JP6007426B2 - Sake production method - Google Patents

Description

The present invention relates to a method for producing sake .

  In recent years, there has been an increase in consumer anxiety about safety due to mislabeling or incorrect labeling of alcoholic and food labels.

  For this reason, the technology of ensuring the reliability of the label display of this food / beverage product is requested | required by confirming the content of the food / beverage product directly, and confirming the origin of the raw material used for this food / beverage product Yes.

  For this purpose, as a technique that can easily and accurately identify the origin of food and beverages such as alcoholic beverages and foods and identify raw materials, for example, identification and discrimination of sake yeast using the AWA1 gene of sake yeast as an index. Proposal to do (Non-Patent Document 2), and in order to distinguish various sake yeasts that are closely related to each other, it is possible to extract a single nucleotide polymorphism (SNPs) region of the genome and amplify it There is a proposal (Non-Patent Document 3).

  Similarly, many methods have been proposed to amplify genes using specific primer pairs for various specific gene regions of yeast in order to discriminate near-species yeast used for sake and beer brewing. For example, in Patent Document 1, a primer pair that specifically amplifies a region including a site where recombination between a Saccharomyces cerevisiae type nucleotide sequence and a Saccharomyces bayanus type nucleotide sequence occurs on the same chromosome of beer yeast is used. There is a disclosure of a technique for discriminating a brewer's yeast strain by performing nucleic acid amplification, and Patent Literature 2 uses a primer for amplifying a DNA gene containing a brewing yeast FLO5 gene, YHR213W gene or YAR062W gene, It is shown that the brewing yeast is discriminated. Patent Document 3 discloses the use of a primer for nucleic acid amplification of a predetermined gene region using yeast transposon, and Patent Document 4 details the primer set for detecting a polymorphic gene. Has been disclosed, and the identification of yeast strains using this primer has been disclosed. Furthermore, Patent Documents 5 to 8 disclose yeast identification and discrimination using other specific gene regions of yeast. Is disclosed.

  Non-Patent Document 2 described above discloses that yeast can be identified by using the AWA1 gene, which makes it easy to distinguish differences between sake yeasts, and Non-Patent Document 3 discloses a single nucleotide sequence of genomic DNA. It is shown by the shape (SNPs) analysis that it is possible to discriminate Japanese sake yeast, and also in Patent Documents 1 to 8, the base sequences of the specific sites between the limited yeasts have diversity. Yeast identification methods using the above are disclosed, and these are also aimed at discrimination of production areas and identification of raw materials.

  Although these techniques of Patent Documents 1 to 8 and Non-Patent Documents 2 and 3 are homologous, they are methods for identifying yeast strains by finding gene portions having polymorphisms. There is a problem that it is limited and difficult to apply to yeast for producing food and drink. In addition, none of these detection methods guarantees the consistency and identity between the composition of the content of the food and drink and the content of the label displayed on the container or bag in which the food or drink is placed.

  On the other hand, as a discriminating method by nucleic acid analysis technology capable of discriminating the origin of food and drinks such as alcoholic beverages and foods and identifying raw materials simply and accurately, this gene part is mutated by imparting serellin resistance to the FAS2 gene of yeast There is also a proposal (Non-patent Document 1) to use this mutated portion for yeast identification.

JP 2008-245636 A JP 2008-193904 A JP 2007-181438 A JP 2007-082431 A JP 2006-340671 A JP 2005-027527 A JP 2004-329086 A JP 2003-245077 A

Akada R. et al., J. Biosci. Bioeng., Vol. 92, p.189-192, 2001. Kishi et al., "Determination of sake yeast in sake mash by PCR-DGGE method", Abstracts of the 62nd Annual Meeting of the Japan Society for Biotechnology (2010), p.43, 2010.10. Hasuda et al., “Distinguishing Sake Sake Yeast by Single Nucleotide Polymorphism Analysis of Genomic DNA,” J. Brew. Soc. Japan, Vol.106, p.706, 2011.

  However, the method of Akada et al. In Non-Patent Document 1 is a discrimination method using the mutation site of the FAS2 gene of yeast by imparting selerin resistance. However, when the FAS2 gene is introduced into yeast, this yeast produces high caproic acid ethyl ester. Therefore, there is a problem that the quality of the product is affected and it is difficult to maintain and manage the quality.

  In addition, it is extremely difficult to distinguish between yeasts of the same species or related species by any of the conventional methods based on the above-mentioned patent documents and non-patent documents. That is, the polymorphic nature of conventional genes is used for identification. In this method, it is possible to discriminate between the No. 7 yeast and the No. 9 yeast. For example, the No. 7 yeasts and the No. 9 yeasts differing in the selected time and place. It cannot be used for more accurate classification and identification of attributes, and it has been difficult to use for identification of attributes such as more detailed properties and functions of yeast.

  An object of the present invention is to provide means for easily discriminating the identity of yeast, and to improve the quality and reliability of foods and drinks produced using yeast.

  The gist of the present invention will be described with reference to the accompanying drawings.

Sake yeast is cultured in a selective medium containing canavanine, arginine and ornithine, and the arginase gene of the sake yeast is mutated to obtain an arginase-deficient yeast. The base sequence of the arginase gene of this arginase-deficient yeast is ascertained and becomes known. Further, the present invention relates to a method for producing sake, characterized in that identification information is associated with the base sequence and sake is produced using an arginase-deficient yeast associated with the identification information.

The method for producing sake according to claim 1, wherein the identification information includes identity management information including at least one of a yeast type, a selection time, and a selection location, a production location, and a production time. And / or a manufacturing lot, manufacturing history, product type, and manufacturing management information including at least one of the manufacturers.

The method for producing sake according to any one of claims 1 and 2, wherein the arginase-deficient yeast has 4 to 10 repeats of 2 to 5 repeats present in the base sequence of the arginase gene before mutation. The present invention relates to a method for producing sake, wherein the base in a repetitive sequence comprising bases is an arginase-deficient yeast in which two or more bases are deleted or substituted by one base.

  Since the present invention is configured as described above and a predetermined gene of yeast is mutated to provide identification information for determining the identity of yeast, yeast identification is improved and yeast identity is easily determined. In contrast to the conventional method for producing yeast for food and drink, which has only been able to identify yeast strains by finding homologous and polymorphic portions of genes, the present invention causes mutations in certain genes in yeast. Accordingly, the mutated portion can be easily and quickly detected, and the yeast can be easily identified by adding the mutation as yeast identification information. That is, since the identity of the yeast used for the production of food and drink can be easily determined by giving identification information to the predetermined gene of yeast, this is determined from the predetermined gene of the yeast of the food and drink produced using yeast. By discriminating the identification information, it is possible to immediately determine whether or not it is a food or drink manufactured using a predetermined yeast for manufacturing food or drink.

  In addition, since the present invention uses the mutated predetermined gene as a gene that does not need to be expressed in the production of food and drink, the food and drink can be produced even if this gene is not expressed, and produced using this yeast. The deterioration of the quality of food and drink can be reduced as much as possible.

Further, to impart a nucleotide sequence pattern was mutated gene as identification information, the case of using the identification information in association with the management information for the classification identification of yeast, they are classified separately in detail the nature of the yeast and functions It becomes possible, and it becomes possible to more strictly and easily discriminate whether or not the yeasts have the same properties and functions. Therefore, when producing foods and drinks using this yeast, the yeast that maintains higher uniformity of the yeast Therefore, the brewing characteristics and fermentation characteristics of yeast during the production of foods and drinks are further stabilized, and it is possible to contribute to stabilizing the quality and improving the quality of foods and drinks produced using these yeasts. Become. In addition, when this identification information is used in association with management information for manufacturing a food or drink manufactured using this yeast, the manufacturing information of the food or drink manufactured using this yeast for manufacturing food or drink is given to the yeast itself. Since the identification information is determined from the predetermined gene of the yeast of the food and drink produced using yeast, the production origin of this food and drink can be discriminated and manufactured using this yeast for producing food and drink. It becomes possible to improve the reliability of food and drink.

Further, food or beverage identity determination of yeast was prepared by using an easy food or beverage for producing yeast, because yeast is used mutated genes that do not need to be expressed in the production of this food product, the gene expression Therefore, it is possible to produce foods and drinks, and therefore the foods and drinks are of good quality with little deterioration or decrease in the brewing characteristics and fermentation characteristics of the yeast. And since the identification information of this yeast can be confirmed by discriminating the predetermined gene of yeast used for manufacture of this food / beverage product, it becomes possible to grasp | ascertain the origin of this food / beverage product correctly. Thus, for example, when this identification information is associated with management information for the production of foods and beverages produced using this yeast, the origin of the yeast used for production can be determined and the traceability of the foods and beverages is improved. It is possible to improve the reliability of this food and drink. In addition, when the identification information is used in association with such management information for classification and identification of yeast, the classification of the yeast can be performed more accurately. For example, production management (microorganism management) such as yeast purity measurement Can be performed quickly and simply, for example, in the process of producing foods and drinks produced using yeast, for example, the purity of yeast in sake mothers and straws can be maintained higher than before, and The stability of the production process is improved, and the quality of food and drink produced using this yeast can also be stabilized.

Further, to obtain the yeast or yeast nucleic acids from in food products produced using the yeast, the nucleotide sequence pattern of a given gene in the yeast, the yeast previously set, i.e., easy to identity determination of yeast It is determined whether or not it matches the base sequence pattern of the predetermined gene of the yeast for food and beverage production, and if it matches, it is only necessary to determine that it is a food and beverage produced with the yeast for food and beverage production, The origin of the food / beverage product can be easily determined, the traceability of the food / beverage product can be improved, and the reliability of the food / beverage product as a product can be increased. In addition, it becomes possible to discriminate foods and beverages that do not use the predetermined yeast for producing foods and beverages. For example, it is possible to identify the authenticity of foods and beverages, thereby producing an effect of improving the reliability of the product label display. In addition, in a yeast for producing foods and drinks in which yeast identification information is given to a predetermined gene of yeast, precise classification and identification of attributes such as finer properties and functions of yeast can be easily performed, and food and drink using this yeast It becomes easy to keep the quality of the product constant, and this is a method for determining the identity of yeast used in the production of food and drink, which can contribute to the stabilization and improvement of product quality.

It is a base sequence pattern of the arginase gene (1st position-480th position) of various yeasts concerning a present Example. It is a base sequence pattern of arginase genes (positions 481 to 960) of various yeasts according to this example. It is a base sequence pattern of the arginase gene (961st-1002nd) of various yeasts concerning a present Example. It is explanatory drawing of the base-sequence analysis of the yeast for food-drinks manufacture (G74NFarg1) by Experiment 3 which concerns on a present Example. It is explanatory drawing of the base sequence analysis of the yeast for food-drinks manufacture (G74NFarg2) by Experiment 3 which concerns on a present Example. It is explanatory drawing of the base-sequence analysis of the yeast for food-drinks manufacturing (G74NFarg3) by Experiment 3 which concerns on a present Example. It is explanatory drawing of the base sequence analysis of the yeast for food-drinks manufacture (G74NFarg4) by Experiment 3 which concerns on a present Example. It is explanatory drawing of the base sequence analysis of the yeast at the time of mixing the yeast for food-drinks manufacture by Experiment 3 which concerns on a present Example. It is an electrophoresis pattern which discriminate | determines the yeast in the sake lees by Experiment 4 which concerns on a present Example. It is an electrophoresis pattern which discriminate | determines the yeast in sake made by the experiment 4 which concerns on a present Example. It is an electrophoresis pattern of the sample of Experiment 5 which concerns on a present Example.

  An embodiment of the present invention which is considered to be suitable will be briefly described with reference to the drawings showing the operation of the present invention.

  The yeast for producing foods and beverages (hereinafter simply referred to as “yeasts for producing foods and beverages”) that allows easy identification of the identity of the yeast of the present invention is performed by mutating a predetermined gene of yeast to determine the identity of the yeast. Since the yeast is given the identification information and mutated a predetermined gene of the yeast to give the identification information to the yeast, the yeast identification is improved and the yeast for food and beverage production that makes it easy to identify the identity of the yeast In contrast to the conventional method of identifying yeast strains by finding a homologous and polymorphic portion of a gene, the present invention makes it easy to quickly mutate a mutated portion by causing a mutation in a given gene. In addition, the yeast can be easily identified by adding this mutation as yeast identification information. Therefore, for example, it is possible to immediately determine whether or not the food is a food or drink manufactured using a predetermined food or drink manufacturing yeast by determining identification information from a predetermined gene of the yeast of the food or drink manufactured using yeast.

  Moreover, since the mutated predetermined gene is a gene that does not need to be expressed in the production of food and drink, the food and drink can be produced even if this gene is not expressed, and the quality of the food and drink produced using this yeast is improved. The reduction can be reduced as much as possible, and for example, a food or drink with reduced deterioration of yeast brewing characteristics and fermentation characteristics as much as possible can be manufactured.

  Therefore, for example, when this identification information is used in association with management information for manufacturing a food or drink manufactured using this yeast, the manufacturing information of the food or drink manufactured using this yeast for manufacturing food or drink is used as the yeast itself. Since the yeast or yeast nucleic acid is obtained from the food or drink manufactured using yeast and the identification information is determined from the predetermined gene of this yeast, the origin of the food or drink can be determined. Therefore, it is possible to immediately determine whether or not the food or drink is manufactured using a predetermined yeast for manufacturing food or drink, thereby improving the traceability of the food or drink and improving the reliability of the food or drink.

  Further, for example, the base sequence pattern of the mutated gene is given as identification information, and different identification information is given depending on attributes such as the properties and functions of the yeast, and this identification information is used for classification and identification of such yeast. When used in association with management information, it is possible to classify yeasts in detail according to the properties and functions of the yeast, and it is possible to classify yeasts having the same properties and functions more strictly and to discriminate them very easily. Therefore, when foods and drinks are manufactured using the yeasts for food and drink manufacturing precisely classified as described above, it is possible to contribute to stabilization and improvement of the quality of foods and drinks manufactured using these yeasts.

  Moreover, according to the food / beverage products of the present invention, the yeast used in the production of food / beverage products such as alcoholic beverages and various foods is a gene that does not need to be expressed in the production of food / beverage products. For example, it is possible to produce a food or drink with as little deterioration or decrease in brewing characteristics and fermentation characteristics as possible. And since the identification information of this yeast can be confirmed by discriminating the predetermined | prescribed gene of yeast used for manufacture of food / beverage products, it becomes possible to grasp | ascertain the origin of this food / beverage products correctly. Thus, for example, when this identification information is associated with management information for the production of foods and beverages produced using this yeast, the origin of the yeast used for production can be determined and the traceability of the foods and beverages is improved. It is possible to improve the reliability of this food and drink, for example, it is possible to distinguish it from food and drink produced without using the yeast for producing food and drink of the present invention. Can be improved. In addition, when the identification information is used in association with the management information for classification and identification of such yeast, since the attributes and classification of the yeast can be performed more accurately, it is only excellent in distinguishability from other yeasts. In addition, for example, since the properties and functions of the yeast are uniform and the yeast purity can be maintained at a higher level, the stability of the production process of these foods and beverages is improved. It becomes easier to keep the quality of the prepared food and drink constant, and it can contribute to stabilizing the quality of the manufactured food and drink and improving the quality.

  Further, according to the yeast identity discrimination method (hereinafter simply referred to as “yeast identity discrimination method”) used in the production of the food and drink according to the present invention, for example, a distribution product produced using yeast. The yeast or yeast nucleic acid is obtained from the food or drink, and the base sequence pattern of the yeast gene is matched with the base sequence pattern corresponding to the identification information of the food or drink production yeast set in advance when the food or drink is manufactured. And if it matches, it is possible to determine that the food or drink is manufactured using the yeast for manufacturing food or drink, and to easily determine the origin of the food or drink. Traceability can be improved and the reliability of this food and drink product can be improved, and food and drink products that do not use the specified yeast can be identified. become, It is possible to conduct a quality check in the distribution process of the food, also produce the effect of increasing the reliability of the product by increasing the reliability of the product labeling. In addition, when using yeast for food and beverage production in which management information for classification and identification of yeast is given as identification information to a predetermined gene of yeast, precise classification and identification of attributes such as finer properties and functions of yeast It is easy to maintain the quality of food and drink using this yeast, and can contribute to stabilization and improvement of product quality. become.

  When the present inventors give a mutation to the arginase gene (CAR1 or YPL111W) of sake yeast, the arginase gene product lacks a predetermined function, and the arginase-deficient yeast (that is, the ability of the arginase gene to mutate to metabolize arginine) (A non-urea-producing yeast with no or reduced pH) is obtained, and the knowledge that this yeast does not affect brewing characteristics in alcoholic beverage production (Kitamoto et al., J. Brew. Soc. Japan, Vol. 87, p. 602) -607, 1992), and the arginase gene of arginase-deficient yeast obtained from CAO medium (Japanese Patent Publication No. 6-97993) containing canavanine, arginine and ornithine has random base substitutions and deletions. And found that there are diversity in mutation sites and mutation types, and using this mutation diversity, the specific arginase-deficient yeast Found that it can be used for individual identification of yeast, and that it can be used for discrimination of yeast identity without affecting the quality of brewing characteristics, etc., and mutations corresponding to each of these mutations The present invention was completed by confirming that a simple and rapid detection system using a detection primer for detecting a site could be designed.

Specifically, it is confirmed that the arginase gene of this yeast is about 1000 bases long, and when the above-mentioned mutation occurs at random, the number of combinations of mutations is 4 ¹⁰⁰⁰ , that is, 10 ⁶⁰² or more. Since there can be 1000 patterns even if only one base substitution is made, this sequence pattern can be used as identification information, specifically, management information for the production of food and drink such as alcoholic beverages and foods, or the conventional It can be used as management information that further identifies and classifies the attributes of the yeast individuals that were the same species and the same strain of the same fermentation characteristics, and as management information for production, for example, for each production region or each brewery, Or it came to the idea that it can allocate for every kind of liquor.

  Therefore, for example, a mutation is given to the arginase gene of yeast, and the mutated yeast contains at least one of identification information of the food and drink, for example, the production place, production time, production lot, product type, and manufacturer of the food and drink When a product is produced using yeast having the same arginase gene of the same mutation for each production management unit assigned as management information for production, the yeast that produces this food and drink is quaternary encoded with the base of the gene. Identification information, that is, production management information can be given, and the yeast in which the arginase gene is mutated becomes a non-urea-producing yeast without impairing the fermentation characteristics, so that extremely high quality food and drink can be obtained.

  Furthermore, when the mutation is imparted to a predetermined gene, the present inventors have lost the predetermined function of the gene product of this gene and are deficient, and the yeast gene described above as a yeast gene that does not affect fermentation characteristics. Carriers with production information of foods and beverages that use the yeast to confirm the presence of the acid phosphatase gene (PHO3 or YBR092C) in addition to the arginase gene (CAR1 or YPL111W) and to mutate this gene region It has also been found that it can function as.

  The present inventors have already developed a method for discriminating between a specific sake yeast and a yeast other than sake yeast using the presence or absence of this acid phosphatase activity (Japanese Patent No. 4627972). Is mutated in the above-mentioned acid phosphatase gene (forming a fusion gene in which the PHO3 gene is fused downstream of the PHO5 gene), and acid phosphatase activity is inactivated while maintaining brewing characteristics and fermentation characteristics In the same manner as the arginase gene, for example, by mutating the acid phosphatase gene of sake yeast having an acid phosphatase (Kyokai No. 8, malic acid high-productive polyacid yeast No. 28, etc.), It discovered that manufacturing information or the identification information of yeast could be provided.

  The yeast used in the present invention is, for example, sake yeast (Japan Brewing Association Kyoto 7 yeast, Kyoto 9 yeast, Kyoto 10 yeast, high ester producing yeast 1701, high ester producing yeast 1801, etc.). , Saccharomyces mainly used in the production of alcoholic beverages and foods such as wine yeast (Japan Brewing Association Grape No. 1 yeast, Grape No. 3 yeast, Grape No. 4 yeast, etc.), beer yeast, baker's yeast etc. A yeast belonging to the genus Saccharomyces cerevisiae.

  Although discrimination between these different kinds of yeasts has been conventionally performed, it was extremely difficult to discriminate between yeasts belonging to the same strain. When using the present invention that imparts a mutation corresponding to yeast identification information to a predetermined gene of this yeast, even yeast belonging to the same species, for example, No. 7 yeast, No. 8 yeast, Identification information unique to the individual can be given as a marker to a yeast individual separated by genetic analysis or selection. Therefore, since the yeast obtained by using this identification information as a mark can be easily identified, the identification and management of the yeast becomes extremely easy and the management of food and drink using this yeast also becomes easy. Quality stabilization and quality improvement are also possible.

  In addition, the yeast may be subjected to natural mutation treatment, mutation treatment, or gene recombination treatment to impart a mutation to a predetermined gene of yeast.

  In addition, since yeast naturally causes mutation with a certain degree of probability without special artificial mutation treatment, it is possible to cause mutation in a predetermined target gene by such natural mutation. Therefore, for example, in arginase-deficient yeast, the yeast is naturally mutated in the above-mentioned CAO medium (medium containing canavanine, arginine and ornithine), and a method for measuring arginase activity in yeast cells by Kitamoto et al. (Kitamoto K. et al, J. Ferment. Bioeng., Vol.75, p.359-363, 1993) or assay method using ornithine medium and arginine medium (Kitamoto et al., J. Brew. Soc. Japan, Vol.87, p. 598-601, 1992) can be used to obtain yeast having no or reduced arginase activity, ie, arginase-deficient yeast.

  As a method by mutation, as a method by physical means, for example, a method by ultraviolet irradiation or radiation irradiation, a method by chemical means, ethylmethanesulfonic acid (EMS), N-methyl N′-nitro, etc. There is a method of suspending bacterial cells in a solution of a mutation agent such as -N-nitrosoguanidine (NTG), and these methods can be appropriately carried out alone or in combination.

  Confirmation of the mutation state of the yeast treated with mutation as described above is carried out by analyzing the base sequence of a predetermined gene of this yeast by the chemical decomposition method (Maxam & Gilbert method) and the chain terminator method (Sanger dideoxy method). This base sequence is compared with the gene sequence of a database of a predetermined gene having no mutation to determine a mutation site, and a yeast having one or more base insertions, deletions or substitutions of one or more bases in the predetermined gene is determined. The yeast can be selected to have a mutation added to a predetermined gene.

  For example, in the case of the arginase gene, the base sequence in the arginase gene is analyzed, and the base sequence of the arginase gene before deletion, as reported by Roberta A. et al. (Roberta A. et al., J. Bacteriol., Vol. 103, p.1079-1087, 1984) and gene databases (Sake Yeast Genome Database (http://nribf1.nrib.go.jp/SYGD/) SYGD ID: e8011600201 (Systematic Name: K7_YPL111W) for sake yeast genomes) ) And the base sequence of the arginase gene of the yeast in which the arginase gene was mutated to determine the mutation site, insertion or deletion of one or more bases in the given arginase gene, A yeast having one or more substitutions may be selected and used as a specific arginase-deficient yeast deficient due to a mutation in the arginase gene. Arginase deficiency may be determined from only the base sequence of the arginase gene and may be a specific arginase-deficient yeast.

  In the present invention, it can be said that this predetermined gene of yeast is used as an information recording area for holding management information for manufacturing food or drink or management information for classification and identification of yeast. In addition, when producing foods and drinks using yeasts with this gene mutated, foods and drinks using only yeasts that have been mutated with the above-mentioned predetermined genes and given management information for production and management information for classification identification However, alcoholic beverages or foods produced using yeast that does not have a mutation in a predetermined gene, such as normal yeast, can be used to produce foods and beverages by mutating the predetermined gene of the present invention. It may be possible to add a suitable amount of the yeast for food and beverage production to which the management information for classification and management information for classification and identification of yeast is added in advance, or a plurality of types of yeast for food and beverage production with different management information. You may make it use.

  In addition, nucleic acid amplification of yeast genes obtained from foods and drinks is performed using at least a pair of primers consisting of a forward primer and a reverse primer that binds to a specific gene region and specifically anneals, for example, polymerase chain reaction ( PCR, LAMP (Loop-mediated isothermal amplification) method, isothermal gene amplification (ICAN) method, SDA (Strand displacement amplification) method, etc. may be used.

  Whether the yeast contained in the food or drink is a yeast having a predetermined gene by comparing the base sequence pattern of the gene obtained by this nucleic acid amplification with a pattern corresponding to the yeast identification information set in advance. Furthermore, in the case of a predetermined yeast, management information for manufacturing the food and drink and management information for classification and identification of the yeast can be obtained from the predetermined gene of the yeast.

  Therefore, since the present invention provides the yeast gene used for the production of food and drink with the identification information of the food and drink or yeast, the yeast is obtained from the appropriate food and drink produced using the yeast, Whether the food / beverage products or yeast identification information set in advance from a predetermined gene is detected, and the origin of the food / beverage products or yeast can be determined from the contents thereof, so that the traceability of the food / beverage products as a product can be improved. It is possible to improve the reliability of the product, and it is possible to discriminate foods and beverages that do not use a predetermined yeast. There is also an effect of improving the reliability of display.

  Further, since it becomes possible to identify and classify attributes such as more detailed properties, brewing characteristics, and fermentation characteristics of yeast, it is possible to perform production management (microorganism management) such as yeast purity measurement quickly and easily. Therefore, since the yeast purity in the production process of foods and drinks produced using yeast such as sake mothers and koji can be maintained higher than before, the stability of the production process of these foods and drinks is improved, and this yeast is used. It becomes easy to keep the quality of the manufactured food and drink constant, and it can contribute to stabilization and improvement of product quality.

  In addition, using the yeast for producing foods and beverages of the present invention, various foods and beverages, for example, sake, fruit wine, other brewed sake, continuous distilled spirits, single distilled spirits, whiskey, brandy, alcohol for raw materials, spirits , Beer, sparkling liquor, other sparkling liquor, synthetic sake, mirin, sweet fruit liquor, liqueur, powdered liquor, miscellaneous liquor, or alcoholic beverages such as these, or bread, liquor, pickles Seasonings such as yeast extract, health foods or supplements containing yeast, or fermented seasonings such as miso, soy sauce, vinegar, or cakes, cookies, sake buns, Chinese buns and other sweets, or cider, juice , Can produce soft drinks such as beer-taste beverages, and may be processed products made from these foods and beverages, to obtain yeast or yeast nucleic acid It may be any food or drink that can be.

  Specific embodiments of the present invention will be described with reference to the drawings.

  This example is a yeast for food and beverage production that is easy to determine the identity of yeast by mutating a predetermined gene of yeast used for the production of food and beverage, and imparting identification information when determining the identity of the yeast, The predetermined gene to be mutated is a gene that does not need to be expressed in food production, and specifically, this gene is the same as that of yeast characterized in that it does not deteriorate the fermentation characteristics of yeast or is less deteriorated. This is a yeast for producing foods and beverages (hereinafter simply referred to as “yeasts for producing foods and beverages”) that is easy to discriminate sex.

  In addition, this example produced sake using this yeast for producing foods and beverages, and sake produced using this yeast for producing foods and beverages was produced using other sakes produced without using this yeast for producing foods and beverages. It is confirmed that it can be easily distinguished.

  The identification information of the present embodiment gives the base sequence pattern of the mutated predetermined gene as yeast identification information, and not only the identity of yeast, but also management information for classification and identification of yeast, or food and drink It is used in association with management information for manufacturing.

  Here, the management information for the production of food and drink includes the production place (country, prefecture, municipality, etc.), production time (year, etc.), production lot, production history, It is the management information for manufacture of the food / beverage products containing at least any one of a product classification, a manufacturer, etc. When this management information for the production of food and drink is used in association with the yeast identification information, the production information of the food and drink is given to the yeast itself, so the yeast is obtained from the food and drink produced using the yeast. Alternatively, by extracting a nucleic acid derived from yeast from a food and drink and discriminating identification information from a predetermined gene of this yeast, it is possible to determine the origin of production of this food and drink, and manufacturing using a predetermined yeast for producing food and drink Since it is possible to immediately determine whether or not the food or drink has been made, it is possible to improve the traceability of the food or drink and improve the reliability of the food or drink.

  Further, management information for classification and identification of yeast includes sake yeast (Japan Brewing Association Kyoto 7 yeast, Kyoto 9 yeast, Kyoto 10 yeast, high ester producing yeast 1701, high ester producing yeast 1801. Etc.), wine yeast (Japan Brewing Association grape wine No. 1, yeast No. 3 yeast, wine No. 4 yeast etc.), beer yeast, baker's yeast, etc. In yeast belonging to the genus Saccharomyces cerevisiae, for example, yeast classification and identification information for classifying yeasts of the same species, such as Kyoto 7 yeast, or closely related yeasts, for example, the selection time or place of yeast, or It can be used for identifying more detailed properties of yeast, such as brewing characteristics and fermentation characteristics. Therefore, by giving management information for classification and identification of yeast, it is possible not only to have excellent discrimination from other yeasts, but also to improve the purity of yeast evenly because the properties and functions of yeast are uniform. Thus, it becomes easier to keep the quality of the food and drink manufactured using this yeast constant, and it is possible to contribute to stabilizing the quality and improving the quality of the manufactured food and drink.

  In addition, the management information for production or the management information for classification and identification of yeast as identification information given to the yeast in this example is obtained by mutating a predetermined gene of yeast previously selected as sake yeast by natural mutation or mutation. The base sequence pattern of the mutated gene is made to correspond to each of the production management information of food and drink or the identification management information of yeast. For example, the base of the yeast gene is assembled by genetic recombination technology. (It is of course possible to recombine a given gene by gene recombination technology).

  In addition, in the yeast for producing foods and beverages, a gene that does not deteriorate the brewing characteristics or fermentation characteristics of the yeast or a gene with little deterioration is selected as the predetermined gene to be mutated. In the yeast for producing foods and drinks of this example, this predetermined gene is a gene that does not deteriorate the brewing characteristics even by mutation, and by adding this mutation, the predetermined function of the gene product is lost or enhanced. It is a gene that can be easily screened for yeast having a mutated gene.

  In addition, a gene that does not degrade the brewing characteristics and fermentation characteristics of yeast or a gene with little deterioration is, in other words, a gene that does not need to be expressed for brewing, particularly in yeast for food and drink such as sake brewing. Therefore, it is a gene that is not expressed for brewing, or a gene that is not desired to be expressed, and more specifically, a gene that can produce food and drink without hindrance even if it is not expressed in yeast.

  Genes that do not need to be expressed in the production of such foods and beverages include carbon dioxide generation that occurs from foods and beverages using yeast or food and beverage products when comparing the production of foods and beverages using yeast before and after mutation A gene that does not decrease the amount, a gene that does not reduce the ability of yeast to grow in a food or beverage product or a food product using yeast is selected.

  Specifically, in sake yeast, when comparing sake production with sake yeast before and after mutation, a gene that does not reduce the amount of carbon dioxide generated in the koji during the fermentation period, a gene that does not reduce the amount of alcohol produced, Genes that do not increase the acidity of sake, genes that do not reduce the alcohol tolerance of yeast, genes that do not reduce the growth ability of yeast during fermentation, genes that do not decrease the concentration of isoamyl acetate or ethyl caproate that are ginjo aromas in sake It is.

  More specifically, for example, in sake production, as described in a report by Akao et al. (Akao T. et al, DNA Res., Vol. 18, p.423-434, 2011) Genes that are expressed in the laboratory yeast of, but are not present or not expressed in sake yeast (Kyokai No. 7), and the aforementioned report of Kitamoto et al. (Kitamoto et al., J. Brew. Soc. Japan, Vol.87, p.602-607, 1992). In bread production, the arginase gene reported by Shima et al. (Shima J. et al., Appl. Environ. Microbiol., Vol. 69, p.715-718, 2003) can be mentioned.

  The present inventors have found an arginase gene and an acid phosphatase gene as such predetermined genes of yeast, and in this example, these genes are naturally mutated to obtain the above-mentioned identification information.

  In this example, the yeast in which a predetermined gene was mutated was obtained by naturally mutating the arginase gene obtained by natural mutation in a selective medium (CAO medium, Japanese Patent Publication No. 6-97993) containing at least canavanine, arginine and ornithine. Thus, the arginase-deficient yeast is deficient in arginase. In addition to the positive selection method using the CAO medium, a replica method by Ouchi et al. (Ouchi et al., Fermentation Engineering, Vol. 61, p.349-352, 1983) may be used.

  In a yeast in which a mutation is imparted to a predetermined gene obtained in the CAO medium of this example, the arginase gene is mutated, and one or more bases in the repetitive sequence of the arginase gene before the mutation are deleted, inserted or substituted. However, in this example, two or more bases in the repetitive sequence consisting of 4 to 10 bases of 2 to 5 repeats existing in the base sequence of the arginase gene before mutation are missing. Arginase-deficient yeast that was lost or substituted by one base was obtained.

  In this example, the mutation site of this arginase gene is a mutation in which base 30 at position 30 of the arginase gene before mutation is replaced with base A, or a mutation in which base G at position 422 is replaced with base A. 448 A mutation in which the base G at the position is replaced with the base A, a mutation in which the base G at the position 490 is replaced with the base A, a mutation in which the base G at the position 491 is replaced with the base A, or a base G at the position 622 Mutation in which base A is substituted, mutation in which base C at position 761 is replaced with base T, mutation in which AG is deleted from base sequence AGAG at positions 817 to 820, or bases at positions 834 to 839 Any of the mutations in which GA has been deleted from the sequence GAGAGA has occurred, but any of these mutations may be mutations in which two or more of these mutations have occurred simultaneously.

  A summary of the base sequence of the arginase gene of yeast used in this example is shown in Table 1. Table 1 shows the base sequence of the arginase gene in which the 501st base is G in the yeast yeast No. 7 whose sequence identifier of the sequence of the arginase gene is represented by SEQ ID NO: 1 in the sequence list (Table 1 and FIG. 1). -3, the strain name is expressed as K7G), and the base sequence of the arginase gene in which the 501st base of the yeast yeast No. 7 whose sequence identifier of the base sequence of the arginase gene is represented by SEQ ID NO: 2 in the sequence listing is T (In Table 1 and FIGS. 1 to 3, the strain name is expressed as K7T), the CAR1 nucleotide sequence of Niigata sake yeast G74NF, in which the sequence identifier of the sequence of the arginase gene is represented by SEQ ID NO: 3 in the sequence listing, and the gene sequence Each of the sequence identifiers has a base sequence represented by SEQ ID NO: 4 to 10 in the sequence listing, and each of the arginase genes of the parent strain G74NF Niigata sake yeast G74NFarg1-7 to which a difference is given, the sequence identifier of the sequence of the arginase gene is shown in SEQ ID NO: 11 of the sequence listing, and the sequence identifier of the gene sequence is the SEQ of the sequence listing Each of the parent strain G9 arginase genes having the base sequences represented by ID NOs: 12 and 13 is mutated, and Niigata sake yeast G9arg1 and 32 are shown. The base sequence data of these genes are shown in the sequence listing. .

  G74NFarg1-7 shown in Table 1 have G74NF as a parent strain, G9arg1 and G9arg32 have G9 as a parent strain, and these G74NFarg1-7, G9arg1 and G9arg32 are located at different base positions of the arginase genes of G74NF and G9, respectively. Mutations are given, and the arginase gene is mutated by these mutations and the ability to metabolize arginine is reduced or reduced, in other words, an arginase-deficient yeast, the arginase gene is mutated to convert arginine to ornithine Yeast that has no or reduced ability to convert and cannot use arginine as a nitrogen source, that is, yeast that does not produce urea in the yeast fermentation product or has a reduced amount of urea produced (non-urea producing yeast) It is. Therefore, since it becomes a urea non-producing yeast and the production of carcinogenic ethyl carbamate is suppressed, the food and drink produced with this arginase-deficient yeast can improve its quality.

  As a method by mutation, as a method by physical means, for example, a method by ultraviolet irradiation or radiation irradiation, a method by chemical means, ethylmethanesulfonic acid (EMS), N-methyl N′-nitro, etc. There is a method of suspending bacterial cells in a solution of a mutation agent such as -N-nitrosoguanidine (NTG), and these methods may be carried out as appropriate, alone or in combination.

  Of course, it is possible to recombine a given gene by genetic recombination technology. Mutation can be introduced according to a conventional method, for example, Methods in Enzymology, Vol.194, Guide to Yeast Genetics and Molecular Biology (1991 by Academic Press, Inc., p.281-301), yeast gene experiment manual ( Maruzen Co., Ltd., issued December 10, 2002). More specifically, the method of Aritomi et al. (Aritomi K. et al., Bioscience. Biotechnology. Biochem., Vol. 68, p.206-214, 2004) and the Aureobasinin A resistant yeast transformation system using pAUR135 DNA ( Takara Bio Co., Ltd.) introduced the arginase gene having the above-mentioned mutation or the arginase gene obtained from the base sequence analysis of the arginase gene of the arginase-deficient strain obtained from the CAO medium into yeast that is not deficient in arginase. A specific arginase-deficient yeast can be obtained. The yeast transformation methods include general methods such as lithium acetate method (Ito H. et al., J. Bacteriol., Vol.153, p.163-168, 1983), spheroplast method and electroporation method. Various methods can be adopted. Furthermore, transformation kits such as S. cerevisiae Direct Transformation Kit Wako (Wako Pure Chemical Industries, Ltd.) and DS Yeast Transformation kit (Dualsystems Biotech) can also be used.

  In addition, “Chapter 2 Transformation Theory and Experiments of Transformation Methods Using PCR Products (for example, Two Hybrids that can be used in Bio Experiment Illustrated Volume 7 (Shyujunsha, published October 30, 2003)) , 5. Gene disruption, 5-1 Gene disruption (replacement) method using PCR, 5-2 Confirmation of substitution ", p.70-74) and Manivasakam et al. (Manivasakam P. et al., Nucleic Acids Res., Vol. 23, p.2799-2800, 1995) can also be used. Specifically, a primer for amplifying a yeast arginase gene containing a mutation site of a specific arginase-deficient yeast arginase gene is prepared by a conventional method. A PCR method (for example, Saiki et al.) Using as a template a yeast genomic DNA prepared using a DNA extraction kit such as Toru-kun (Takara Bio Inc.) or a PCR product derived from an arginase gene prepared as described in the Examples. After obtaining a PCR product derived from the arginase gene containing the mutation site of the arginase gene by the method of (Saiki RK et al., Science, Vol. 230, p.1350-1354, 1985), the arginase was used using this PCR product. The yeast that is not deficient is transformed by a conventional method, and a yeast that can grow on the above-mentioned CAO medium is selected from the transformant, and described in the Examples. By detecting the arginase mutation site and analyzing the base sequence of the arginase gene, confirming that the mutation has been introduced into this yeast, a specific arginase-deficient yeast may be used. Alternatively, a base sequence corresponding to identification information such as management information for manufacturing the food or drink is given in the primer sequence or subjected to the PCR method as a part of the template, and is easily transformed by the obtained PCR product. It is also possible to add identification information to the arginase gene, and the yeast to which the gene is introduced by genetic recombination is preferably haploid, and the yeast culture method, haploid acquisition method, spore formation method, etc. As for the general operation, a conventional method described in Yeast Molecular Genetics Experimental Method (edited by Taiji Oshima, Academic Publishing Center, published in April 1996) may be used.

  Further, when the predetermined gene to be mutated is an acid phosphatase gene, the yeast in which the predetermined gene is mutated is selected containing at least a phosphate ion, 5-bromo-4-chloro-3-indolephosphate phosphate salt By mutating in a medium, an acid phosphatase-deficient yeast deficient in acid phosphatase can be obtained. The sake yeast deficient in this acid phosphatase will also have distinction from the wild yeast having this acid phosphatase.

  Confirmation of the mutation state of the yeast treated as described above is performed by analyzing the base sequence of a predetermined gene of this yeast by the chemical decomposition method (Maxam & Gilbert method) and the chain terminator method (dideoxy method by Sanger et al.). Compare this base sequence with the gene sequence of the database of a given gene without mutation to determine the mutation location, and there are one or more base insertions, deletions and substitutions in the given gene. What is necessary is just to select yeast and make it the yeast for food-drinks manufacture by which the variation | mutation was added to the predetermined gene.

  Thus, when a predetermined gene is mutated, specific identification information can be given to the yeast itself as a marker, and the acquired yeast can be easily identified by using this identification information as a marker. Management becomes extremely easy, and further, management of food and drink using this yeast is facilitated, and furthermore, the production quality of this food and drink can be stabilized and improved.

  Moreover, the food / beverage products of a present Example are the food / beverage products manufactured using this yeast for food-drinks manufacture, Comprising: As the yeast used for manufacture of food / beverage products, the variation | mutation was made to the predetermined gene as mentioned above Manufacture of food and drink using yeast based on the respective regular methods, and management information for the production of food and drink by discriminating a predetermined gene of the yeast contained in the food or drink, or the production of this food and drink Management information for classification and identification of the used yeast can be confirmed.

  Here, this food and drink includes sake, fruit liquor, other brewed liquors, continuous distilled shochu, single distilled shochu, whiskey, brandy, raw alcohol, spirits, beer, sparkling liquor, other sparkling liquors, Contains synthetic sake, mirin, sweet fruit liquor, liqueur, powdered liquor, miscellaneous liquor, liquor such as liquor, or food such as bread, sake lees, pickles, seasoning such as yeast extract, yeast Health foods or supplements, fermented seasonings such as miso, soy sauce, vinegar, or sweets such as cakes, cookies, sake buns, Chinese buns, or soft drinks such as cider, juice, beer-taste beverages, and Processed products made from these foods and drinks may be used as long as they are foods and drinks from which yeast or yeast nucleic acids can be obtained.

  It should be noted that these foods and drinks may use a plurality of types of yeasts for producing foods and drinks with different mutations in a predetermined gene, and food and beverage products having a plurality of types of identification information, for example, management information for production. May be produced using two types of yeast, ie yeast for food and beverage production having management information for classification and identification of yeast, or a plurality of yeast for food and beverage production having management information for different production You may manufacture using a kind, and you may manufacture using multiple types of yeast for food-drinks manufacture which has the management information for classification identification of a different yeast.

  When the food and drink of this example is fermented using rice, rice bran, and yeast to produce koji, at the time of charging a liquor produced with rice and rice koji, or at the time of charging the koji, or the above Sake produced using the yeast for producing foods and drinks during the fermentation of the sake mother and the koji, and the sake production is a conventional method and not a special production method. It has distinctiveness such as discrimination and classification of yeast. In addition, steamed rice is usually used as the rice to be used for koji manufacture and sake mother preparation. However, alpha rice may be used, and when koji is produced, brewed in addition to rice, rice koji, and yeast. You may use auxiliary materials, such as alcohol and glucose.

  Moreover, the identity discrimination | determination method of the food / beverage products of a present Example is the identity discrimination method of the food / beverage products which discriminate | determines the identity of the food / beverage products manufactured using yeast, Comprising: Yeast from the food / beverage products manufactured using yeast Alternatively, the yeast nucleic acid is obtained, and it is determined whether or not the base sequence pattern of the predetermined gene of this yeast matches the sequence pattern of the predetermined gene of the yeast for producing food and drink of this example. It is the method of discriminating that the food / beverage products manufactured using this yeast are the same as the food / beverage products manufactured using the yeast for food-drinks manufacture of a present Example. That is, the identity determination method of the food / beverage products of a present Example is a method of determining the identity of food / beverage products by confirming the identity of the yeast currently used for this food / beverage product.

  In the method for determining the identity of yeast used in this food and drink, the base sequence pattern of a predetermined gene of yeast binds to a predetermined region of the predetermined gene of this yeast and specifically anneals, forward primer and reverse It is obtained by nucleic acid amplification using at least a pair of primers consisting of primers.

  The primer of this example is a primer that amplifies a sequence containing a mutation site of the yeast arginase gene. In designing this primer, the mutation site may be obtained as a DNA amplification fragment, or the mutation site. May be the primer sequence. When this mutation site is used as a primer sequence, the mutation site can be confirmed by an allele PCR method or a real-time PCR method.

  Specifically, in this example, a predetermined gene of yeast is detected after extracting or concentrating nucleic acid by an appropriate method such as using a food or drink as it is as a nucleic acid sample or using a commercially available nucleic acid extraction kit. After obtaining a nucleic acid sample and amplifying the nucleic acid by coexistence with a predetermined primer, as a combination of primers consisting of a forward primer and a reverse primer, the base sequences are SEQ ID NO: 18 and 19, SEQ ID NO: 18 and 19, SEQ ID The use of one or more of the following methods (1) to (5) using a pair of primers of the base sequence indicated by the sequence identifier of NO: 20 and 21 or SEQ ID NO: 22 and 23 The identity of the food and drink is determined by determining the identity of the yeast used for production (details are given in detail in Experiment 3).

(1) a forward primer having a base sequence represented by SEQ ID NO: 18 in the sequence listing;
Using the reverse primer of the base sequence shown in SEQ ID NO: 19, the arginase gene of the yeast was amplified by PCR, and this amplified fragment was restricted to restriction enzyme B.
After treatment with stXI, agarose gel electrophoresis is performed, and the identity of the yeast is discriminated based on the difference in the electrophoresis pattern.

(2) a forward primer having a base sequence represented by SEQ ID NO: 20 in the sequence listing;
Using the reverse primer having the base sequence shown in SEQ ID NO: 21, the arginase gene of the yeast was nucleic acid amplified by the PCR method, and the amplified fragment was restricted to the restriction enzyme C.
After treatment with viKI-1, agarose gel electrophoresis is performed, and the identity of the yeast is determined by the difference in the electrophoresis pattern.

(3) a forward primer having a base sequence represented by SEQ ID NO: 18 in the sequence listing;
Using the reverse primer of the base sequence shown in SEQ ID NO: 19, the arginase gene of the yeast was amplified by PCR, and this amplified fragment was restricted to restriction enzyme B.
After treatment with siHKAI, agarose gel electrophoresis is performed, and the identity of the yeast is determined by the difference in the electrophoresis pattern.

(4) a forward primer having a base sequence represented by SEQ ID NO: 20 in the sequence listing;
Using the reverse primer of the base sequence shown in SEQ ID NO: 21, the arginase gene of the yeast was nucleic acid amplified by the PCR method, and this amplified fragment was digested with restriction enzyme B
After treatment with slI, agarose gel electrophoresis is performed, and the identity of the yeast is discriminated based on the difference in the electrophoresis pattern.

(5) a forward primer having a base sequence represented by SEQ ID NO: 22 in the sequence listing;
Using the reverse primer of the base sequence shown in SEQ ID NO: 23, the arginase gene of the above-mentioned yeast was nucleic acid amplified by PCR, and this amplified fragment was restricted to restriction enzyme B
After treatment with slI, agarose gel electrophoresis is performed, and the identity of the yeast is discriminated based on the difference in the electrophoresis pattern.

  The composition of the reaction solution and cycling conditions for nucleic acid amplification performed by the PCR (polymerase chain reaction) method using at least the primer of any one of (1) to (5) are the nucleic acid amplification method used, the Tm value of the primer, and the thermal cycler. Determine according to the specifications. Specific examples of nucleic acid amplification using the PCR method are shown in Experiments 3 to 5 below.

  In this embodiment, the nucleic acid sample is DNA. However, when RNA is amplified by nucleic acid, for example, complementary DNA may be synthesized by reverse transcription and then amplified.

  In this example, the production area of a food or drink such as alcoholic beverages or foods using yeast is determined from the base sequence pattern of the amplification product obtained as a result of the nucleic acid amplification described above. The base sequence pattern of the amplification product is any one of the molecular weight size, base length, base sequence, and abundance (relative or absolute amount) of each nucleic acid amplification fragment contained in the reaction solution after completion of the nucleic acid amplification reaction. The pattern.

  Specifically, the band pattern obtained by electrophoresis of the reaction solution after completion of the nucleic acid amplification reaction directly or further treated with a restriction enzyme and the relative intensity of each band. In addition, as electrophoresis, for example, detection of single-base deletion or insertion using a Spreadex gel (Elchrom Scientific), or SSCP (single-stranded conformation type) using a GMA gel (Elchrom Scientific) Detection of base substitution such as a method, or a method of treating a PCR product with a restriction enzyme can be used. Further, a quantitative PCR method or a real-time PCR method may be used.

  When using the method for determining the identity of yeast in food and drink according to this example, in the normal yeast that is not deficient in arginase, the arginase gene of the nucleic acid amplification product obtained as described above has a large number of strains. A common result is obtained. On the other hand, in the arginase-deficient yeast of this example (for example, G74NFarg1-7, G9arg1 and G9arg32), there is a clear partial difference in the base sequence of the arginase gene region. Therefore, a specific arginase-deficient yeast can be detected by comparing the sequence patterns of the arginase genes of the gene product as described above. In addition, such a specific arginase-deficient yeast can be reliably discriminated from other normal yeasts.

  The experiment which confirmed the above is shown below. Hereinafter, the arginase gene is mainly referred to as CAR1.

1 Experiment 1 Acquisition of arginase-deficient yeast 1-1 Experimental method (1) Yeast and medium used Niigata sake yeast G74NF strain, G9 owned by Niigata Brewing Experiment Station
Is a stock. The medium is CAO plate medium (0.17% yeast nitrogen base w / oa
mino acid and ammonium sulfate (Difco), 20ppm canavanine (Sigma)
1 mM arginine hydrochloride, 5 mM ornithine hydrochloride, 2% glucose, 2% agar), arginine plate medium (0.17% yeast nitrogen base w / o amino acid
and ammonium sulfate (Difco), 5 mM arginine hydrochloride, 2% glucose,
2% agar), ornithine plate medium (0.17% yeast nitrogen base w / o ami)
no acid and ammonium sulfate (Difco), 5 mM ornithine hydrochloride, 2% glucose, 2% agar), YPD liquid medium (1% yeast extract, 2% polypeptone, 2%
Glucose).

(2) Acquisition of arginase-deficient yeast using CAO plate medium (a) Acquisition by natural mutation
Kitamoto et al. (Kitamoto et al., J. Brew. Soc. Japan, Vol.87, p.598-601, 1992)
The arginase-deficient strain was selected by Specifically, G74NF and G9 strains
After inoculating each of the above in 5 mL of YPD liquid medium and allowing them to stand at 30 ° C. for 3 days.
Apply directly to CAO plate medium without mutation treatment and incubate at 30 ° C for 14 days.
The colonies that were removed from both the arginine plate and the ornithine plate.
Apply each and grow on ornithine plate, but not on arginine plate.
Are selected as candidates for arginase-deficient strains.
Perform single colony isolation again using CAO plate medium and remove arginase deficiency.
Lost stock was obtained.

(B) Acquisition by mutation processing
G74NF strain is inoculated into 5 mL of YPD liquid medium, and statically cultured at 30 ° C. for 3 days
After the G74NF strain was prepared by the method of Oda et al. (Oda et al., “Lysine required from brewing yeast
Isolation of electrophilic mutants ", J. Brew. Soc. Japan, Vol.83, p.614-617, 1988)
Mutation treatment with ethyl methanesulfonate (EMS) (Sigma)
Arginase-deficient strain from CAO plate medium by the same method as in (a) above
Got.

1-2 Experimental results Each of the G74NF and G9 strains was applied to a CAO plate medium, a relatively large colony was fished, and those that could not grow on an arginine plate medium but could grow on an ornithine plate medium were selected. .

In the method by natural mutation, 1,115 grown on CAO plate medium from G74NF strain.
The strain was assayed, and the strain that could not grow on the arginine plate medium but could grow on the ornithine plate medium, and one arginase-deficient strain (hereinafter, this strain is referred to as G74NFarg1 strain).
Obtained. Similarly, 160 strains grown on a CAO plate medium were tested from the G9 strain, and 2 arginase-deficient strains (hereinafter referred to as G9arg1 and G9arg32 strains, respectively).
Obtained.

In addition, in the mutation-treated method, 96 colonies grown on the CAO plate medium were tested, and 6 strains (hereinafter referred to as arginase-deficient strains) that could not grow on the arginine plate medium but could grow on the ornithine plate medium were used. This is expressed as G74NF2-7 strain).

      A summary of these results is shown in Table 1 above.

2 Experiment 2 Nucleotide sequence analysis of CAR1 of arginase-deficient yeast 2-1 Experimental method (1) Extraction of yeast genomic DNA G74NF strain, G9 strain, and G74NFarg1-7, G9a obtained in Experiment 1
This is a DNA extraction and purification kit for yeast from cells obtained by inoculating rg1 and G9arg32 strains in 5 mL of YPD liquid medium, standing at 30 ° C. for 2 days, and then centrifuging the culture. Gen Toru-kun (for yeast) (Takara Bio Inc.)
NA was extracted. The extracted genomic DNA is TE so that the concentration is 3 ng / μL.
It was dissolved in a buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0, Nippon Gene Co., Ltd.) and stored at −20 ° C.

(2) Analysis of the base sequence of CAR1 The base sequence analysis of each yeast was performed by the cloning sequence method or the direct sequence method.

(A) Nucleotide sequence analysis by cloning sequence method
The reagents used in the PCR method include EmeraldAmp PCR Master Mix (Takara Bio (
Co.)) was used. In addition, the forward primer used in this PCR method and
As a reverse primer, SEQ ID NO: 14 shown in Table 2 above
CAR1-4F and SEQ ID NO: 15 CAR1-12R
Primer set, and CAR1-14F of SEQ ID NO: 16
And primer set consisting of CAR1-3R with SEQ ID NO: 17
CAR1 was amplified.

In addition, the PCR reaction solution was prepared to the composition shown in Table 3 and Table 4, and the thermal cycle was performed.
Using Clar (TAKARA PCR Thermal cycler MP, Takara Shuzo Co., Ltd.) at 98 ° C
After heat denaturation for 1 minute, 98 ° C for 10 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute
With a cycling profile of 30 cycles with a temperature profile of 1 cycle
A CR reaction was performed.

The PCR product obtained was purified with QIAquick Gel Extraction Kit (QIAGEN)
The purified DNA fragment was converted into DNA Ligation kit Ver.2.1 (Takara Bio Inc.)
) Was inserted into the plasmid vector pT7 Blue T-vector (Novagen)
. The plasmid into which this DNA fragment was inserted was Escherichia coli (E. coli, DH5α strain).
Using the conventional method, and the cloned plasmid is
Purification was performed using Plus SV minipreps (Promega). The purified plasmid is
, BigDye Terminator Ver.3.1 (Applied Biosystems)
For Kens reaction, Applied Biosystems 3130xl Genetic Analyzer (
The base sequence was determined by Applied Biosystems.

(B) Base sequence analysis by direct sequence method
The amplification of CAR1 by the PCR method was performed in the same manner as in the previous section (a). P obtained
The CR product was run on a 2% agarose gel (SeaKem GTG Agarose, Lonza) 10
After electrophoresis at 0V for 40 minutes and staining with Gel Red (Biotium),
An agarose gel containing the DNA fragment was excised under ultraviolet irradiation. Obtained
PCR products are extracted from the QIAquick Gel Extraction Kit (QIAGEN)
After that, DN using BigDye Terminator Ver.3.1 (Applied Biosystems)
Applied to A sequence reaction, Applied Biosystems 3730xl Genetic Analyzer
The nucleotide sequence was determined by Isa (Applied Biosystems).

2-2 Experimental Results G74NF strain, G9 strain, G74NFarg1 to 7 strains, G obtained by sequencing
9arg1 and G9arg32 and Sake Yeast Genome Database (http: // nribf
1. Carriage 7 (K7 strain) CAR1 (SYG) published on 1.nrib.go.jp/SYGD/)
D ID: e8011600201, Systematic Name: K7_YPL111W).

The results are shown in FIGS. As a result of comparing the base sequences, the K7 strain, the G74NF strain, and the G9 strain had the same base sequence. On the other hand, G74NFarg1 strain is K7 strain and G
As a result of comparison with the 74NF strain, there was a deletion mutation of 2 bp in one location, and G74NFarg2
-7 strains were confirmed to have one base substitution at a different location in each strain. G9arg1
As a result of comparing the strain with the K7 strain and the G9 strain, there is one base substitution at one position, and the G9arg32
The strain was confirmed to have a 2 bp deletion mutation at one site.

Furthermore, in the deletion mutation, the G74NFarg1 has 8 from the 817th position of CAR1.
A mutation in which AG has been deleted from the 20th nucleotide sequence AGAG, and in G9arg32, a mutation has been confirmed in which GA has been deleted from the 834th to 839th nucleotide sequences GAGAGA of CAR1.
A slip mutation found in a microsatellite sequence at a repetitive sequence site in the base sequence of CAR1 (for example, JP 2009-219451 A, Special Table 2010-521156)
No., Agrafioti I., Nucleic Acids Res., Vol.35 (Database issue), D71-D75, 2
007) -like deletion was shown to occur.

From the above results, CAR1 of arginase-deficient yeast obtained using CAO medium.
The mutations in (1) do not depend on the specific nucleotide sequence site, and there are variations in the mutation site and mutation type. For example, when a single base substitution such as that obtained in this experiment occurs in CAR1, the substitution There are about 1000 patterns. When multiple substitutions occur, arginase-deficient yeast having a variety of CAR1 mutation patterns can be obtained.

3 Experiment 3 Construction of discrimination method between arginase deficient yeast 3-1 Experimental method (1) Yeast used and culture medium As yeast used for discrimination, G74NF strain and G74NFar obtained in Experiment 1
g1-4 strains were selected and used.

(2) Extraction of yeast genomic DNA Extraction of genomic DNA from each yeast was performed in the same manner as in Experiment 2.

(3) Discrimination between arginase-deficient yeasts Using the prepared yeast genomic DNA sample as a template, the yeast CAR by PCR method
Amplification of DNA from No. 1 was performed. For this PCR reaction, a primer set shown in Table 5, which was a combination of a forward primer and a reverse primer corresponding to each strain, was used. The sequence of each primer is the same as in Table 2 above.

The PCR method uses EmeraldAmp PCR Master Mix (Takara Bio Inc.) and P
Prepare a CR reaction solution with the composition shown in Tables 6-9, and use a thermal cycler (TKARA PCR Th
ermal cycler MP, Takara Shuzo Co., Ltd.) and heat-denatured at 98 ° C. for 1 minute, then 9
8 ° C. for 10 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 30 seconds were reacted under a cycling condition of 30 cycles as a temperature profile of one cycle.

The obtained PCR product was purified by QIAquick Gel Extraction Kit (QIAGEN), and TE buffer (10 mM Tris-HCl was added so that the sample volume after purification was 40 μL.
, 1 mM EDTA, pH 8.0, Nippon Gene Co., Ltd.)
The product was eluted. The purified PCR product was subjected to restriction enzyme treatment under the reaction solution conditions shown in Tables 10 to 13, and then purified with a QIAquick Gel Extraction Kit (QIAGEN).

All restriction enzymes were manufactured by New England Biolabs. The obtained purified sample was placed on a 2.5% agarose gel (Agalose XP, Nippon Gene) at 100 V,
After electrophoresis for 30 minutes and staining with Gel Red (Biotium), DNA fragments were detected under ultraviolet light irradiation.

3-2 Experimental result The discrimination | determination result of each arginase deficient yeast was shown to FIGS. As shown in these figures, in the G74NFarg strains 1 to 4, the pattern of DNA fragments obtained by each restriction enzyme was different from that of the G74NF strain due to the mutation of CAR1.

Therefore, sake yeast genomic DNA is PCR-amplified using the above primers, the amplified product is subjected to restriction enzyme treatment using the above restriction enzymes, and the pattern of the DNA fragment appearing by electrophoresis is determined, so that G74NF It was possible to easily and accurately determine whether the strain was one of the four strains.

In addition, the base number of the DNA fragment by the restriction enzyme treatment shown in FIGS. 4 to 7 is Saccharomyce
s Yeast Genome restriction Analysis (http: //www.yeastgenome.
Analysis result by org / cgi-bin / PATMATCH / RestrictionMapper) Sorted Fragment Size (bp
) Value.

4 Experiment 4 Discrimination Test in Sake Production 4-1 Experimental Method (1) Sake Sake Preparation Sake was produced in a three-stage preparation using the charge formulation shown in Table 14. Yeast is G74N
F strain and G74NFarg1 strain were used for each preparation. The raw material rice is Yue Tanrei (rice polishing rate 4
0%) was used. For the sake-making, we used the one that produced the Toru from the sake mother lees and stored at 0 ° C. The liquor is a high-temperature saccharified liquor, according to the general method for nurturing ginjo liquor.
Used on the day. Each sake mother inoculated one platinum loop of fungus in 300 mL of Baume 6 broth culture medium and left to stand at 25 ° C. for 7 days, and each of them used 2 sake mothers. Also,
The salmon is charged at 10 ° C for feeding, 12.5 ° C for dancing, 7 ° C for intermediate feeding, and 6 ° C for distilling.
In accordance with general ginjo sake preparation methods, the temperature is controlled so that the maximum product temperature is 11 ° C.
The 4NF strain was fermented for 26 days, and the G74NFarg1 strain was fermented until the 27th, and then placed in an upper tank using a Kamata filter press (Iwata Sangyo Co., Ltd.) to obtain a sake. In the present example, the sake produced means unconditioned sake immediately after the upper tank.

(2) Discrimination of Yeast in Sake Sake Yeast used for sake production was determined from each koji on the 25th day. For the PCR reaction for nucleic acid amplification, a primer set combining re74NF-1 and re74NF-2 shown in Table 2 was used. Details of the detection system using this primer set are shown in FIG. In addition, the base number of the DNA fragment by the restriction enzyme treatment shown in FIG.
haromyces Genome Database Yeast Genome restriction Analysis (http://www.ye
Analysis result by Sortgen Fragment (astgenome.org/cgi-bin/PATMATCH/RestrictionMapper)
This is the value of ment Size (bp).

In PCR method, MightyAmp DNA Polymerase Ver.2 (Takara Bio Inc.)
As a nucleic acid sample, sputum was used as it was. A PCR reaction solution was prepared as shown in Table 15 and a thermal cycler (TKARA PCR Thermal cycler MP, Takara Shuzo Co., Ltd.).
) And heat-denatured at 99 ° C. for 2 minutes, and then reacted at 98 ° C. for 10 seconds, 60 ° C. for 15 seconds, and 68 ° C. for 30 seconds under a cycle condition of 30 cycles. PCR product is QIAquick Gel Extraction Kit (QIAG
EN buffer) so that the sample volume after purification is 40 μL.
The PCR product was eluted from the spin column with mM Tris-HCl, 1 mM EDTA, pH 8.0, Nippon Gene Co., Ltd.). The purified PCR product is converted into a restriction enzyme BslI.
(New England Biolabs) was subjected to restriction enzyme treatment according to the reaction conditions shown in Table 13, and then purified with QIAquick Gel Extraction Kit (QIAGEN). The purified sample obtained was run on a 2.5% agarose gel (Agalose XP, Nippon Gene) at 100 V, 4
After electrophoresis for 0 minutes and staining with Gel Red (Biotium), DNA fragments were detected under UV irradiation.

(3) Distinguishing yeast in sake lees by culture method The purity of yeast in each lees of G74NF and g74NFarg1 strains on the 25th day of brewing was determined by Kitamoto et al. (Kitamoto et al., J. Brew. Soc. .87, p.602-607, 1992)
It was measured.

(4) Analysis of ingredients in Sake Sake The general characteristics of each Sake are as follows: National Tax Agency Predetermined Analysis Method (National Tax Agency Predetermined Analysis Method, 3 Sake, National Tax Agency Instruction No. 6, Supplement, June 22, 2007, http: //www.nta.go.jp/shiraberu/zei
ho-kaishaku / tsutatsu / kobetsu / sonota / 070622 / 01.htm). Also,
Urea in sake is produced by the diacetyl monooxime method (DAMO method, Courombe J. et al.,
Clin. Chem., Vol. 9, p. 102-108, 1963).

(5) Discrimination of produced liquor Extraction of nucleic acid samples from each produced liquor is performed by the method of Hotzel et al. (Hotzel H. et al, Eur. Foo
d Res. Technol., Vol.209, p.192-196, 1999)
100 μL of nucleic acid extract from sake was obtained using Invisorb Genomeic DNA kit III (STRATEC Molecular).

Subsequently, using the nucleic acid sample prepared as described above as a template, the sake was identified. For this discrimination, 60F discriminates the G74NFarg1 nucleic acid constructed in Experiment 3.
-A detection system using a primer set of Car1 and 60R-CAR1 was used.

In addition, EmeraldAmp PCR Master Mix (Takara Bio Inc.) was used for the PCR method, and a PCR reaction solution was prepared with the composition shown in Table 16, and a thermal cycler (TAKARA P) was prepared.
CR Thermal cycler MP, Takara Shuzo Co., Ltd.) was heat-denatured at 98 ° C for 1 minute, and then 30 cycles of 98 ° C for 10 seconds, 58 ° C for 30 seconds, 72 ° C for 30 seconds as one cycle temperature profile. The reaction was performed under cycling conditions.

The obtained PCR product is purified by QIAquick Gel Extraction Kit (QIAGEN), and TE buffer (10 mM Tris-H is used so that the sample volume after purification is 40 μL.
Cl, 1 mM EDTA, pH 8.0, Nippon Gene Co., Ltd.)
The CR product was eluted. The purified PCR product was digested with restriction enzyme BstXI (New England Bi
olabs) under the conditions shown in Table 10, and after the restriction enzyme treatment, QIAquick Gel Extrac
Purification kit (QIAGEN). The purified sample obtained was electrophoresed on a 2.5% agarose gel (Agalose XP, Nippon Gene) at 100 V for 30 minutes, stained with Gel Red (Biotium), and then subjected to DNA fragmentation under UV irradiation. Was detected.

4-2 Experimental Results (1) Discrimination of yeast in sake lees FIG. 9 shows the results of yeast discrimination by nucleic acid amplification from sake lees. G74NFarg
From the cocoon using 1 strain, only the 118 bp DNA fragment derived from the G74NFarg1 strain is 98 bp DNA derived from the G74NF strain from the cocoon using the G74NF strain.
Only detected. Further, when the purity of each sputum was determined by a culture method, G74
Only arginase-deficient yeast was detected from the cocoon derived from the NFarg1 strain, and G74NF
Since no arginase-deficient yeast was detected in the cocoons of the strain, the results were consistent. From this result, it became clear that the purity of yeast in sake lees can be determined by this discrimination method.

The PCR product before restriction enzyme treatment was purified by gel electrophoresis using a conventional method, and BigD
ye Terminator Ver.3.1 (Applied Boisystems) was used for DNA sequencing reaction, and Applied Biosystems 3170xl Genetic Analyzer (Applied Biosystem
The nucleotide sequence was determined by S), and it was confirmed that the PCR amplification product was derived from sake yeast CAR1.

(2) Discrimination from Sake Sake The results of yeast discrimination by nucleic acid amplification from Sake are shown in FIG. G74NFar
A pattern of DNA fragments derived from G74NFarg1 was detected from the sake made using the g1 strain, and a pattern of DNA fragments derived from G74NF was detected from the sake made using the G74NF strain. Therefore, it was found that sake can be discriminated between sake products by producing sake using a specific arginase-deficient yeast and detecting the mutation site of CAR1 derived from this used sake. .

        In addition, Table 17 shows general components and urea concentrations of each sake.

Urea was not detected in the sake made using G74NFarg1 strain, and G74NFa
The rg1 strain was a urea non-producing strain. Therefore, the pattern of DNA fragments derived from the G74NFarg1 strain is an indicator to support the use of non-urea producing yeast.

The PCR product before restriction enzyme treatment was purified by gel electrophoresis using a conventional method, and BigD
DNA sequencing using ye terminator Ver.3.1 (Applied Biosystems) and Applied Biosystems 3170xl generic analyzer (Applied Biosystems)
And the PCR amplification product was confirmed to be derived from sake yeast CAR1.

5 Experiment 5 Liquor production area and production site identification test 5-1 Experimental method (1) Liquor products The products used for discrimination were purchased from a liquor store in Niigata City and manufactured by a brewery manufacturer in Kobe City, Hyogo Prefecture. Sake (foaming) (hereinafter referred to as Company A product), liqueur manufactured by Niigata Prefecture Shibata brewing manufacturer (hereinafter referred to as Company B product), Niigata Prefecture Amagasaki City brewing manufacturer The produced sake (hereinafter referred to as “C company product”) and the sake produced using G74NFarg1 obtained in Experiment 4 (manufactured at Niigata Brewing Experiment Station (Niigata City, Niigata Prefecture)) were used.

(2) Identification of liquor products' production area and production site Liquors were identified by the same method as in Experiment 4-1 (5). Only the product of Company A was subjected to PCR using Mighty Amp DNA Polymerase Ver.2 (Takara Bio Inc.) using the product as it was. The PCR reaction solution was prepared to the composition shown in Table 18, and heat-denatured at 99 ° C for 2 minutes using this PCR reaction solution, then 98 ° C for 10 seconds, 60 ° C for 15 seconds,
The reaction was performed under cycling conditions of 30 cycles with a temperature profile of 68 ° C. for 30 seconds as one cycle. The operation after PCR was carried out in the same manner as in Experiment 4-1 (5).

5-2 Experimental results The results of discrimination of alcoholic beverage products by yeast-derived CAR1 are shown in FIG. G74NFar
From the sake made using the g1 strain, a pattern of DNA fragments derived from the G74NFarg1 strain was detected. On the other hand, from the products of each company A, B, and C, a DNA fragment pattern different from that produced using G74NFarg1 was obtained. Therefore, by producing alcoholic beverages using a specific arginase-deficient yeast and detecting the yeast-derived CAR1 mutation site used for the production from this sake, it is possible to discriminate the production area and the production site.

  As described above, in this example, a predetermined gene was spontaneously mutated or mutated, but it can also be performed by genetic recombination. It is possible to easily divide into a plurality of sections and assign the above-described identification information, that is, identification information for production or identification information for classification of yeast to one or more of the divided areas. In such a case, since it becomes possible to appropriately give a plurality of identification information to a predetermined gene of the yeast for producing food and drink, the types of yeast for producing food and drink used for one food and drink can be reduced. Therefore, the management of the yeast for producing foods and beverages and the management of the production process of the foods and beverages produced using the yeasts for producing foods and beverages can be simplified.

  Note that the present invention is not limited to this embodiment, and the specific configuration of each component can be designed as appropriate.

Claims (3)

Sake yeast is cultured in a selective medium containing canavanine, arginine, and ornithine, and the arginase gene of the sake yeast is mutated to obtain an arginase-deficient yeast. A method for producing sake, comprising associating identification information with the base sequence and producing sake using arginase-deficient yeast associated with the identification information . The method for producing sake according to claim 1, wherein the identification information includes identity management information including at least one of a yeast type, a selection time, and a selection location, a production location, a production time, and a production. A method for producing sake, which is one or both of production management information including at least one of a lot, a production history, a product type, and a manufacturer . The method for producing sake according to any one of claims 1 and 2, wherein the arginase-deficient yeast is composed of 4 to 10 bases having 2 to 5 repeats present in the base sequence of the arginase gene before mutation. A method for producing sake, wherein the base in the repetitive sequence is an arginase-deficient yeast in which two or more bases are deleted or substituted by one base.