimination.

According to the present invention, it is possible to discriminate the kind of raw material plant only from a sample of brewed liquor such as sake, wine, beer, etc., and it is possible to scientifically determine the difference between the display and the content raw material.
Further, since the brewed liquor is used as a sample and the varieties of the raw material plants are discriminated, disguise of the brewed liquor raw material display is prevented.
Furthermore, the relationship between the quality of the brewed liquor and the raw material plant is clarified, and the quality of the alcohol can be improved by selecting a high-quality raw material plant.

  The brewed liquor in the present invention refers to an alcoholic beverage produced by subjecting plant seeds and fruits as raw materials to alcohol fermentation by microorganisms such as yeast. The brewed liquor is not limited to the following, but includes, for example, Japanese sake, beer, wine, old sake, apple wine and the like.

  Sake is made by adding koji molds to rice to make the “source”, and adding mash mother and salt and water to yeast, and saccharification of starch and conversion of sugar to alcohol proceed in parallel. Refers to brewed liquor produced by fermentation. Brewing alcohol may be added for seasoning and cost reduction.

Beer refers to sake made from barley malt, water, and hops as main ingredients, and fermented by adding starch, rice, and other ingredients.
Wine refers to sake made by fermenting grape fruits with yeast.

The microorganism in the present invention refers to a microorganism that performs fermentation for converting a saccharide into alcohol, such as wine yeast in wine, koji mold and yeast in sake, beer yeast in beer, and examples include Saccharomyces cerevisiae , Saccharomyces carlsbergensis (Saccharomyces carlsbergensis), Saccharomyces Roukushi (Saccharomyces rouxii), Aspergillus oryzae (Aspergillus oryzae), Aspergillus niger (Aspergillus niger), Aspergillus kawachii (Aspergilluskawachii), Aspergillus Usami (Aspergillus usamii), etc. Can be mentioned.

  The raw material plant in the present invention refers to a plant that provides a saccharide raw material to fermenting microorganisms such as yeast and koji mold and serves as a raw material for alcohol production. The raw material plant is not limited to the following, and includes, for example, rice, barley, grape, wheat, corn, apple, hop and the like.

  The plant gene-derived DNA in the present invention refers to DNA that is peculiar to DNA such as rice and grapes and is not contained in microorganisms such as koji molds and yeasts, and examples thereof are related to starch synthase and plant proteins. Examples thereof include repetitive sequences in gene DNA, glycosyltransferase DNA, and plant DNA, particularly repetitive sequences in mycotondoria and chloroplast DNA.

  Below, the brewing liquor raw material discrimination | determination method of this invention is demonstrated in detail.

  The brewing liquor raw material discrimination method of the present invention comprises (A) a step of preparing DNA by extracting and purifying DNA from brewing liquor, (B) a step of performing PCR in the presence of a primer derived from a plant gene, and (C) amplification. A step of discriminating the type or variety of the source plant based on the DNA polymorphism.

Hereinafter, each step will be described.
(A) A step of preparing template DNA by extracting and purifying DNA from brewed sake
(a) Concentration of brewed sake The majority of brewed sake such as sake, beer and wine is water, about 82-84% for sake, 88-93% for beer, 87-89% for wine, Shaoxing ( About 79% of old liquor is water, and the solids content is extremely low (5th Japanese Food Standard Composition Table, Science and Technology Agency Resource Research Committee, 2000). Moreover, since most of the DNA in the raw plant and seeds is degraded by the DNA-degrading enzymes of Aspergillus or yeast during the production process, the template DNA at the concentration required for PCR primer binding (annealing) is obtained. For this purpose, a concentration step under conditions that do not cause denaturation or degradation of DNA is required.

  Therefore, in order to obtain a high concentration of the raw material plant-derived DNA or fragment thereof that remains slightly in the brewed liquor, the brewed liquor is dried using, for example, any one of freeze drying, airflow drying, and spray drying. Powder form.

  Freeze-drying is a method in which a frozen sample solution is decompressed under a high vacuum of, for example, about 0.133 Pa (0.1 Torr) or less, and is dried as a solid by sublimating water. For example, a freeze dryer such as Eyela FD-1 can be used.

  Airflow drying is airflow crushing, which is a method in which a sample is vibrated at a high frequency by pressure fluctuation caused by a high-speed flow of air and self-pulverized.

  Spray drying is a method of increasing the surface area by spraying a sample solution into a high-temperature air stream to dry it in a short time.

  Alternatively, the brewed liquor may be concentrated into a thin film by any one of cold air drying, cold air vacuum drying, and vacuum centrifugal drying.

  Cold air drying is a method of evaporating and drying a sample by sending air at normal temperature without heating the sample.

  Cold air vacuum drying is a method of promoting dryness of a sample by reducing the pressure using a water flow pump or a vacuum pump during cold air drying.

  The reduced-pressure centrifugal drying is a method of accelerating the drying of the sample by adding a centrifugal operation with a centrifuge instead of standing the sample in the reduced-pressure drying.

  According to these drying methods, water can be removed to form a powder or thin film without increasing the product temperature of the sample brewed liquor, so that the quality of the trace amount of template DNA remaining in the brewed liquor is not impaired, and It is suitable because it suppresses the production of browning substances such as melanoidin by heating, and it can be extracted with extremely high concentration efficiency. In particular, lyophilization is most suitable because DNA is not denatured in the concentrated powdering step because moisture is removed from the frozen brew sample by sublimation.

(b) Treatment with enzyme The brewed liquor concentrated in (a) is first treated with an amylolytic enzyme (amylase). Enzymes are used alone or in combination of two or more. This treatment can reduce the molecular weight of the starch component.

The amylase may be either prokaryotic or eukaryotic, and examples thereof include α-amylase, β-amylase, and glucoamylase. Any of these can be used in the present invention, but thermostable amylase is preferable, and amylase having an optimum temperature of 60 ° C. or higher, for example, α-amylase derived from Bacillus licheniformis is particularly preferable. This is because the reaction temperature can be increased, the reaction time can be shortened, the reaction yield can be increased, and other enzyme reactions, for example, mixed DNA degrading enzymes can be inhibited to suppress DNA degradation.

  When normal amylase is used, for example, sample 1 in which 100 to 1000 U / mg amylase is converted to an aqueous solution of 0.1 to 5 mg / ml and an appropriate buffer (for example, 0.1 M Tris-HCl buffer) is added to the powdered brew. 0.005 to 0.1 volume, preferably 0.01 to 0.05 volume, is added to the volume. The starch is decomposed by reacting at a reaction temperature of 30 to 45 ° C. for 10 minutes or longer, preferably 30 to 120 minutes.

  In the case of the present invention, the removal of starch and starch degradation products contained in the brewed liquor solids is extremely important in preparing a high-quality template DNA. Accordingly, it is preferable to use a heat-resistant α-amylase such as α-amylase derived from Bacillus licheniformis, and the reaction can be similarly performed at 60 to 85 ° C., for example. This inhibits the activity of the mixed DNA degrading enzyme and makes it possible to prepare a high-quality template DNA.

  Furthermore, in order to extract and purify high-quality template DNA from brewed liquor solids, the conventional template DNA extraction method (enzyme method) from cooked rice or rice cake has been improved. It is preferable to make it about 3 times as long as the case of the finished product. This improves DNA extraction even from brewed liquor solids that have undergone a fermentation process. The reaction temperature is, for example, 60 to 85 ° C, more preferably 80 ° C. As a result, the mixed DNA-degrading enzyme is more strongly inhibited and starch degradation by amylase is promoted. A reaction time of 15 minutes may be insufficient, and is preferably 30 minutes or more. However, even if the reaction is continued for 10 hours or more, the amount of starch degradation does not increase so much, and instead heat denaturation of DNA may be promoted.

  Next, an enzyme treatment is performed with a proteolytic enzyme (protease). Enzymes are used alone or in combination of two or more. This treatment can reduce the molecular weight of the protein component.

Examples of proteases include proteases derived from microorganisms such as bacteria, yeast and fungi, proteases derived from prokaryotes such as proteases derived from plants and proteases derived from animals. Examples of microorganism-derived proteases include subtilisin derived from Bacillus subtilis and milk-clotting enzyme derived from Rhizomucor pusillus . Examples of plant-derived proteases are papain, bromelain and ficin. Examples of animal-derived proteases are pepsin, trypsin, chymotrypsin, chymosin, cathepsin. In the present invention, any of these can be used, but a protease derived from Tritirachium album having high activity even in the presence of a surfactant (trade name protease K, manufactured by Worthington Biochemical) Is preferably used. There is an advantage that the activity is expressed even in the presence of sodium lauryl sulfate (SDS), agarose, acrylamide and the like. By reacting with a protease, a protein that interferes with DNA extraction in a brew sample can be decomposed and removed, and a DNA-degrading enzyme that is a protein is also decomposed by the protease to prevent DNA from being decomposed.

  When protease is used, for example, 10 to 1000 U / mg protease is made into an aqueous solution of 5 to 50 mg / ml, and 0.001 to 0.1 volume, preferably 0.02 to 0.05 volume, is added to 1 volume of the sample. The protein is degraded by reacting at -60 ° C for 10 minutes or longer, preferably 30-120 minutes. In order to solubilize proteins and improve the efficiency of enzyme action, a surfactant such as SDS or a pH stabilizer such as Tris-HCl buffer may be used in combination.

(c) DNA extraction and purification
DNA extraction can be performed by (b) phenol extraction method, phenol / chloroform extraction method, CTAB method, etc. following the treatment with enzyme. For example, ethyl alcohol or the like is added to the supernatant obtained by centrifuging the sample treated with the amylase and protease, solid-liquid separation such as centrifugation is performed, the degradation product is removed as a supernatant fraction, and the DNA is precipitated. Get as. Impurities such as proteins and lipids can be obtained by dissolving the precipitate fraction with Tris buffer in the presence of ethylenediaminetetraacetic acid (EDTA) and treating it with an organic solvent such as phenol or phenol / chloroform / isoamyl alcohol mixed solution (PCI solution). Remove. When using brewed liquor as a sample, it is preferable to thoroughly remove the mixture by repeating this phenol treatment and PCI treatment.

  When extracting and purifying template DNA from brewed sake, an organic solvent consisting of methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, isoamyl alcohol, benzyl alcohol, acetone, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide, chloroform and phenol It is preferred to remove the dye component using one or more organic solvents of the group. By adding this step, enzyme activity such as DNA polymerase, which plays an important role in PCR, is drastically reduced by pigment components, and a lot of amplified DNA can be obtained to distinguish plant types and varieties. Become.

  When the CTAB method is used following the enzyme method, for example, the 2 × CTAB solution (2% CTAB 100 mM Tris-HCl, pH 8, 1.4 M NaCl, 20 mM EDTA) previously heated to 65 ° C. is used. Transfer the protease treatment solution, for example, after incubation at 65 ° C. for 1 hour, cool to room temperature, add chloroform / isoamyl alcohol (24: 1), and mix with a rotator for 20 minutes. Next, the upper layer obtained by centrifugation at 10,000 rpm for 10 minutes is transferred to a new tube, and chloroform / isoamyl alcohol is added and mixed for 20 minutes. Next, the upper layer liquid that has been centrifuged at 10,000 rpm for 10 minutes is transferred to a new tube. Add 2-propanol to this solution and mix gently, then centrifuge at 15000 rpm for 5 minutes, remove the supernatant, add 70% ethanol, and wash the inner wall of the tube. Centrifuge for 5 minutes, remove the supernatant, and dry under reduced pressure. After drying, add 50 μl of TE buffer and place in a constant temperature bath at 55 ° C to completely dissolve the pellet.

  As described above, DNA is purified in a state that hardly contains starch and its degradation product, protein and its degradation product, lipid and its degradation product, by adding a phenol method, CTAB method, etc. following the enzymatic method. Can do.

(B) PCR with plant gene-derived primers
Using the template DNA obtained in (A) above, DNA is amplified by PCR. PCR is an abbreviation for Polymerase Chain Reaction. Double-stranded DNA is denatured at a high temperature of about 96 ° C. and separated into single strands (denaturation step), and then a primer is added to the single-stranded DNA. The DNA is bound to DNA (annealing step), and then DNA polymerase is used to extend the DNA from the binding portion using the single-stranded DNA as a template (extension step), and these three steps of denaturation, annealing, and extension are repeated n times. Thus, it refers to a reaction in which the number of molecules of the first DNA is amplified to 2 ⁿ molecules of the molecular length sandwiched between the primer binding sites. The PCR reaction conditions are, for example, about 90 to 98 ° C. for about 15 seconds to 5 minutes in the denaturation step, about 36 to 65 ° C. for about 30 seconds to 10 minutes in the annealing step, about 65 to 75 ° C. and about 30 in the extension step. The second to 10 minutes may be cycled about 30-50 times, but can be varied depending on the target DNA. Also, for example, 10-50 ng template DNA, 0.5-10 ng primer, 0.5-5 U DNA polymerase, 50-500 μM deoxyribonucleotide triphosphate mixture (dNTP), 1-5 μM magnesium chloride and 10-50 mM Use Tris-HCl buffer (pH 8). Commercially available products can be used as the DNA amplification device, such as PC-700 (manufactured by Astech Co., Ltd.), thermal cycler MP (manufactured by Takara Shuzo Co., Ltd.), thermal cycler dice (manufactured by Takara Bio Inc.), GeneAmp PCR System 9700 (AppliedBiosystems Co., Ltd.) etc. can be used.

Here, preparation of primers used for PCR will be described.
A primer refers to a DNA fragment used in the PCR reaction, and the primer fragment is based on the law of complementation in which A, which is a base constituting DNA, binds only to T and G only binds to single-stranded DNA. Since it only binds to the part of the base sequence that is complementary to the base sequence and DNA extension begins from there, the length of the DNA molecule amplified by PCR is determined by the part that is complementary to the primer used. In other words, by comparing the molecular lengths of the amplified DNAs by electrophoresis, it is possible to detect differences in the base sequence of the primer binding portion between plant species or cultivars. The primer used in the present invention is a primer derived from a pair of plant genes that can amplify a base sequence that shows distinction between species or varieties, and is obtained by performing PCR from DNA obtained from the brewed sake. It is designed based on a base sequence that is discriminated between species or varieties.

  Arbitrary random primers are used in PCR for obtaining primers. Random primer is a template that uses a DNA extracted from a brewed liquor sample as a template DNA to amplify by PCR and forms a double-stranded structure by binding complementary to single-stranded DNA. A primer that is the starting point for DNA replication. Usually a 10-20 mer nucleotide, a synthetic primer in which adenine (A), thymine (T), guanine (G) and cytosine (C) are bound to a random sequence. In the present invention, a commonly used random primer may be selected. For example, a 10-mer random primer commercially available from Operon or a DNA oligomer set commercially available from Wako Pure Chemical Industries, Ltd. (12 (Mer) and the like.

  Subsequently, electrophoresis of the amplified DNA obtained by PCR is performed. This is to detect a band indicating a base sequence that shows the distinguishability between species or varieties, which is a base for designing a target primer, among amplification products. Here, electrophoresis means that DNA amplified by PCR is separated by the difference in molecular weight of DNA when it moves in an agarose gel or polyacrylamide gel by being attracted by direct current charge, and this is brominated. An operation for detecting differences in amplified DNA as a band pattern by staining with ethidium and irradiating with ultraviolet rays.

  A pair of primers is prepared as follows from the band in which discrimination between the varieties detected in this way appears. That is, the band showing the distinctiveness is cut out from the gel, DNA is extracted and collected, and this is incorporated into Escherichia coli and proliferated. Next, a plasmid is extracted by the alkali miniprep method or the like, and is propagated by the PCR method using this as a template DNA, and the base sequence is determined by a DNA automatic sequencer.

  Primers are designed from the determined base sequence. In the PCR method using the random primer, the portion of the DNA derived from the brewed liquor sample that is the template should have the same or complementary (homologous) sequence to the random primer. . In other words, a DNA base sequence that is cut out from this template DNA and extracted and has high species discriminating ability (this is the base of the primer in PCR) has the same or homologous sequence as the random primer at both ends. Become. Therefore, it is possible to design a primer useful for determining the type or variety of a raw material plant in a brewed liquor sample having an appropriate sequence and length from the forward side and the reverse side of this highly discriminating DNA base sequence. it can.

  Alternatively, the primer sequence can be determined as follows. For example, a DNA microarray for genes of plants such as rice, barley, grapes, corn, soybean, etc., preferably plant seeds, is prepared or obtained and identified by hybridization of labeled DNA samples from brewed liquor with known genes on the array To screen for genes. The base sequence of the gene is obtained from a sequence database such as GenBank, and the primer sequence is determined based on this sequence. In addition, in order to determine an identifiable sequence between species or varieties of the same plant, the nucleotide sequences of the genes screened as described above are aligned for each plant type or variety obtained from the sequence database. It is also possible to prepare and select a sequence portion having a difference, and determine a primer sequence based on the selected sequence portion.

  When designing a primer using random primers as described above, or when designing a primer using a DNA microarray or sequence database, complementary to the genes of microorganisms (gonococci, yeast, etc.) contained in the brewed sake Design not to be. Alternatively, after the design, it is confirmed by PCR or the like that it is not complementary to the microorganism gene.

  In the present invention, a plant-derived repetitive sequence such as a tandem repeat (VNTR, variable number of tandem repeat) or a microsatellite-derived primer may be used. Primers derived from chloroplast DNA may be used.

  Unlike nuclear DNA, mitochondrial DNA has a ring-like structure, has a larger number of DNA per cell, has a faster base substitution rate than nuclear DNA, and adopts a mode of inheritance different from nuclear DNA called maternal inheritance. Therefore, sequence polymorphism analysis in the control region (D loop) of human mitochondrial DNA has become a powerful tool for human evolutionary research and forensic personal identification.

  Examples of mitochondrial genes used in the present invention include, for example, mitochondrial cytochrome gene, mitochondrial protein synthesis gene, mitochondrial heat shock protein gene, mitochondrial transcription factor, mitochondrial ribosomal protein gene, mitochondrial D loop sequence, mitochondrial molecular chaperone gene, apoptosis Examples include inducers, FZO protein genes and mitochondrial repeats.

Chloroplast DNA is the DNA contained in chloroplasts, which are plant-specific photosynthetic organs. By using this, it is possible to eliminate discrimination inhibition due to the coexistence of yeast-derived DNA and koji mold-derived DNA in brewed sake. However, it is possible to discriminate the brewed liquor raw material plant.
Examples of chloroplast DNA used in the present invention include repetitive sequences contained in chloroplasts.

  A repetitive sequence refers to a portion where a certain sequence of constituent bases A, T, G, and C appears repeatedly in DNA, and these repetitive sequences are concentrated in one place when they are scattered in DNA. Among the latter, a repetitive sequence vertically connected at one place is called a tandem repetitive sequence (VNTR), and it has been used for personal identification because there are individual differences in the number of repeats.

  Microsatellite is classified into one of tandem repeat sequences, and is a sequence in which several to several tens of repeat units of 2 to 5 base pairs are repeated, and is also used as a polymorphic marker. In the present invention, tandem repeats and microsatellite are not particularly distinguished.

  In the present invention, by selecting a repetitive sequence peculiar to the source plant from the above-mentioned repetitive sequences and using it as a primer for PCR, it is possible to eliminate discrimination interference due to DNA derived from koji molds or yeasts. Examples can include mitochondrial repeats in rice, microsatellite repeats in barley and grapes, and the like.

Primers derived from tandem repeats and microsatellite can be prepared in the same manner as described above.
Furthermore, in the present invention, a single nucleotide polymorphism (SNP) detection primer may be used.
SNP refers to a difference due to substitution of one base in the base sequence of DNA, and may cause individual disease resistance, alcohol metabolism characteristics, and the like.

  SNPs in plant genes have a very high frequency of appearance, and individual identification based on them is extremely promising. By focusing on the SNPs in the aforementioned plant genes, it is brewed while distinguishing from DNA derived from Neisseria gonorrhoeae and yeast. It becomes possible to identify sake. In addition, since SNPs exist among closely related varieties, it is possible to distinguish varieties efficiently and accurately, and examples include SNPs such as glutelin gene.

  In the present invention, for example, a primer may be designed so as to include a SNP site present in the raw plant and prepared in the same manner as in the above method. If the tested variety has the SNP, DNA is amplified by PCR.

  It is desirable that the primer in the present invention has a completely or almost complementary sequence. The primer size is preferably in the range of 10 to 50 bases, more preferably 15 to 30 bases. By selecting the primer, the PCR method selectively binds only to the base sequence portion that becomes an identification band useful for variety identification.

  As a result of performing the PCR, the present inventors obtained several types of bands that show distinction between plant species or varieties, and obtained various primers derived from these bands.

  Examples of primers used when the brew is sake, A6 (SEQ ID NO: 1 and 2), A7 (SEQ ID NO: 3 and 4), A65 (SEQ ID NO: 5 and 6), B1 (SEQ ID NO: 7 and 8) , G28 (SEQ ID NO: 9 and 10), B43 (SEQ ID NO: 11 and 12), E30 (SEQ ID NO: 13 and 14), F6 (SEQ ID NO: 15 and 16), F30 (SEQ ID NO: 17 and 18), G4 (SEQ ID NO: 19 and 20), J6 (SEQ ID NO: 21 and 22), BL3 (SEQ ID NO: 23 and 24), M11 (SEQ ID NO: 25 and 26), Zein (SEQ ID NO: 27 and 28), P5 (SEQ ID NO: 29 and 30), S13 (SEQ ID NO: 31 and 32), M2CG (SEQ ID NO: 33 and 34), T16 (SEQ ID NO: 35 and 36), WK9 (SEQ ID NO: 37 and 38), P3 (SEQ ID NO: 39 and 40), SS1 (SEQ ID NO: 41) And 42), SS2 (SEQ ID NO: 43 and 44), SS2-2 (SEQ ID NO: 45 and 46), GBSS (SEQ ID NO: 47 and 48), DB (SEQ ID NO: 49 and 50), Pro10 (SEQ ID NO: 51 and 52) , Pro13 (SEQ ID NOs: 53 and 54), Glu ( (Column numbers 55 and 56), Mochi (SEQ ID NOs: 57 and 58), BL3-3 (SEQ ID NOs: 59 and 60), BL1 (SEQ ID NOs: 61 and 62), BL2 (SEQ ID NOs: 63 and 64), BL4 (SEQ ID NO: 65) And 66), BL4-2 (SEQ ID NO: 67 and 68), BL65 (SEQ ID NO: 69 and 70), BL81 (SEQ ID NO: 71 and 72), BL6 (SEQ ID NO: 73 and 74), Mitol (SEQ ID NO: 75 and 76) , Chlo1 (SEQ ID NO: 77 and 78), GluSNP (SEQ ID NO: 79 and 80), BARGBSS (SEQ ID NO: 83 and 84), Ipoamy (SEQ ID NO: 87 and 88), Sugacss (SEQ ID NO: 89 and 90), B7 (SEQ ID NO: 91 and 92), G22 (SEQ ID NOs: 93 and 94) and SBE (SEQ ID NOs: 100 and 101).

  Examples of primers used when the brew is beer are A6 (SEQ ID NOs: 1 and 2), A7 (SEQ ID NOs: 3 and 4), A65 (SEQ ID NOs: 5 and 6), B1 (SEQ ID NOs: 7 and 8). ), G28 (SEQ ID NO: 9 and 10), B43 (SEQ ID NO: 11 and 12), E30 (SEQ ID NO: 13 and 14), F6 (SEQ ID NO: 15 and 16), F30 (SEQ ID NO: 17 and 18), G4 (sequence) No. 19 and 20), J6 (SEQ ID No. 21 and 22), BL3 (SEQ ID No. 23 and 24), M11 (SEQ ID No. 25 and 26), Zein (SEQ ID No. 27 and 28), P5 (SEQ ID No. 29 and 30) , S13 (SEQ ID NO: 31 and 32), M2CG (SEQ ID NO: 33 and 34), T16 (SEQ ID NO: 35 and 36), WK9 (SEQ ID NO: 37 and 38), P3 (SEQ ID NO: 39 and 40), SS1 (SEQ ID NO: 41 and 42), SS2 (SEQ ID NO: 43 and 44), SS2-2 (SEQ ID NO: 45 and 46), GBSS (SEQ ID NO: 47 and 48), DB (SEQ ID NO: 49 and 50), Pro10 (SEQ ID NO: 51 and 52) ), Pro13 (SEQ ID NOs: 53 and 54), G lu (SEQ ID NO: 55 and 56), Mochi (SEQ ID NO: 57 and 58), BL3-3 (SEQ ID NO: 59 and 60), BL1 (SEQ ID NO: 61 and 62), BL2 (SEQ ID NO: 63 and 64), BL4 (sequence) No. 65 and 66), BL4-2 (SEQ ID No. 67 and 68), BL65 (SEQ ID No. 69 and 70), BL81 (SEQ ID No. 71 and 72), BL6 (SEQ ID No. 73 and 74), BARGBSS (SEQ ID No. 83 and 84), MAIZGBSS (SEQ ID NO: 85 and 86), B7 (SEQ ID NO: 91 and 92), G22 (SEQ ID NO: 93 and 94), SBE (SEQ ID NO: 100 and 101) and primers derived from soybean protein glycinin (SEQ ID NO: 106 and 107).

  Examples of primers used when the brew is wine are A6 (SEQ ID NOs: 1 and 2), A7 (SEQ ID NOs: 3 and 4), A65 (SEQ ID NOs: 5 and 6), B1 (SEQ ID NOs: 7 and 8). ), G28 (SEQ ID NO: 9 and 10), B43 (SEQ ID NO: 11 and 12), E30 (SEQ ID NO: 13 and 14), F6 (SEQ ID NO: 15 and 16), F30 (SEQ ID NO: 17 and 18), G4 (sequence) No. 19 and 20), J6 (SEQ ID No. 21 and 22), BL3 (SEQ ID No. 23 and 24), M11 (SEQ ID No. 25 and 26), Zein (SEQ ID No. 27 and 28), P5 (SEQ ID No. 29 and 30) , S13 (SEQ ID NO: 31 and 32), M2CG (SEQ ID NO: 33 and 34), T16 (SEQ ID NO: 35 and 36), WK9 (SEQ ID NO: 37 and 38), P3 (SEQ ID NO: 39 and 40), SS1 (SEQ ID NO: 41 and 42), SS2 (SEQ ID NO: 43 and 44), SS2-2 (SEQ ID NO: 45 and 46), GBSS (SEQ ID NO: 47 and 48), DB (SEQ ID NO: 49 and 50), Pro10 (SEQ ID NO: 51 and 52) ), Pro13 (SEQ ID NOs: 53 and 54), G lu (SEQ ID NO: 55 and 56), Mochi (SEQ ID NO: 57 and 58), BL3-3 (SEQ ID NO: 59 and 60), BL1 (SEQ ID NO: 61 and 62), BL2 (SEQ ID NO: 63 and 64), BL4 (sequence) No. 65 and 66), BL4-2 (SEQ ID No. 67 and 68), BL65 (SEQ ID No. 69 and 70), BL81 (SEQ ID No. 71 and 72), BL6 (SEQ ID No. 73 and 74), VITGT (SEQ ID No. 81 and 82), B7 (SEQ ID NOs: 91 and 92), G22 (SEQ ID NOs: 93 and 94), SBE (SEQ ID NOs: 100 and 101), primers derived from the grape mitochondrial cytochrome gene (SEQ ID NOs: 102 and 103) and grape sucrose-binding protein gene Derived primers (SEQ ID NOs: 104 and 105).

  For example, if you use rice-derived A6, barley-derived BARGBSS, corn-derived MAIZGBSS, grape-derived VITGT, soybean glycinin-derived primers, rice, barley, corn, grape, soybean are included as raw materials, respectively. Can be determined.

  In the exemplified primers, SEQ ID NOS: 1 to 94 are odd sequence numbers as forward primers, even sequence numbers are reverse primers, and from SEQ ID NO: 100, even numbers are forward primers and odd numbers are reverse primers. is there.

  Appropriate ones are selected from the primers and used in the PCR method. Only one primer may be used, but two or more can be used simultaneously (multiplex PCR method), the number of PCR and electrophoresis can be reduced, and the plant or variety of raw material can be easily and quickly and accurately. Can be determined. Some of the PCR primers are commercially available (Takara Bio, Wako Pure Chemicals, Operon, Espec Oligo Service, etc.).

  In order to advance the multiplex PCR method efficiently, it is necessary to adjust the melting temperature of each primer to be used and the annealing temperature of the PCR in consideration of the melting temperature. Here, the melting temperature (Tm) refers to a temperature at which the double strands of DNA are separated into single strands and the double strand and the single strand are present in a ratio of 1: 1. In general, the annealing temperature for PCR is usually suitable around the Tm of the primer used. In the present invention, when using primers together, by selecting and mixing primers having similar Tm, and selecting an appropriate annealing temperature close to Tm, an identification band obtained when each primer is used alone Can be obtained by one or a few PCRs.

  Specifically, it is preferable that the difference between the average value of Tm of the primers used and the Tm of each primer is within 15 ° C., and the annealing temperature of PCR is also in that range. When PCR is performed at an annealing temperature lower than 36 ° C., it is not preferable because an identification band may appear even in varieties that originally do not show an identification band, making identification difficult. On the other hand, if the elongation step is performed at a temperature higher than 80 ° C., an identification band that should appear originally does not appear, which is inappropriate.

(C) Discrimination between types or varieties of raw material plants based on polymorphisms of amplified DNA Polymorphism means that there are diversity in traits and morphology among normal individuals in the same species. In the present invention, discrimination of a raw material variety or a raw material plant variety based on the polymorphism of the amplified DNA is performed by comparing the molecular weights of the PCR amplified DNA detected by electrophoresis. When DNA amplified by PCR using the above-mentioned primers is electrophoresed, DNA migrates according to its molecular weight and forms a band. Compared by its molecular weight or band formation position, it compares between plant species or varieties. Identify differences. When a plurality of bands are formed using a plurality of primers, different band patterns are observed depending on the kind or variety of plants.

  Specific methods for analyzing the amplified DNA polymorphism include conventional methods such as agarose gel electrophoresis and acrylamide gel electrophoresis. That is, after completion of PCR, separation by molecular weight distribution is performed using agarose gel electrophoresis, acrylamide gel electrophoresis, or the like, and after completion of electrophoresis, the gel is stained with ethidium bromide. Thereafter, the stained gel is irradiated with ultraviolet rays, and each DNA band pattern (electrophoresis image) that appears thereby is photographed. Next, by comparing the band pattern of each sample shown in the electrophoretic photograph with the band pattern of the sample whose varieties are known in advance, it is possible to determine which cultivar is the raw rice. . Whether or not a certain plant is used as a raw material in brewed liquor can be determined by the presence or absence of a DNA band amplified by a primer derived from the raw material plant. Furthermore, like quantitative PCR that has recently been used, in parallel with PCR, the amount of a monomer combined with a fluorescent primer or fluorescent dye is quantified and compared with a fluorescence detector, thereby making it possible to determine the type or variety of plants. It is also possible to make a determination.

  Examples of the present invention are shown below, but the scope of the present invention is not limited by these Examples.

(Comparison by PCR of commercial ginjo sake and rice suitable for brewing)
The rice samples of Yamada Nishiki, Hachiso, Hyogo Yumenishiki, and Omachi, which are suitable for sake brewing, are crushed with a test rice grinder manufactured by Kett Science Laboratory and used with a rice mill for testing (parest) manufactured by Kett Science Laboratory. A polished rice sample having a rice yield of 90% was obtained. This polished rice sample was pulverized using a miller (IFM-100) manufactured by Iwatani to obtain a powder sample. 0.4 g of a polished rice powder sample was extracted with an aqueous solution of 0.2 mL of 2 × CTAB and 0.2 mL of sterilized water at 65 ° C. for 30 minutes, and an equal amount of chloroform-isoamyl alcohol was added to the same solution and purified with a rotator for 15 minutes. Next, the same amount of chloroform-isoamyl alcohol and 10% CTAB solution are added to the supernatant obtained by centrifugation, and the mixture is purified again. Approximately 2.5 times the volume of precipitation buffer is added to the supernatant after centrifugation, and the mixture is added at −80 ° C. for 5 minutes. Cooled for a minute to form a precipitate. The precipitate was collected by centrifugation, dissolved in TE containing 1M NaCl, and an equal amount of isopropyl alcohol was added. After mixing by inversion, the mixture was centrifuged and the precipitate was dissolved in TE and treated with RNase at 55 ° C. for 30 minutes. Equal volume of neutral phenol is added to this and purified. After centrifugation, 0.2M NaCl and 2.5 volumes of cold ethanol are added to precipitate DNA, washed with 70% ethanol, dissolved in TE and cast into template. DNA.

To 0.2g of powder freeze-dried 25ml of Japanese sake (commercially pure rice ginjo sake, polished rice ratio 55%), add 300μL of 0.1M Tris-HCl buffer (pH 8.0), and derived from Bacilluslichenformis 100 μL of thermostable α-amylase (Sigma, 790 U / mg solid, 1 mg / ml) was added and reacted at 80 ° C. for 1 hour to decompose starch. Next, 30 μL of 10% SDS was added to 100 μL of protease K (derived from Tritirachiumalbum , 20 mg / mL manufactured by Worthington Biochemical), and reacted at 55 ° C. for 1 hour to decompose the protein. Chilled ethyl alcohol was added to the centrifuged supernatant, and a precipitate was formed on ice for 15 minutes. The precipitate after centrifugation is dissolved in 300 μL of TE (Tris-HCl buffer containing EDTA), and after RNAse treatment, the protein is separated and removed three times with an equal amount of phenol, and then with chloroform / isoamyl alcohol. The purified DNA for template was prepared by purification and precipitation with ethyl alcohol. A method for preparing template DNA from sake is shown in FIG.

DNA was amplified by the PCR method using the template DNA prepared from the polished rice and sake as described above. Prepared by mixing 10.8 μL of sterilized water, 0.2 μL of Taq Polymerase (5 U / μL), 2.0 μL of reaction buffer, 2.0 μL of 25 mM MgCl ₂ , 1 μL of template DNA (400 ng / μL), and 2 μL of dNTPs. Add 2 μL of the set (primer WK9 of SEQ ID NOs: 37 and 38, primer B43 of SEQ ID NOs: 11 and 12, primer M11 of SEQ ID NOs: 25 and 26, primer G22 of SEQ ID NOs: 93 and 94, manufactured by Takara Bio, 5 pmol / μL) The volume was adjusted to 20 μL and PCR was performed. The PCR conditions were denaturation at 96 ° C. for 1 minute, annealing at 62 ° C. for 1 minute, and extension at 72 ° C. for 2 minutes, and this was repeated 40 times.

The results of PCR and electrophoresis are shown in FIG.
As shown in FIG. 2, polymorphs of amplified DNA appear in PCR performed using the sake-brewed rice and DNA extracted and purified from sake by the method of the present invention as a template. It has become clear that Yamada Nishiki or Hyogo Yumenishiki is being used instead of Yatan and Omachi.

  For comparison, in the case of DNA extracted and purified using a commercially available simple DNA extraction kit (manufactured by Takara Bio, DNA extraction kit, product code 9098) instead of the method of the present invention, the raw rice is Although the same result as FIG. 2 was obtained, the direction from Japanese sake could not obtain an amplified DNA band, and it was impossible to compare with raw rice.

(Preprocessing)
5g brewed liquor is heated and dried (dried at 135 ° C for 3 hours by Advantech's heat dryer FC-610), cold-air dried (48 hours at 20 ° C by the above-mentioned heated dryer), and cold-air dried under reduced pressure (using an Ira rotary evaporator) Then, it was concentrated and dried into a thin film by four types of drying methods: vacuum drying with a water pump while blowing a nitrogen stream, and vacuum drying (vacuum drying at 30 ° C. for 24 hours using a centrifugal evaporator CVE200D made by Ira). To 0.2 g of the dried thin film, add 300 μL of 0.1 M Tris-HCl buffer (pH 8.0), add 100 μL of Bacilluslichenformis- derived heat-resistant α-amylase (Sigma, 790 U / mg solid, 1 mg / ml), and add 1 at 80 ° C. The starch was decomposed by reacting for a period of time. Next, 30 μL of 10% SDS was added to 100 μL of protease K (derived from Tritirachiumalbum , 20 mg / mL manufactured by Worthington Biochemical), and reacted at 55 ° C. for 1 hour to decompose the protein. Chilled ethyl alcohol was added to the centrifuged supernatant, and a precipitate was formed on ice for 15 minutes. The precipitate after centrifugation is dissolved in 300 μL of TE (Tris-HCl buffer containing EDTA), and after RNAse treatment, the protein is separated and removed three times with an equal amount of phenol, and then with chloroform / isoamyl alcohol. The purified DNA for template was prepared by purification and precipitation with ethyl alcohol. PCR was performed in the same manner as in Example 1 using 1 μL of these template DNAs as samples. The results are shown in Table 1.

Using 5 g of brewed liquor, dried by heating (dried at 105 ° C for 18 hours with Advantech's heat dryer FC-610) and then pulverized, freeze-dried, and spray-dried to obtain powder. To the 0.2 g obtained, add 300 μL of 0.1 M Tris-HCl buffer (pH 8.0), add 100 μL of thermostable α-amylase derived from Bacilluslichenformis (Sigma, 790 U / mg solid, 1 mg / ml), and continue at 80 ° C. for 1 hour Reacted to decompose starch. Next, 30 μL of 10% SDS was added to 100 μL of protease K (derived from Tritirachiumalbum , 20 mg / mL manufactured by Worthington Biochemical), and reacted at 55 ° C. for 1 hour to decompose the protein. Chilled ethyl alcohol was added to the centrifuged supernatant, and a precipitate was formed on ice for 15 minutes. The precipitate after centrifugation is dissolved in 300 μL of TE (Tris-HCl buffer containing EDTA), and after RNAse treatment, the protein is separated and removed three times with an equal amount of phenol, and then with chloroform / isoamyl alcohol. The purified DNA for template was prepared by purification and precipitation with ethyl alcohol. PCR was performed in the same manner as in Example 1 using 1 μL of these template DNAs as samples. The results are shown in Table 2. The sample turned brown by heat drying, and no amplified DNA appeared after PCR. On the other hand, in the case of freeze drying or spray drying, amplified DNA appeared after PCR. In particular, in the case of lyophilization, since water was removed from the frozen brewed liquor sample by sublimation, denaturation of DNA in the concentrated powdering process did not occur, and the most favorable result was obtained.

  In the same manner as in Example 1 in the presence of primer A65 (SEQ ID NOs: 5 and 6) using the four types of template DNAs of Example 2 and Example 3 (heat drying, centrifugal vacuum drying, freeze drying, spray drying). PCR was performed. The annealing temperature was 42 ° C. The result of electrophoresis after PCR is shown in FIG. As is clear from FIG. 3, no amplified DNA appeared in the case of heat drying. In the case of the other three preparation methods, the appearance of amplified DNA by PCR was observed.

(Search for suitable primers)
Samples of Yamada Nishiki, Hyakumangoku, Hachiman, Hyogo Yumenishiki, and Omachi rice, which are suitable for sake brewing, were used as samples, and DNA was extracted and purified by the same method as in Example 1 to obtain template DNA. PCR was performed in the presence of various primers for distinguishing varieties. The result is shown in FIG. As shown in FIG. 4, when using S13 (SEQ ID NOs: 31 and 32) and F30 (SEQ ID NOs: 17 and 18) as primers for discriminating rice varieties, it is possible to discriminate between rice brewers. there were.

(Search for suitable primers)
Samples of raw rice (rice cake) from Yamada Nishiki, Hachiso, Hyogo Yumenishi, and Omachi, which are suitable for sake brewing, were extracted and purified as the template DNA in the same manner as in Example 1, and used as primers for discriminating rice varieties. PCR was carried out in the presence of. The result is shown in FIG.

  As shown in FIG. 5, when rice blast resistance gene-related primers BL3 (SEQ ID NOs: 23 and 24) and A65 (SEQ ID NOs: 5 and 6) are used as primers, rice brewing suitable rice is identified. Was possible. On the other hand, in the case of template DNA extracted from yeast, amplified DNA by these primers did not appear.

(Mutual identification of suitable rice for brewing)
Samples of Yamada Nishiki, Hyakumangoku, Omachi, Hyogo Yumenishiki, and Hachiman's raw rice (rice cake), which are suitable for sake brewing, were extracted and purified as the template DNA by the same method as in Example 1. Primers for variety identification (primer WK9 of SEQ ID NOs: 37 and 38, primer B43 of SEQ ID NOs: 11 and 12, primer M11 of SEQ ID NOs: 25 and 26, primer G22 of SEQ ID NOs: 93 and 94, S13 of SEQ ID NOs: 31 and 32, PCR was performed in the presence of A65) of SEQ ID NOs: 5 and 6. The results are shown in FIG.

  As shown in Table 3, it became possible to distinguish rice brewing suitable rice by the presence or absence of six kinds of amplified DNA bands.

(Example of identification of raw rice using sake as a sample)
Extraction and purification of template DNA and PCR were carried out in the same manner as in Example 1, using fodder, Yamada Nishiki, and sake made from 500 million stones as samples. As the primers, primer WK9 of SEQ ID NOs: 37 and 38, primer B43 of SEQ ID NOs: 11 and 12, primer M11 of SEQ ID NOs: 25 and 26, primer G22 of SEQ ID NOs: 93 and 94 were used. Two types of methods were used for extracting and purifying the template DNA from sake produced by ferry, which is a rice suitable for sake brewing. That is, (1) is the method of Japanese Patent No. 3048149 , that is, 20 mg of lyophilized powder is placed in a microtube, 300 μL of Tris-HCl buffer (100 mM, pH 8.0, 100 mM NaCl) is added, and then the same as in Example 1 After adding 5 μL of thermostable amylase and reacting at 60 ° C. for 1 hour, adding 5 μL of protease K derived from Tritirachiumalbum , reacting at 37 ° C. for 2 hours, and then adding 1 mL of ethyl alcohol at −20 ° C., − After standing at 20 ° C. for 15 minutes, the precipitate obtained by centrifugation is dissolved in 300 μL of TE solution, added with 400 μL of phenol, DNA is extracted with a rotary stirrer for 30 minutes, centrifuged and the supernatant is collected. Add an equal volume of PCI, extract for 30 minutes, add 6 mL of 5 M NaCl to the supernatant, then add 400 μL of cold ethyl alcohol, wash the centrifuged precipitate twice with 70% ethyl alcohol, and add the final precipitate Is dissolved in 40 μL of 10-fold diluted TE solution to make template DNA. The method is the method of the present invention example described in Example 1. The result is shown in FIG.

  As shown in Fig. 7, when ferry is used as raw rice, an amplified DNA band due to G22 appears and does not appear in Nishiki Yamada and Hyakumangoku. It became possible to distinguish it from sake made from Mangoku. The ferry shown in lane 1 is an example in which DNA extracted and purified by the method of Japanese Patent No. 3048149 was used as a template. In this case, no amplified DNA band appeared.

(Example of identification of raw rice using sake as a sample)
Extraction and purification of template DNA and PCR were performed by the method shown in Example 1, using sake made from 500 million stones, Nishiki Yamada, and Omachi as samples. As the primer, B43 of SEQ ID NOs: 11 and 12 was used. The result is shown in FIG.

  As shown in FIG. 8, when 500 million stones were used as the raw material rice, a low-molecular specific amplified DNA band appeared and did not appear in Yamada Nishiki and Omachi. In addition, since the amplified DNA band by B43 appears only in 500 million stones and Yamada Nishiki, it does not appear in Omachi, so sake made from 500 million stones and sake made from Yamada Nishiki and Omachi Mutual identification is now possible.

(Identification example using raw rice and sake as samples)
Using the raw rice and sake of Yamada Nishiki, Hyakumangoku, Omachi and Koshihikari as samples, template DNA was extracted from the rice and sake in the same manner as in Example 1, and PCR was performed. The result is shown in FIG.

  As shown in FIG. 9, the amplification bands by B43 obtained from Yamada Nishiki, Hyakumangoku, Omachi, and Koshihikari sake were different from each other and could be distinguished from each other. In addition, the amplified DNA band was included in the amplified DNA band of each raw rice.

(Determination of whether or not corn is used in beer)
Two commercially available beers (Sapporo Black Label (trademark) and Sapporo Ebisu Beer (trademark)) were lyophilized, and PCR template DNA was prepared in the same manner as in FIG. 1, and Zein, the main protein of corn, was prepared. PCR was performed in the presence of primers (SEQ ID NOs: 27 and 28) derived from these genes, and the amplified DNA was compared by electrophoresis. The result is shown in FIG.

  As shown in FIG. 10, amplification of zein-derived DNA was observed with the black label, but not with Ebisu beer. This was a result consistent with the labeling of each beer.

(Determination of whether or not corn is used in beer)
Using commercially available giraffe sparkling wine and beer (Kirin Ichiban Shibori (trademark)) and corn (sweet corn) as samples, DNA was extracted in the same manner as in Example 8, and derived from corn starch granule-bound starch synthase (GBSS) PCR was carried out in the presence of the primers (SEQ ID NOs: 85 and 86), and the amplified DNA was compared by electrophoresis. The result is shown in FIG.

  As shown in FIG. 11, amplified DNA derived from corn GBSS was observed at the first giraffe, and it was confirmed that corn was added to the raw material. This was consistent with the display.

(Distinguishing wine varieties)
DNA was extracted from two types of grape seeds (Gross Coleman and Red Grove) in the same manner as in Example 1 to obtain a template. FIG. 12 shows the results of performing PCR using these template DNAs together with primers (VITGT, SEQ ID NOs: 81 and 82) derived from the Vitis Glucosyltransferase gene and comparing the amplified DNAs by electrophoresis.

  As shown in FIG. 12, it was shown that grape cultivars Gross Coleman and red glove varieties can be distinguished by PCR using primers derived from the Vitis Glucosyltransferase gene.

(Distinguishing between microbial DNA and rice using mitochondrial DNA-derived primers)
Template DNA was prepared in the same manner as in Example 1, using Aspergillus oryzae , yeast ( Saccharomyces cerevisiae ) and sake rice (Omachi) as samples. PCR conditions were the same as in Example 1 except that 20 ng of template DNA was used, the annealing temperature was 38 ° C., and 0.8 μl each of a mitochondrial DNA repetitive sequence (SSR) -derived primer (Mitol) shown in SEQ ID NOs: 75 and 76 was used. The same apparatus and method were used. The results are shown in FIG.

  As shown in FIG. 13, no amplified DNA band appeared in Neisseria gonorrhoeae. In the case of sake rice, clear amplified DNA appeared in the low molecular region around 300 bp. In yeast, an amplified band appeared in the high molecular region near 1 kbp, which is completely different from rice. From this, it was shown that the primer derived from the repetitive sequence of mitochondrial DNA is very useful as a primer for PCR for distinguishing the kind of raw plant from the DNA derived from the microorganism for fermentation of brewing sake.

(Example of alcohol discrimination by mitochondrial DNA primer)
Three types of sake (B: 500 million stones, A and C are not shown for sake rice varieties) were used as samples, and template DNA was prepared in the same manner as in Example 1. PCR conditions were the same as in Example 1 except that 20 ng of template DNA was used, the annealing temperature was 38 ° C., and 0.8 μl each of the mitochondrial DNA repetitive sequence (SSR) -derived primer (Mito1) shown in SEQ ID NOs: 75 and 76 was used. The same apparatus and method were used. The results are shown in FIG.

  As shown in FIG. 14, in the case of template DNA prepared from sake of sake rice A and C, no amplified DNA band appeared. In the case of sake rice B (500 million stones), clear amplified DNA appeared in a low molecular region around 300 bp. From this, it was shown that the primer derived from the repetitive sequence of mitochondrial DNA is extremely useful as a primer for PCR for discriminating the kind of raw rice for brewing sake.

(Example with repetitive sequence primer derived from chloroplast DNA)
Template DNA was prepared in the same manner as in Example 1 using three types of sake (Omachi, 500 Million Stone, Yamada Nishiki) and Aspergillus oryzae as samples. PCR conditions were as follows except that 20 ng of template DNA was used, the annealing temperature was 38 ° C., and 0.8 μl each of the chloroplast DNA repetitive sequence (SSR) -derived primer (Chlo1) shown in SEQ ID NOs: 77 and 78 was used. 1 and the same apparatus and method. The results are shown in FIG.

  As shown in FIG. 15, in the case of template DNA prepared from sake rice, an amplified DNA band appeared clearly in a low molecular region around 400 bp. On the other hand, in the case of template DNA prepared from Aspergillus, no amplified DNA band appeared. Therefore, the primer derived from the repetitive sequence of chloroplast DNA is extremely useful as a primer for PCR to discriminate brewer's raw material rice without being affected by the template DNA of koji mold remaining in the brewer's sake. It has been shown.

(Example of primer development by SNP)
Washida et al. (H. Washida, C.-Y. Wu, A. Suzuki, U. Yamanouchi, T. Akihara, K. Harada and F. Takaiwa: Plant Molecular Biology, 40, 1-12, 1999) The PCR was carried out using M7 (tgcaaagt) within the regulatory gene of the glutelin gene as a primary primer from the structural gene of 620 bp, using 9 types of sample rice DNA as templates, and the amplified DNA was cloned to determine the nucleotide sequence. The sequence is shown in FIG. 16 (in FIG. 16, the base sequences of 61 to 180 and 241 to 300 are omitted) (SEQ ID NO: 95: LGC-1, SEQ ID NO: 96: IR2061, SEQ ID NO: 97: Casalas, SEQ ID NO: 98: WITA 7, dream ten colors). The single base substitution (SNP) which shows the difference of sample rice was found from the base sequence shown in FIG. That is, in the morning light and Koshihikari, the base sequence which is AGTTTT was TGTTTT in IR2061 and Yumejiro. Based on this SNP, primers of SEQ ID NOs: 79 and 80 were designed.

(Example of identification by SNP primer)
The template DNA was extracted and purified from rice samples from around the world by the method described in Example 1, and PCR was performed in the presence of the SNP primer (Gluterin SNP (GluSNP) shown in SEQ ID NOs: 79 and 80) designed in Example 14. Went. The apparatus and conditions used for PCR were the same as in Example 1. The amount of template DNA used was 1 μl (concentration 400 ng / μl), and the amount of primers used was 0.6 μl for both forward (SEQ ID NO: 79) and reverse (SEQ ID NO: 80). (Concentration 5 pmol / μl) and the annealing temperature was 50 ° C. The electrophoresis results after PCR are shown in FIG. As is clear from FIG. 17, it was possible to discriminate the variety of the sample rice based on the single nucleotide polymorphism (SNP), and the usefulness as a primer for discriminating the raw rice of the brewed sake was shown.

(Example of primer development specific to plant DNA that is distinguishable from microorganisms)
Extraction of chromosomal DNA from yeast and filamentous fungi was performed as follows. (1) Yeast extract (Difco) 5 g / l, tryptone 10 g / l, NaCl 5 g / l (pH 6.0) as a medium for filamentous fungi, (2) Yeast extract 5 g / l, tryptone 5 g / l as a medium for yeast l, sucrose 5 g / l, (pH 7.0) were prepared, each 10 ml was dispensed into a test tube with a cotton plug, and sterilized by an autoclave.

The strains were obtained from the microbial bank of the National Food Research Institute, National Agriculture and Food Research Organization. Aspergillus oryzae (NFRI 1130) was cultured at 30 ° C for 40 hours, and Saccharomycescerevisiae (NFRI 3069) was cultured at 30 ° C for 22 hours. Cultured. About 9 ml of these culture solutions were centrifuged (microcentrifuge tube 8,000 rpm for 10 minutes), and the cells were collected.

  Add 1.8 ml of Isoplant (nippon gene) solutionI to these cells, vortex, add 0.9 ml of solutionII (Lysisbuffer), vortex, heat at 50 ° C for 15 minutes, add 0.9 ml of 3M sodium acetate, Left for 15 minutes. Next, the mixture was centrifuged at 8,000 rpm for 10 minutes to recover the aqueous layer. This was transferred to a microcentrifuge tube and centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. Add 0.6 ml of ethanol to 0.3 ml of this supernatant, leave it overnight at -20 ° C, centrifuge at 12000 rpm for 15 minutes the next day, wash the resulting precipitate with 70% ethanol, dissolve in 0.45 ml of TEbuffer, 50ul of 3M sodium acetate was added, 0.8ml of isopropanol was added, and the mixture was allowed to stand at -20 ° C for 1 hour. Subsequently, the mixture was centrifuged at 12,000 rpm for 15 minutes, the precipitate was washed once with 70% ethanol, and the obtained DNA precipitate was dissolved in 0.2 ml of TE buffer to obtain template DNA.

  Template DNA was also prepared from sake rice (Yamada Nishiki, Hyakumangoku, Omachi) in the same manner as in Example 1. Using these template DNAs, PCR was performed using a thermal cycler dice manufactured by Takara Bio in the presence of various random primers. The annealing temperature was 36 ° C. The result of the electrophoresis is shown in FIG.

  As shown in FIG. 18, in the case of each primer of A65, BL4-2, B7, A7, BL81, G22, B43, and G4, amplified DNA different from koji mold or yeast is obtained, and these DNAs are cut out from the gel. In this way, plant-specific STS primers can be designed. The sequences of these primers are SEQ ID NO: 5 and 6, SEQ ID NO: 67 and 68, SEQ ID NO: 91 and 92, SEQ ID NO: 3 and 4, SEQ ID NO: 71 and 72, SEQ ID NO: 93 and 94, SEQ ID NO: 11 and 12, SEQ ID NO: Shown in 19 and 20.

(Example of template DNA preparation from red wine and PCR using it)
Commercially available red wine (Meijiya imported 2005 Beaujolais Nouveau, JJM Beaujolais V Ospice Lyon, Domaine des HospicesDivils de Lyon), white wine (Chabli) and beer (Black Label (trademark) made by Sapporo Beer) FD-1 made by Ira Dissolve 0.2 g of the lyophilized powder sample in 400 μl of Tris-HCl buffer (pH 8) containing 0.1 M NaCl, and add 100 μL of heat-resistant α-amylase derived from Bacilluslichenformis (Sigma, 790 U / mg solid, 1 mg / ml) And reacted at 80 ° C. for 2 hours to decompose starch. Subsequently, 30 μL of 10% SDS was added to 100 μL of protease K (derived from Tritirachiumalbum , 20 mg / mL manufactured by Worthington Biochemical), and reacted at 55 ° C. for 1 hour to decompose the protein. Chilled ethyl alcohol was added to the centrifuged supernatant, and a precipitate was formed on ice for 15 minutes. This centrifugation and washing with 70% ethyl alcohol were repeated three times to remove the red dye. The precipitate after centrifugation is dissolved in 300 μL of TE (Tris-HCl buffer containing EDTA), and after RNAse treatment, the protein is separated and removed three times with an equal amount of phenol, and then with chloroform / isoamyl alcohol. The purified DNA for template was prepared by purification and precipitation with ethyl alcohol. A spectrum of template DNA extracted and purified from red wine is shown in FIG. Coexisting components such as proteins are reduced, and a 260 nm DNA peak clearly appears. The primers of SEQ ID NOs: 75 and 76 (mitochondrial repetitive sequence, Mito1) and SEQ ID NOs: 77 and 78 (chloroplast-derived repetitive sequence, Chlo1) were coexisted in these template DNAs, and the annealing temperature was set to 36 ° C. FIG. 20 shows the result of performing PCR using GeneAmp PCR System 9700 manufactured by the company and comparing amplified DNA by electrophoresis. As shown in FIG. 20, an amplification band did not appear only in white wine, but an amplification DNA band appeared clearly in red wine and beer.

  When template DNA was prepared without performing washing three times with 70% ethyl alcohol and removing protein three times with phenol, the DNA was not amplified by PCR.

(Increase in rice identification primers)
A template DNA for PCR was extracted and purified from various raw rices in the same manner as in Example 1, and the distinguishability was examined using various primers. As a result, primer A6 shown in SEQ ID NOS: 1 and 2,
Primer B1 shown in SEQ ID NOs: 7 and 8, primer G28 shown in SEQ ID NOs: 9 and 10, primer E30 shown in SEQ ID NOs: 13 and 14, primer F6 shown in SEQ ID NOs: 15 and 16, primer J6 shown in SEQ ID NOs: 21 and 22. Primer P5 shown in SEQ ID NOs: 29 and 30, primer M2CG shown in SEQ ID NOs: 33 and 34, primer T16 shown in SEQ ID NOs: 35 and 36, primer P3 shown in SEQ ID NOs: 39 and 40, primer SS1 shown in SEQ ID NOs: 41 and 42, Primer SS2 shown in SEQ ID NOs: 43 and 44, primer SS2-2 shown in SEQ ID NOs: 45 and 46, primer DB shown in SEQ ID NOs: 49 and 50, primer Pro10 shown in SEQ ID NOs: 51 and 52, primers shown in SEQ ID NOs: 53 and 54 Pro13, primer Mochi shown in SEQ ID NOs: 57 and 58, primer BL1 shown in SEQ ID NOs: 61 and 62, primer BL2 shown in SEQ ID NOs: 63 and 64, primer BL65 shown in SEQ ID NOs: 69 and 70, sequence number In primer BL6 shown in 73 and 74, primer (derived from barley) shown in SEQ ID NOs: 83 and 84, BARGBSS, primer Ipoamy shown in SEQ ID NOs: 87 and 88, primer Sugacss shown in SEQ ID NOs: 89 and 90, as shown in Table 4, It was possible to identify various varieties of raw rice. BARGBSS can be used, for example, to determine whether barley is used in a sample sake.

(Comparison of PCR amplified DNA sequences using primer A7)
Japanese sake with raw material labeling of various types of sake brewing suitable rice (Yamada Nishiki, Hyakumangoku, Omachi, Miyama Nishiki) was freeze-dried into powder samples, and template DNA was extracted and purified by the enzymatic method described in Example 1 did.

  PCR was carried out in the same manner as in Example 1 in the presence of primer A7 (SEQ ID NOs: 3 and 4) on this template DNA, and amplified DNA was detected by electrophoresis. The result is shown in FIG. The amplified DNA of Miyama Nishiki detected in FIG. 21 was cut out from the gel and incorporated into Escherichia coli, and the nucleotide sequence was determined. The result is shown in FIG. 22 (SEQ ID NO: 99). As shown in FIG. 22, the amplified DNA had exactly the same sequence as the DNA amplified from the sake brewing rice by PCR in the presence of primer A7. From this, by the method of the present invention, DNA unique to sake brewing can be amplified and distinguished from other types of rice by PCR using template DNA extracted and purified from sake in the presence of appropriate primers. Became clear.

(Example of detection of rice contained in beer ingredients)
Five types of commercially available beers (A, B, C, D, E) were pulverized using an Ira freeze dryer (FD51). Of these powder samples, template DNA was extracted and purified by the same enzymatic method as in Example 1 for four types A, B, C, and D. For two types of samples D and E, the polysaccharide was removed by the CTAB method following the enzyme method to increase the degree of purification. Specifically, the protease treatment solution was transferred to 2 × CTAB solution (2% CTAB 100 mM Tris-HCl, pH 8, 1.4 M NaCl, 20 mM EDTA) that had been heated to 65 ° C., and incubated at 65 ° C. for 1 hour. After cooling to room temperature, chloroform / isoamyl alcohol (24: 1) was added and mixed with a rotator for 20 minutes. Next, the upper layer obtained by centrifugation at 10,000 rpm for 10 minutes was transferred to a new tube, and chloroform / isoamyl alcohol was added and mixed for 20 minutes. Next, the upper layer solution obtained by centrifuging at 10000 rpm for 10 minutes is transferred to a new tube, and 2-propanol is added to this solution and gently mixed, and then centrifuged at 15000 rpm for 5 minutes to remove the supernatant. % Ethanol was added to wash the inner wall of the tube. Then, after centrifuging for 5 minutes, the supernatant was removed and dried under reduced pressure. After drying, 50 μl of TE buffer was added, placed in a constant temperature bath at 55 ° C., and the pellet was completely dissolved to obtain a template aqueous solution. Thus, DNA could be purified in a state containing almost no starch and its degradation product, protein and its degradation product, lipid and its degradation product, by adding the CTAB method following the enzymatic method.

  DB (SEQ ID NOs: 49 and 50) was used as a primer for detecting rice in the raw material. PCR conditions were 600 ng of template DNA, 12 μl each of 5 pM primer concentration, and an annealing temperature of 38 ° C.

The result of electrophoresis after PCR is shown in FIG.
As shown in FIG. 23, in the case of beer, by combining the enzyme method followed by the CTAB method to increase the purity of the template DNA, amplification of the identification DNA by PCR is improved, and a small amount of rice in the raw material is detected. It became possible to do. Only B and D indicated the use of rice. In the case of B, even though rice was used, template DNA was prepared only by the enzymatic method, and thus could not be detected by PCR. On the other hand, in the case of D, the enzymatic method alone could not be detected as in the case of B, but it became possible to detect the use of rice by combining the enzymatic method with the CTAB method.

  Template DNA for PCR was prepared by enzymatic method from four types of commercially available sake freeze-dried powder. PCR was carried out in the presence of primers SBE (SEQ ID NOs: 100 and 101, 0.5 μl of 5 pM added) for 6 types of template DNAs together with the same molds of bacillus and yeast as in Example 18. The annealing temperature was 36 ° C. The results of subjecting the PCR product to agarose gel electrophoresis are shown in FIG. As shown in FIG. 24, a common amplification band appeared in sake different from gonococci and yeast. The amplified band DNA was excised from this gel, purified by the glass bead method, incorporated into Escherichia coli and amplified, and the base sequence was determined. Its base sequence showed high homology of 99.6% with the amylopectin branching enzyme related to rice starch synthesis.

(Preparation of template DNA from wine by enzyme / CTAB two-step method)
Three types of red wine and three types of white wine were freeze-dried to obtain powder samples. The template DNA was extracted and purified by applying the enzyme method to this as in Example 1. Furthermore, purification of the template DNA was repeated by adding the CTAB method to these template DNA solutions. FIG. 25 shows the state of DNA precipitation upon addition of ethanol and the DNA precipitation after centrifugation. As shown in FIG. 25, by performing purification by the CTAB method following the enzyme method, it was possible to prepare a high-quality template DNA from which red pigment was removed even from red wine.

(Example of identification of raw grapes using wine as a sample)
Using only the enzyme method prepared in Example 24 and the DNA prepared by the enzyme / CTAB two-step method as a template, using as a primer a primer derived from a grape mitochondrial cytochrome gene (SEQ ID NOs: 102 and 103) and a primer derived from a grape sucrose-binding protein gene (sequence) PCR was carried out in the presence of numbers 104 and 105). The annealing temperature was 36 ° C. The result of electrophoresis after PCR is shown in FIG.

  As shown in FIG. 26, in the case of wine, since there are many impurities such as polyphenols, a high-quality PCR template DNA cannot be obtained only by the enzymatic method, and no amplified DNA appeared after PCR (FIG. 26, B). -1 and B-2). On the other hand, a good PCR result can be obtained for the first time by using as a template DNA prepared by the two-step method combining the CTAB method with the enzyme method. The primer of grape mitochondrial cytochrome gene or grape sucrose-binding protein gene is used for the raw grapevine. Discrimination became possible (FIG. 26, A-1 and A-2).

  Six types of commercially available beer were freeze-dried to obtain powder samples, and template DNA was extracted and purified by an enzyme / CTAB two-step method. PCR was performed using primers derived from soybean protein glycinin (SEQ ID NOs: 106 and 107) or primers derived from corn starch synthase (GBSS) (SEQ ID NOs: 85 and 86) as primers. The annealing temperature was 42 ° C. The electrophoresis result after PCR is shown in FIG. As shown in FIG. 27A, use of soybean was detected in samples No. 3, 4 and 6, and only sample No. 5 was confirmed not to use corn.

  Since the present invention makes it possible to discriminate the type or variety of the raw material plant using only brewed liquor as a sample, it is useful for preventing impersonation of the display and may be industrially used in inspection institutions, mass retailers, etc. high.

  In addition, the present invention has a high possibility of clarifying the relationship between the brewing plant and the quality of the brewed sake. It is extremely likely to be used industrially for the purpose of improving the quality of brewed sake.

  Furthermore, since the present invention provides a means for obtaining an index indicating the relationship between the varietal characteristics of raw material plants and the quality of sake for brewing companies, selection of raw materials, investigation of raw materials of other companies' products, consumer preference and brewing raw materials It can be used for grasping the relationship between the two, and since the practical advantage of the present invention is great, the possibility of industrial use is very high.

The preparation method of template DNA from sake is shown. An electrophoresis image of DNA amplified using raw rice and sake as samples is shown. Electrophoretic images of DNA amplified using various types of dried brewed sake are shown. An electrophoretic image of DNA amplified using raw rice as a sample is shown. An electrophoretic image of DNA amplified using raw rice as a sample is shown. An electrophoretic image of DNA amplified using raw rice as a sample is shown. An electrophoresis image of DNA amplified using sake as a sample is shown. An electrophoresis image of DNA amplified using sake as a sample is shown. An electrophoresis image of DNA amplified using raw rice and sake as samples is shown. An electrophoresis image of DNA amplified using beer as a sample is shown. An electrophoresis image of DNA amplified using beer or the like as a sample is shown. An electrophoresis image of DNA amplified using a grape seed as a sample is shown. An electrophoresis image of DNA amplified using microorganisms and raw rice as a sample is shown. An electrophoresis image of DNA amplified using sake as a sample is shown. An electrophoresis image of DNA amplified using sake and koji mold as samples is shown. The base sequence of each variety of rice is shown. An electrophoretic image of DNA amplified using raw rice as a sample is shown. An electrophoresis image of DNA amplified using microorganisms and raw rice as a sample is shown. A graph of the DNA spectrum of red wine is shown. An electrophoresis image of DNA amplified using wine and beer as samples is shown. An electrophoresis image of DNA amplified using sake as a sample is shown. The base sequence of DNA obtained from sake is shown. An electrophoresis image of DNA amplified using beer as a sample is shown. An electrophoresis image of DNA amplified using sake and microorganisms as samples is shown. Preparation of template DNA from red wine and white wine by enzyme / CTAB two-step method is shown. An electrophoresis image of DNA amplified using wine as a sample is shown. An electrophoresis image of DNA amplified using beer as a sample is shown.

Claims (12)

This includes decomposing starch and protein by causing heat-resistant α-amylase and protease to act on brewed sake, followed by ethanol treatment, followed by phenol treatment to remove the protein, and further treatment with a chloroform / isoamyl alcohol mixed solvent. After decomposing starch and protein by causing heat-resistant α-amylase and protease to act on DNA or brewed liquor extracted and purified by the process, polysaccharides are removed by cetyltrimethylammonium bromide (CTAB) treatment. further chloroform / isoamyl alcohol mixed solvent to treated extracted and purified by a process comprising the DNA of the cast type, PCR was carried out with primers presence of plant gene-derived, the raw material plant based on polymorphic amplified DNA A method for discriminating the type of brewed sake. This includes decomposing starch and protein by causing heat-resistant α-amylase and protease to act on brewed sake, followed by ethanol treatment, followed by phenol treatment to remove the protein, and further treatment with a chloroform / isoamyl alcohol mixed solvent. After decomposing starch and protein by causing heat-resistant α-amylase and protease to act on DNA or brewed liquor extracted and purified by the process, polysaccharides are removed by cetyltrimethylammonium bromide (CTAB) treatment. further chloroform / isoamyl alcohol mixed solvent to treated extracted and purified by a process comprising the DNA of the cast type, PCR was performed with the primers presence of plant gene-derived raw material plant variety based on polymorphic amplified DNA A method for discriminating brewed sake ingredients.   The method according to claim 1 or 2, wherein the brewed sake is sake, beer or wine.   The method according to any one of claims 1 to 3, further comprising a step of pulverizing the brewed liquor by freeze drying, airflow drying or spray drying.   The method according to any one of claims 1 to 3, further comprising a step of concentrating the brewed liquor into a thin film by cold air drying, cold air vacuum drying, or vacuum centrifugal drying.   In the DNA extraction / purification, 1 selected from the group consisting of methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, isoamyl alcohol, benzyl alcohol, acetone, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, chloroform and phenol The method of any one of Claims 1-5 which removes a pigment | dye component using a kind or two or more types of organic solvents. When the brewed sake is sake, the primers are A6 (SEQ ID NOs: 1 and 2), A7 (SEQ ID NOs: 3 and 4), A65 (SEQ ID NOs: 5 and 6), B1 (SEQ ID NOs: 7 and 8), G28 (SEQ ID NO: 9 and 10), B43 (SEQ ID NO: 11 and 12), E30 (SEQ ID NO: 13 and 14), F6 (SEQ ID NO: 15 and 16), F30 (SEQ ID NO: 17 and 18), G4 (SEQ ID NO: 19 and 20), J6 (SEQ ID NO: 21 and 22), BL3 (SEQ ID NO: 23 and 24), M11 (SEQ ID NO: 25 and 26), Zein (SEQ ID NO: 27 and 28), P5 (SEQ ID NO: 29 and 30), S13 ( SEQ ID NO: 31 and 32), M2CG (SEQ ID NO: 33 and 34), T16 (SEQ ID NO: 35 and 36), WK9 (SEQ ID NO: 37 and 38), P3 (SEQ ID NO: 39 and 40), SS1 (SEQ ID NO: 41 and 42) ), SS2 (SEQ ID NO: 43 and 44), SS2-2 (SEQ ID NO: 45 and 46), GBSS (SEQ ID NO: 47 and 48), DB (SEQ ID NO: 49 and 50), Pro10 (SEQ ID NO: 51 and 52), Pro13 (SEQ ID NO: 53 and 54), Glu (SEQ ID NO: 5 5 and 56), Mochi (SEQ ID NO: 57 and 58), BL3-3 (SEQ ID NO: 59 and 60), BL1 (SEQ ID NO: 61 and 62), BL2 (SEQ ID NO: 63 and 64), BL4 (SEQ ID NO: 65 and 66) ), BL4-2 (SEQ ID NO: 67 and 68), BL65 (SEQ ID NO: 69 and 70), BL81 (SEQ ID NO: 71 and 72), BL6 (SEQ ID NO: 73 and 74), Mitol (SEQ ID NO: 75 and 76), Chlo1 (SEQ ID NO: 77 and 78), GluSNP (SEQ ID NO: 79 and 80), BARGBSS (SEQ ID NO: 83 and 84), Ipoamy (SEQ ID NO: 87 and 88), Sugacss (SEQ ID NO: 89 and 90), B7 (SEQ ID NO: 91 and 92), which is one or more kinds of primers are selected from the group consisting of G22 (SEQ ID NO: 93 and 94) and SBE (SEQ ID NO: 100 and 101), according to any one of claims 3-6 the method of. When the brew is beer, the primers are A6 (SEQ ID NOs: 1 and 2), A7 (SEQ ID NOs: 3 and 4), A65 (SEQ ID NOs: 5 and 6), B1 (SEQ ID NOs: 7 and 8), G28 (SEQ ID NO: 9 and 10), B43 (SEQ ID NO: 11 and 12), E30 (SEQ ID NO: 13 and 14), F6 (SEQ ID NO: 15 and 16), F30 (SEQ ID NO: 17 and 18), G4 (SEQ ID NO: 19 and 20), J6 (SEQ ID NO: 21 and 22), BL3 (SEQ ID NO: 23 and 24), M11 (SEQ ID NO: 25 and 26), Zein (SEQ ID NO: 27 and 28), P5 (SEQ ID NO: 29 and 30), S13 ( SEQ ID NO: 31 and 32), M2CG (SEQ ID NO: 33 and 34), T16 (SEQ ID NO: 35 and 36), WK9 (SEQ ID NO: 37 and 38), P3 (SEQ ID NO: 39 and 40), SS1 (SEQ ID NO: 41 and 42) ), SS2 (SEQ ID NO: 43 and 44), SS2-2 (SEQ ID NO: 45 and 46), GBSS (SEQ ID NO: 47 and 48), DB (SEQ ID NO: 49 and 50), Pro10 (SEQ ID NO: 51 and 52), Pro13 (SEQ ID NO: 53 and 54), Glu (SEQ ID NO: 5 5 and 56), Mochi (SEQ ID NO: 57 and 58), BL3-3 (SEQ ID NO: 59 and 60), BL1 (SEQ ID NO: 61 and 62), BL2 (SEQ ID NO: 63 and 64), BL4 (SEQ ID NO: 65 and 66) ), BL4-2 (SEQ ID NO: 67 and 68), BL65 (SEQ ID NO: 69 and 70), BL81 (SEQ ID NO: 71 and 72), BL6 (SEQ ID NO: 73 and 74), BARGBSS (SEQ ID NO: 83 and 84), MAIZGBSS (SEQ ID NO: 85 and 86), B7 (SEQ ID NO: 91 and 92), G22 (SEQ ID NO: 93 and 94), SBE (SEQ ID NO: 100 and 101) and a primer derived from soybean protein glycinin (SEQ ID NO: 106 and 107) The method according to any one of claims 3 to 6 , wherein the primer is one type or two or more types of primers selected from the group. When the brew is wine, the primers are A6 (SEQ ID NOs: 1 and 2), A7 (SEQ ID NOs: 3 and 4), A65 (SEQ ID NOs: 5 and 6), B1 (SEQ ID NOs: 7 and 8), G28 (SEQ ID NO: 9 and 10), B43 (SEQ ID NO: 11 and 12), E30 (SEQ ID NO: 13 and 14), F6 (SEQ ID NO: 15 and 16), F30 (SEQ ID NO: 17 and 18), G4 (SEQ ID NO: 19 and 20), J6 (SEQ ID NO: 21 and 22), BL3 (SEQ ID NO: 23 and 24), M11 (SEQ ID NO: 25 and 26), Zein (SEQ ID NO: 27 and 28), P5 (SEQ ID NO: 29 and 30), S13 ( SEQ ID NO: 31 and 32), M2CG (SEQ ID NO: 33 and 34), T16 (SEQ ID NO: 35 and 36), WK9 (SEQ ID NO: 37 and 38), P3 (SEQ ID NO: 39 and 40), SS1 (SEQ ID NO: 41 and 42) ), SS2 (SEQ ID NO: 43 and 44), SS2-2 (SEQ ID NO: 45 and 46), GBSS (SEQ ID NO: 47 and 48), DB (SEQ ID NO: 49 and 50), Pro10 (SEQ ID NO: 51 and 52), Pro13 (SEQ ID NO: 53 and 54), Glu (SEQ ID NO: 5 5 and 56), Mochi (SEQ ID NO: 57 and 58), BL3-3 (SEQ ID NO: 59 and 60), BL1 (SEQ ID NO: 61 and 62), BL2 (SEQ ID NO: 63 and 64), BL4 (SEQ ID NO: 65 and 66) ), BL4-2 (SEQ ID NO: 67 and 68), BL65 (SEQ ID NO: 69 and 70), BL81 (SEQ ID NO: 71 and 72), BL6 (SEQ ID NO: 73 and 74), VITGT (SEQ ID NO: 81 and 82), B7 (SEQ ID NO: 91 and 92), G22 (SEQ ID NO: 93 and 94), SBE (SEQ ID NO: 100 and 101), a primer derived from the grape mitochondrial cytochrome gene (SEQ ID NO: 102 and 103) and a primer derived from the grape sucrose binding protein gene (sequence) The method according to any one of claims 3 to 6 , which is one kind or two or more kinds of primers selected from the group consisting of Nos. 104 and 105). The method according to any one of claims 1 to 9 , wherein the primer is a part of a repetitive sequence derived from a plant. The method according to any one of claims 1 to 10 , wherein the polymorphism is discriminated by an SNP discrimination method. Extraction and purification of DNA from brewed liquor, using as a template, PCR in the presence of plant gene-derived primers, and determining the type or varieties of raw plant based on the polymorphism of the amplified DNA. A method using the primer according to any one of claims 7 to 9 as the primer.

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