JP2021090398A - Yeast having high ornithine productivity, and method for producing liquors or food products - Google Patents
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Abstract
Description
本発明は、オルニチン高生産酵母、及び酒類又は食品の製造方法に関する。 The present invention relates to ornithine high-producing yeast and a method for producing alcoholic beverages or foods.
アミノ酸は、清酒及びワインを含む酒類の呈味成分の一つであり、また、代謝の促進等に関与する機能性成分でもあるため、酒類の品質を左右する重要な要素であると考えられている。そのため、酒類中のアミノ酸含有量をコントロールする方法が検討されている。 Amino acids are one of the taste components of alcoholic beverages including sake and wine, and are also functional components involved in the promotion of metabolism, etc., and are therefore considered to be important factors that affect the quality of alcoholic beverages. There is. Therefore, a method for controlling the amino acid content in alcoholic beverages is being studied.
特許文献１には、酒類（特にワイン）の製造において、アミノ酸の含有量を改善する方法として、酵母変異株Saccharomyces cerevisiae ＭＡ３９１１（ＦＥＲＭ Ｐ−１７８３９号）を用いることが記載されている。 Patent Document 1 describes that a yeast mutant Saccharomyces cerevisiae MA3911 (FERM P-17839) is used as a method for improving the amino acid content in the production of alcoholic beverages (particularly wine).
特許文献２には、酒類の製造においてアミノ酸含有量を改善する方法として、Saccharomyces cerevisiaeに属し、アミノ酸の取り込に関与する遺伝子の変異としてｇａｐ１及びａｐｆ１（ｓｈｒ３）の変異を有する酵母変異株ＭＡ２５０４（ＦＥＲＭ Ｐ−１７４０７号）を用いる酒類の製造方法が記載されている。 In Patent Document 2, as a method for improving the amino acid content in the production of alcoholic beverages, a yeast mutant strain MA2504 which belongs to Saccharomyces cerevisiae and has mutations of gap1 and apf1 (shr3) as mutations of genes involved in amino acid uptake (shr3). A method for producing alcoholic beverages using FERM P-17407) is described.
非特許文献１には、次の記載がある。甲州種ワインのアミノ酸含有量の増加を目的にワイン酵母のgeneral amino acid permeaseをコードするＧＡＰ１、arginine permeaseをコードするＣＡＮ１、およびER integral membrane componentをコードするＡＰＦ１（ＳＨＲ３）の遺伝子に着目して、ｇａｐ１とａｐｆ１（ｓｈｒ３）あるいはｇａｐ１とｃａｎ１の二重変異を有する菌株をそれぞれ分離し、これらを用いてワイン醸造を行なった。 Non-Patent Document 1 has the following description. Focusing on the genes of GAP1, which encodes the general amino acid permease of wine yeast, CAN1 which encodes the arginine permease, and APF1 (SHR3), which encodes the ER integral membrane component, for the purpose of increasing the amino acid content of Koshu wine. Strains having double mutations of gap1 and apf1 (shr3) or gap1 and can1 were isolated, and winemaking was carried out using these.
非特許文献２には、次の記載がある。ＧＡＢＡ含有量の多いワインの醸造を目的に、炭酸ガス処理によりＧＡＢＡを蓄積したブドウを原料として、ワイン酵母から分離したｇａｐ１ ｐｕｔ４ ｕｇａ４の三重変異株を用いてワイン醸造を行った。 Non-Patent Document 2 has the following description. For the purpose of brewing wine with a high GABA content, wine brewing was carried out using grapes having GABA accumulated by carbon dioxide treatment as a raw material and a triple mutant strain of gap1 put4 uga4 isolated from wine yeast.
非特許文献３には、ａｐｆ遺伝子座の変異が、Saccharomyces cerevisiaeのアミノ酸透過酵素の活性を選択的に抑制することが記載されている。
Non-Patent
非特許文献４には、ｐｕｔ４変異が、Saccharomyces cerevisiaeのプロリン輸送システムに欠陥をもたらすことが記載されている。 Non-Patent Document 4 describes that the put4 mutation causes a defect in the proline transport system of Saccharomyces cerevisiae.
また、アミノ酸のうちオルニチンは、肝臓の代謝を助け、肝臓障害を改善する作用を有する可能性がある物質として知られており、オルニチンを添加した機能性の酒類及び食品等の製品が見られる。 Among the amino acids, ornithine is known as a substance that assists liver metabolism and may have an action of improving liver damage, and products such as functional alcoholic beverages and foods to which ornithine is added can be seen.
従来、酒類中のアミノ酸含有量をコントロールするため、酵母の改変を行うことが検討されてきたが、外添することなく、オルニチンの含有量が高い酒類及び食品等の製品を製造することはできなかった。 Conventionally, it has been considered to modify yeast in order to control the amino acid content in alcoholic beverages, but it is possible to produce products such as alcoholic beverages and foods having a high content of ornithine without adding an external substance. There wasn't.
本開示は、オルニチンの含有量が高い酒類及び食品を製造できるオルニチン高生産酵母を提供することを目的とする。 An object of the present disclosure is to provide an ornithine high-producing yeast capable of producing alcoholic beverages and foods having a high content of ornithine.
本開示の第一は、サッカロミセス・セルビシエ（Saccharomyces cerevisiae）に属し、ＳＨＲ３遺伝子に変異を有し、ＧＡＰ１遺伝子に変異を有しない、オルニチン高生産酵母に関する。 The first of the present disclosure relates to ornithine high-producing yeasts belonging to Saccharomyces cerevisiae, having a mutation in the SHR3 gene and no mutation in the GAP1 gene.
前記オルニチン高生産酵母における、前記変異が、ＳＨＲ３遺伝子がコードするタンパク質の第４４番グルタミンに対応するコドンを終始コドンにした変異、又は第１２６番グルタミン酸に対応するコドンを終始コドンにした変異を有することが好ましい。 In the ornithine high-producing yeast, the mutation has a mutation in which the codon corresponding to glutamine No. 44 of the protein encoded by the SHR3 gene is a stop codon, or a mutation in which the codon corresponding to glutamic acid No. 126 is a stop codon. Is preferable.
前記オルニチン高生産酵母が、ＳＭＤ−３（受託番号ＮＩＴＥ Ｐ−０３０６９）であることが好ましい。 The ornithine high-producing yeast is preferably SMD-3 (accession number NITE P-0369).
前記オルニチン高生産酵母が酒類製造用酵母であることが好ましい。 It is preferable that the ornithine high-producing yeast is a yeast for producing alcoholic beverages.
本開示の第二は、前記オルニチン高生産酵母を用いて製造する、酒類又は食品の製造方法に関する。 The second aspect of the present disclosure relates to a method for producing alcoholic beverages or foods produced by using the ornithine high-producing yeast.
前記製造方法において、前記酒類が清酒であることが好ましい。 In the production method, it is preferable that the liquor is sake.
本開示によれば、オルニチンの含有量が高い酒類及び食品を製造できるオルニチン高生産酵母を提供することができる。 According to the present disclosure, it is possible to provide an ornithine high-producing yeast capable of producing alcoholic beverages and foods having a high content of ornithine.
［オルニチン高生産酵母］
本開示のオルニチン高生産酵母は、サッカロミセス・セルビシエ（Saccharomyces cerevisiae）に属し、ＳＨＲ３遺伝子に変異を有し、ＧＡＰ１遺伝子に変異を有しないものである。
[Ornithine high production yeast]
The ornithine high-producing yeast of the present disclosure belongs to Saccharomyces cerevisiae, has a mutation in the SHR3 gene, and does not have a mutation in the GAP1 gene.
オルニチン高生産酵母の遺伝子に変異を有するかどうかを判断する場合、変異とはきょうかい酵母９号あるいはＯＣ−２を参照株とした変異をいう。また、変異を有しないというのは、完全に塩基配列が同じであること、及びオルニチン高生産酵母の遺伝子がコードするタンパク質のアミノ酸配列が同じであることを含む。 When determining whether or not there is a mutation in the gene of ornithine high-producing yeast, the mutation means a mutation using Kyokai Yeast No. 9 or OC-2 as a reference strain. In addition, having no mutation means that the base sequence is completely the same and that the amino acid sequence of the protein encoded by the gene of the ornithine high-producing yeast is the same.
ＳＨＲ３遺伝子は、小胞体シャペロンであり、アミノ酸透過酵素のプロセシング及び輸送に関与するタンパク質（Super high Histidine Resistant 3）をコードする遺伝子である。 The SHR3 gene is an endoplasmic reticulum chaperone, a gene encoding a protein (Super high Histidine Resistant 3) involved in the processing and transport of amino acid permeating enzymes.
ＳＨＲ３遺伝子の変異としては、ＳＨＲ３遺伝子がコードするタンパク質の第４４番グルタミンに対応するコドンを終始コドンにした変異、又はＳＨＲ３遺伝子がコードするタンパク質の第１２６番グルタミン酸に対応するコドンを終始コドンにした変異が好適である。 As the mutation of the SHR3 gene, the codon corresponding to the 44th glutamine of the protein encoded by the SHR3 gene was used as a stop codon, or the codon corresponding to the 126th glutamic acid of the protein encoded by the SHR3 gene was used as a stop codon. Mutations are preferred.
本開示において、遺伝子がコードするタンパク質のアミノ酸配列の番号は、Ｎ末端のメチオニンを１としてＮ末端からＣ末端に向けて１ずつ増加する番号を付与するものである。また、遺伝子の塩基配列の番号は、開始コドンＡＴＧのＡを１として５’末端から３’末端方向に向けて１ずつ増加する番号を付与するものである。 In the present disclosure, the amino acid sequence number of the protein encoded by the gene is assigned a number in which the N-terminal methionine is set to 1 and the number increases by 1 from the N-terminal to the C-terminal. Further, the number of the base sequence of the gene is given by setting A of the start codon ATG as 1 and increasing the number by 1 from the 5'end to the 3'end.
ＧＡＰ１遺伝子は、アミノ酸の膜透過に関わるタンパク質（General amino-acid permease）をコードする遺伝子である。ＧＡＰ１遺伝子の具体的な配列としては、配列番号１３にて示される配列が挙げられる。本開示のオルニチン高生産酵母は、このＧＡＰ１遺伝子に変異を有しない。 The GAP1 gene is a gene encoding a protein (general amino-acid permease) involved in membrane permeation of amino acids. Specific sequences of the GAP1 gene include the sequence shown in SEQ ID NO: 13. The ornithine high-producing yeast of the present disclosure does not have a mutation in this GAP1 gene.
ＧＡＰ１遺伝子に変異を有する場合としては、きょうかい酵母９号あるいはＯＣ−２に対して、ＧＡＰ１遺伝子の配列が、コードするタンパク質のアミノ酸の置換、欠失又は挿入を伴う変異を有する場合が挙げられる。具体的には、例えば、配列番号２３に示されるＭＡ３９１１のＧＡＰ１遺伝子の配列が挙げられる。 Examples of cases where the GAP1 gene has a mutation include a case where the sequence of the GAP1 gene has a mutation associated with amino acid substitution, deletion or insertion of the encoding protein with respect to Kyokai Yeast No. 9 or OC-2. .. Specifically, for example, the sequence of the GAP1 gene of MA3911 shown in SEQ ID NO: 23 can be mentioned.
本開示のオルニチン高生産酵母は、一倍体であっても二倍体であってもよいが、二倍体の当該酵母は培養中に変異が起きにくく、酒類又は食品の安定した製造ができる。また、本開示のオルニチン高生産酵母のうち二倍体のものとして、具体的には、ＳＭＤ−３（受託番号ＮＩＴＥ Ｐ−０３０６９）が好適であるが、これに限定されるものではない。 The ornithine high-producing yeast of the present disclosure may be diploid or diploid, but the diploid yeast is less likely to cause mutation during culturing and can stably produce alcoholic beverages or foods. .. Further, as the diploid yeast among the ornithine high-producing yeasts of the present disclosure, specifically, SMD-3 (accession number NITE P-0369) is suitable, but the present invention is not limited thereto.
本開示のオルニチン高生産酵母は、従来のサッカロミセス・セルビシエ（Saccharomyces cerevisiae）に属する酵母の用途に用いることができる。例えば、酒類及び食品の製造におけるアルコール発酵に用いることができる。特に、本開示のオルニチン高生産酵母は、アルコール生産能に優れるため、酒類製造用酵母、特に清酒製造用酵母として好適である。 The ornithine high-producing yeast of the present disclosure can be used for yeasts belonging to the conventional Saccharomyces cerevisiae. For example, it can be used for alcoholic fermentation in the production of alcoholic beverages and foods. In particular, the high-producing ornithine-producing yeast of the present disclosure is suitable as a yeast for producing alcoholic beverages, particularly a yeast for producing sake, because it is excellent in alcohol-producing ability.
本開示のオルニチン高生産酵母は、オルニチン生産能に優れるため、当該酵母によれば、外添することなく、オルニチンの含有量が高い酒類及び食品等の製品を製造することができる。 Since the ornithine high-producing yeast of the present disclosure is excellent in ornithine-producing ability, according to the yeast, products such as alcoholic beverages and foods having a high ornithine content can be produced without addition.
［オルニチン高生産酵母の取得方法］
本開示のオルニチン高生産酵母は、Saccharomyces cerevisiaeに属する酵母を親株として用いて取得することができる。親株としては、きょうかい酵母７号及びきょうかい酵母９号等が挙げられる。
[How to obtain ornithine high-producing yeast]
The ornithine high-producing yeast of the present disclosure can be obtained by using a yeast belonging to Saccharomyces cerevisiae as a parent strain. Examples of the parent strain include Kyokai Yeast No. 7 and Kyokai Yeast No. 9.
一倍体として用いる場合、本開示のオルニチン高生産酵母は、次の方法により取得することができる。親株であるサッカロミセス・セルビシエ（Saccharomyces cerevisiae）に属する酵母を一倍体化する工程（１）；前記一倍体化した酵母を変異処理する工程（２）；アゼチジン−２−カルボン酸（L-Azetidine-s-carboxylic acid、ＡＺＣ）を含む選択培地で培養し、増殖した株（ＡＺＣ耐性株）を採取するスクリーニングを行う工程（３）；及びプロリンを唯一の窒素源とする培地で培養し、増殖しにくい株（プロリンを資化しにくい株）を採取するスクリーニングを行う工程（４）を含む方法により取得することができる。 When used as a haploid, the ornithine high-producing yeast of the present disclosure can be obtained by the following method. A step of haploidizing yeast belonging to the parent strain Saccharomyces cerevisiae (1); a step of mutating the haploid yeast (2); L-Azetidine -Culturing in a selective medium containing (s-carboxylic acid, AZC) and screening to collect the grown strain (AZC resistant strain) (3); and culturing in a medium containing proline as the sole nitrogen source and growing. It can be obtained by a method including the step (4) of performing a screening for collecting a strain that is difficult to assimilate (a strain that does not easily assimilate proline).
二倍体として用いる場合、本開示のオルニチン高生産酵母は、次の方法により取得することができる。前記のプロリンを唯一の窒素源とする培地で培養し、増殖しにくい株を採取するスクリーニングを行う工程（４）の後に、親株であるサッカロミセス・セルビシエ（Saccharomyces cerevisiae）に属する酵母由来の一倍体と交雑する工程（５）を含む方法により、取得することができる。 When used as a diploid, the ornithine high-producing yeast of the present disclosure can be obtained by the following method. After the step (4) of culturing the above-mentioned proline in a medium containing proline as the sole nitrogen source and screening for collecting a strain that is difficult to grow, a yeast-derived monopoly that belongs to the parent strain Saccharomyces cerevisiae. It can be obtained by a method including the step (5) of crossing with.
親株であるサッカロミセス・セルビシエ（Saccharomyces cerevisiae）に属する酵母を一倍体化する工程（１）としては、例えば、具体的には、ランダムスポア法が挙げられる。 Specific examples of the step (1) of haploidizing yeast belonging to the parent strain Saccharomyces cerevisiae include the random spor method.
前記酵母を変異処理する工程（２）について、変異処理の方法としては、メタンスルホン酸エチル（ＥＭＳ）、紫外線、放射線、及び／又はＮ−メチル−Ｎ’−ニトロ−Ｎ−ニトロソグアジニン（ＮＴＧ）等を用いる方法が挙げられるが、ＥＭＳを用いる方法が好ましい。 Regarding the step (2) of mutating the yeast, the method of mutating is ethyl methanesulfonate (EMS), ultraviolet rays, radiation, and / or N-methyl-N'-nitro-N-nitrosoguadinin (NTG). ) And the like, but the method using EMS is preferable.
ＡＺＣ耐性株を採取するスクリーニングを行う工程（３）は、ＡＺＣを含む培地で培養し、増殖した株を取得する方法を用いることができる。 In the step (3) of screening for collecting the AZC-resistant strain, a method of culturing in a medium containing AZC and obtaining the grown strain can be used.
プロリンを資化しにくい株を採取するスクリーニングを行う工程（４）は、プロリンを唯一の窒素源とする培地で培養し、増殖しにくい株を取得すればよい。当該工程によりＡＺＣ耐性株からＳＨＲ３変異株を絞り込むことができる。プロリンを資化できる株は、ＳＨＲ３遺伝子以外の変異によりＡＺＣ耐性を獲得したか、ＳＨＲ３遺伝子の機能性を残存した変異株である可能性がある。なお、プロリンは、ピロリジン−２−カルボン酸と言い換えることができる。また、ＡＺＣはプロリンの類似物質である。 In the step (4) of screening for collecting a strain that is difficult to assimilate proline, the strain that is difficult to grow may be obtained by culturing in a medium containing proline as the sole nitrogen source. By this step, SHR3 mutant strains can be narrowed down from AZC resistant strains. The strain capable of assimilating proline may have acquired AZC resistance due to a mutation other than the SHR3 gene, or may be a mutant strain in which the functionality of the SHR3 gene remains. In addition, proline can be paraphrased as pyrrolidine-2-carboxylic acid. AZC is a similar substance to proline.
［用途］
本開示のオルニチン高生産酵母は、酒類又は食品の製造に用いることができる。酒類としては、清酒、ワイン及びビール等の麦芽アルコール飲料が挙げられる。本開示のオルニチン高生産酵母は、アルコール生産能に優れるため、特に清酒の製造に好適である。
[Use]
The ornithine high-producing yeast of the present disclosure can be used in the production of alcoholic beverages or foods. Examples of alcoholic beverages include malt alcoholic beverages such as sake, wine and beer. The ornithine high-producing yeast of the present disclosure is particularly suitable for the production of sake because it has excellent alcohol-producing ability.
清酒の製造方法としては、酵母、米（掛米とも言い換えることができる）、麹、及び水を配合して醪を仕込む工程、前記醪を発酵させる工程、及び前記発酵させた醪から清酒を分離する工程、を含む方法が挙げられる。醪を仕込む仕込工程、及び前記醪を発酵させる発酵工程を、共に２回以上繰り返してよい。 As a method for producing sake, a step of mixing yeast, rice (which can be rephrased as kakemai), koji, and water to prepare mash, a step of fermenting the mash, and a step of separating sake from the fermented mash. A method including the step of performing is mentioned. The preparation step of charging the mash and the fermentation step of fermenting the mash may be repeated twice or more.
例えば、醪を仕込む工程を３回繰り返す場合は、これを三段仕込みと呼ぶことができる。各回の工程は、それぞれ初添処理、仲添処理、及び留添処理と呼ぶことができる。 For example, when the process of mashing mash is repeated three times, this can be called three-stage mashing. Each step can be referred to as an initial addition process, an intermediate process, and a reference process, respectively.
初添処理は、酵母を含む酒母、米、麹及び水を配合して醪を仕込む工程である。酒母の代わりに培養した酵母菌体を用いてもよい。酒母は、酵母、米、麹及び水を配合して、酵母を培養することにより調製できる。 The initial mashing process is a process of mixing yeast-containing liquor mother, rice, koji and water to prepare mash. Cultured yeast cells may be used instead of the liquor mother. Sake mother can be prepared by blending yeast, rice, koji and water and culturing the yeast.
仲添処理は、初添処理後の醪に、さらに米、麹及び水を配合して、醪を仕込む工理である。 The mashing process is a process in which rice, jiuqu and water are further mixed with the mash after the initial mashing process to prepare the mash.
留添処理は、仲添処理後の醪に、さらに米、麹及び水を配合して、醪を仕込む工程である。 The mashing process is a step of adding rice, jiuqu and water to the mash after the mashing process to prepare the mash.
醪を発酵させる工程は、仕込んだ醪（三段仕込みの場合、留添処理後の醪）を温度調節と加水により酵母の活性を制御して目標の酒質となるように管理することが好ましい。 In the process of fermenting the mash, it is preferable to control the mash (the mash after the addition treatment in the case of three-stage mash) by controlling the activity of yeast by controlling the temperature and adding water to achieve the target liquor quality. ..
前記発酵させた醪から清酒を分離する工程は、例えば、圧搾機等を用いてろ過する方法が挙げられる。当該工程により、醪を清酒及び酒粕に分離することができる。 Examples of the step of separating sake from the fermented mash include a method of filtering using a squeezer or the like. By this process, mash can be separated into sake and sake lees.
配列番号１：きょうかい酵母９号のＳＨＲ３遺伝子の配列
配列番号２：きょうかい酵母９号のＳＨＲ３遺伝子がコードするタンパク質の配列
配列番号３：ＳＭ１のＳＨＲ３遺伝子の配列
配列番号４：ＳＭ１のＳＨＲ３遺伝子がコードするタンパク質の配列
配列番号５：ＳＭ３のＳＨＲ３遺伝子の配列
配列番号６：ＳＭ３のＳＨＲ３遺伝子がコードするタンパク質の配列
配列番号７：ＳＭＤ−３のＳＨＲ３遺伝子の配列
配列番号８：ＳＭＤ−３のＳＨＲ３遺伝子がコードするタンパク質の配列
配列番号９：ＯＣ−２のＳＨＲ３遺伝子の配列
配列番号１０：ＯＣ−２のＳＨＲ３遺伝子がコードするタンパク質の配列
配列番号１１：ＭＡ３９１１のＳＨＲ３遺伝子の配列
配列番号１２：ＭＡ３９１１のＳＨＲ３遺伝子がコードするタンパク質の配列
配列番号１３：きょうかい酵母９号のＧＡＰ１遺伝子の配列
配列番号１４：きょうかい酵母９号のＧＡＰ１遺伝子がコードするタンパク質の配列
配列番号１５：ＳＭ１のＧＡＰ１遺伝子の配列
配列番号１６：ＳＭ１のＧＡＰ１遺伝子がコードするタンパク質の配列
配列番号１７：ＳＭ３のＧＡＰ１遺伝子の配列
配列番号１８：ＳＭ３のＧＡＰ１遺伝子がコードするタンパク質の配列
配列番号１９：ＳＭＤ−３のＧＡＰ１遺伝子の配列
配列番号２０：ＳＭＤ−３のＧＡＰ１遺伝子がコードするタンパク質の配列
配列番号２１：ＯＣ−２のＧＡＰ１遺伝子の配列
配列番号２２：ＯＣ−２のＧＡＰ１遺伝子がコードするタンパク質の配列
配列番号２３：ＭＡ３９１１のＧＡＰ１遺伝子の配列
配列番号２４：ＭＡ３９１１のＧＡＰ１遺伝子がコードするタンパク質の配列
SEQ ID NO: 1: Sequence of SHR3 gene of Kyokai Yeast No. 9 SEQ ID NO: 2: Sequence of protein encoded by SHR3 gene of Kyokai Yeast No. 9 SEQ ID NO: 3: Sequence of SHR3 gene of SM1 SEQ ID NO: 4: SHR3 gene of SM1 SEQ ID NO: 5: Sequence of SHR3 gene of SM3 SEQ ID NO: 6: Sequence of protein encoded by SHR3 gene of SM3 SEQ ID NO: 7: Sequence of SHR3 gene of SMD-3 SEQ ID NO: 8: Sequence of SMD-3 Sequence of protein encoded by SHR3 gene SEQ ID NO: 9: Sequence of SHR3 gene of OC-2 SEQ ID NO: 10: Sequence of protein encoded by SHR3 gene of OC-2 SEQ ID NO: 11: Sequence of SHR3 gene of MA3911 SEQ ID NO: 12: Sequence of protein encoded by SHR3 gene of MA3911 SEQ ID NO: 13: Sequence of GAP1 gene of Kyokai yeast No. 9 SEQ ID NO: 14: Sequence of protein encoded by GAP1 gene of Kyokai yeast No. 9 SEQ ID NO: 15: GAP1 gene of SM1 SEQ ID NO: 16: Sequence of protein encoded by GAP1 gene of SM1 SEQ ID NO: 17: Sequence of GAP1 gene of SM3 SEQ ID NO: 18: Sequence of protein encoded by GAP1 gene of SM3 SEQ ID NO: 19: GAP1 gene of SMD-3 SEQ ID NO: 20: Sequence of protein encoded by GAP1 gene of SMD-3 SEQ ID NO: 21: Sequence of GAP1 gene of OC-2 SEQ ID NO: 22: Sequence of protein encoded by GAP1 gene of OC-2 SEQ ID NO: 23: Sequence of GAP1 gene of MA3911 SEQ ID NO: 24: Sequence of protein encoded by GAP1 gene of MA3911
＜一般成分＞
アルコール度数、日本酒度、酸度及びアミノ酸度は、それぞれ国税庁所定の分析法に従い、測定した。
<General ingredients>
Alcohol content, sake content, acidity and amino acid content were measured according to the analytical methods prescribed by the National Tax Agency.
＜アミノ酸合計及びオルニチンの含有量＞
アミノ酸合計及びオルニチンの含有量は、島津高速液体クロマトグラフ「ＬＣ−１０Ａ高速アミノ酸分析システム」を用いて測定した。測定条件は次のとおりである。
カラム：Ｓｈｉｍ−ｐａｃｋ Ａｍｉｎｏ−Ｌｉ
トラップカラム：ＩＳＣ−３０Ｌｉ
オーブン温度：３９℃
移動相：島津アミノ酸分析用移動相キットＬｉ型 ０．６ｍｌ／ｍｉｎ
反応試薬：島津アミノ酸反応液ＯＰＡキット
検出器：ＲＦ−１０ＡＸ 励起波長３５０ｎｍ 蛍光波長４５０ｎｍ
アミノ酸合計に含まれるアミノ酸：アスパラギン酸、ヒドロキシプロリン、トレオニン、セリン、アスパラギン、グルタミン酸、グルタミン、プロリン、グリシン、アラニン、バリン、メチオニン、イソロイシン、ロイシン、チロシン、フェニルアラニン、γ-アミノ酪酸、ヒスチジン、オルニチン、リシン、アルギニン
<Total amino acids and ornithine content>
The total amino acids and the content of ornithine were measured using a Shimadzu high performance liquid chromatograph "LC-10A high performance amino acid analysis system". The measurement conditions are as follows.
Column: Sim-pack Amino-Li
Trap column: ISC-30Li
Oven temperature: 39 ° C
Mobile phase: Shimadzu mobile phase kit for amino acid analysis Li type 0.6 ml / min
Reaction reagent: Shimadzu amino acid reaction solution OPA kit Detector: RF-10AX Excitation wavelength 350 nm Fluorescence wavelength 450 nm
Amino acids included in total amino acids: aspartic acid, hydroxyproline, threonine, serine, aspartic acid, glutamic acid, glutamine, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, γ-aminobutyric acid, histidine, ornithine, Ricin, arginine
＜酵母の製造＞
酵母を一倍体化する工程（１）
Saccharomyces cerevisiaeに属する酵母として、きょうかい酵母９号を親株とし、これをランダムスポア法にて一倍体化（α型）した。きょうかい酵母９号のＳＨＲ３遺伝子及びＧＡＰ１遺伝子の配列は、それぞれ配列番号１及び１３に示すとおりである。
<Yeast production>
Step of haploidizing yeast (1)
As a yeast belonging to Saccharomyces cerevisiae, Kyokai yeast No. 9 was used as a parent strain, and this was haploidized (α type) by the random spor method. The sequences of the SHR3 gene and the GAP1 gene of Kyokai Yeast No. 9 are as shown in SEQ ID NOs: 1 and 13, respectively.
酵母を変異処理する工程（２）
一倍体化（α型）したきょうかい酵母９号を、ＹＰＤ培地で、３０℃２４時間培養し、集菌後滅菌蒸留水で洗浄した。これを１０ｍｌの０．１Ｍリン酸バッファー（ｐＨ７）に懸濁し、３００μｌのメタンスルホン酸エチル（ＥＭＳ）を加えて７０分間振とうした。集菌し、１０ｍｌの５％チオ硫酸ナトリウムで中和し、滅菌蒸留水にて洗浄した。
Step of mutating yeast (2)
Haploidized (α-type) yeast No. 9 was cultured in YPD medium at 30 ° C. for 24 hours, collected, and washed with sterile distilled water. This was suspended in 10 ml of 0.1 M phosphate buffer (pH 7), 300 μl of ethyl methanesulfonate (EMS) was added, and the mixture was shaken for 70 minutes. The cells were collected, neutralized with 10 ml of 5% sodium thiosulfate, and washed with sterile distilled water.
ＡＺＣ耐性株を採取するスクリーニングを行う工程（３）
アゼチジン−２−カルボン酸（L-Azetidine-s-carboxylic acid、ＡＺＣ）を含む選択培地（０．５ｍＭのＡＺＣ、０．６７％のDifco Yeast Nitrogen Base w/o Amino Acids（Becton, Dickinson社製）、２％のGlycerolのＳＧ寒天培地）で、３０℃１週間培養し、コロニーを０．５ｍＭのＡＺＣを含むＳＧ寒天培地に植え継ぎ、シングルコロニーを単離した。
Step of screening to collect AZC resistant strain (3)
Selective medium containing azetidine-2-carboxylic acid (AZC) (0.5 mM AZC, 0.67% Difco Yeast Nitrogen Base w / o Amino Acids (Becton, Dickinson)) The cells were cultured in SG agar medium containing 2% Glycerol at 30 ° C. for 1 week, and the colonies were subcultured on SG agar medium containing 0.5 mM AZC to isolate single colonies.
プロリンを資化しにくい株を採取するスクリーニングを行う工程（４）
単離したシングルコロニーの菌株をＰＤ培地（０．１７％のDifco Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate（Becton, Dickinson社製）、２％のGlucose、０．１％のProline）で、３０℃４８時間培養して、増殖の悪い菌株を選抜した。
Step of screening to collect strains that are difficult to assimilate proline (4)
Strains of isolated single colonies were cultivated in PD medium (0.17% Difco Yeast Nitrogen Base w / o Amino Acids and Ammonium Sulfate (Becton, Dickinson), 2% Glucose, 0.1% Proline). Strains with poor growth were selected by culturing at 30 ° C. for 48 hours.
選抜した菌株に野性型ＳＨＲ３遺伝子（きょうかい酵母９号のＳＨＲ３遺伝子）を持つプラスミドを導入した。そして、プラスミドを導入した菌株をＰＤ培地で、３０℃４８時間培養して、増殖した菌株を選抜した。選抜した菌株は４株であった。プラスミドの導入により増殖が回復したことから、ＳＨＲ３遺伝子に変異が存在することが分かる。 A plasmid having a wild-type SHR3 gene (SHR3 gene of Kyokai Yeast No. 9) was introduced into the selected strain. Then, the strain into which the plasmid was introduced was cultured in PD medium at 30 ° C. for 48 hours, and the grown strain was selected. The selected strains were 4 strains. Since the growth was restored by the introduction of the plasmid, it can be seen that the SHR3 gene has a mutation.
選抜した菌株についてＤＮＡシークエンス解析を行い、４株全てのＳＨＲ３遺伝子に変異があることを確認した。具体的には、４株の内、２株（うち１株を「ＳＭ１」と称する）が、ＳＨＲ３遺伝子がコードするタンパク質の第１２６番グルタミン酸に対応するコドンを終始コドンにした変異を有し；他の２株（うち１株を「ＳＭ３」と称する）が、ＳＨＲ３遺伝子がコードするタンパク質の第４４番グルタミンに対応するコドンを終始コドンにした変異を有することを確認した。なお、これらの変異は、いずれもナンセンス突然変異である。 DNA sequence analysis was performed on the selected strains, and it was confirmed that all four strains had mutations in the SHR3 gene. Specifically, two of the four strains (one of which is referred to as "SM1") have a stop codon mutation in the codon corresponding to protein 126 glutamic acid encoded by the SHR3 gene; It was confirmed that the other two strains (one of which is referred to as "SM3") have a stop codon mutation in the codon corresponding to glutamine No. 44 of the protein encoded by the SHR3 gene. All of these mutations are nonsense mutations.
ＳＭ１のＳＨＲ３遺伝子及びＧＡＰ１遺伝子の配列は、それぞれ配列番号３及び１５に示すとおりである。また、ＳＭ３のＳＨＲ３遺伝子及びＧＡＰ１遺伝子の配列は、それぞれ配列番号５及び１７に示すとおりである。 The sequences of the SHR3 gene and the GAP1 gene of SM1 are as shown in SEQ ID NOs: 3 and 15, respectively. The sequences of the SHR3 gene and the GAP1 gene of SM3 are as shown in SEQ ID NOs: 5 and 17, respectively.
ＳＭ１のＳＨＲ３遺伝子は、第３７６番グアニンをチミンにした変異を有する。また、ＳＭ３のＳＨＲ３遺伝子は、第１３０番シトシンをチミンにした変異を有する。ＳＭ１及びＳＭ３のＧＡＰ１遺伝子はいずれも変異を有しない。 The SHR3 gene of SM1 has a mutation in which 376th guanine is thymine. In addition, the SHR3 gene of SM3 has a mutation in which the 130th cytosine is thymine. Neither the GAP1 gene of SM1 nor SM3 has a mutation.
ＳＭ１及びＳＭ３を、それぞれ４−フルオロ−Ｌ−フェニルアラニン（4-Fluoro-L-phenylalanine、ＦＰＡ）を含む選択培地（２５μｇ／ｍｌのＦＰＡを含むＳＧ寒天培地）で３０℃１週間培養して、増殖することを確認した。なお、親株であるきょうかい酵母９号は、ＦＰＡを含む選択培地では増殖できない。 SM1 and SM3 are cultured at 30 ° C. for 1 week in a selective medium (SG agar medium containing 25 μg / ml FPA) containing 4-Fluoro-L-phenylalanine (FPA), respectively, and grown. Confirmed to do. The parent strain, Kyokai Yeast No. 9, cannot grow in a selective medium containing FPA.
また、ＳＭ１及びＳＭ３を、それぞれカナバニン（Canavanine、ｃａｎ）を含む選択培地（４０μｇ／ｍｌのｃａｎを含むＳＧ寒天培地）で３０℃１週間培養して、増殖することを確認した。なお、きょうかい酵母９号は、ｃａｎを含む選択培地では増殖できない。 Moreover, it was confirmed that SM1 and SM3 were cultured in a selective medium containing canavanine (can), respectively (SG agar medium containing 40 μg / ml can) at 30 ° C. for 1 week to grow. In addition, Kyokai Yeast No. 9 cannot grow in a selective medium containing can.
ＳＭ１及びＳＭ３にそれぞれ野性型ＳＨＲ３遺伝子を持つプラスミドを導入した。そして、プラスミドを導入したＳＭ１を、ＦＰＡを含む選択培地（２５μｇ／ｍｌのＦＰＡを含むＳＧ寒天培地）及びｃａｎを含む選択培地（４０μｇ／ｍｌのｃａｎを含むＳＧ寒天培地）で３０℃１週間培養して、増殖できないことを確認した。プラスミドを導入したＳＭ３についても同様に培養して、増殖できないことを確認した。このように、プラスミドの導入によって増殖できなくなったことからも、ＳＨＲ３遺伝子に変異が存在することが分かる。 A plasmid having a wild SHR3 gene was introduced into SM1 and SM3, respectively. Then, SM1 into which the plasmid was introduced is cultured at 30 ° C. for 1 week in a selective medium containing FPA (SG agar medium containing 25 μg / ml FPA) and a selective medium containing can (SG agar medium containing 40 μg / ml can). And confirmed that it could not grow. The plasmid-introduced SM3 was also cultured in the same manner, and it was confirmed that it could not be propagated. As described above, it can be seen that the SHR3 gene has a mutation because it cannot be propagated due to the introduction of the plasmid.
親株であるサッカロミセス・セルビシエ（Saccharomyces cerevisiae）に属する酵母由来の一倍体と交雑する工程（５）
次に、ＳＭ１及びＳＭ３にそれぞれきょうかい酵母９号由来の一倍体（ａ型）と接合させ二倍体化した。この二倍体化した株をランダムスポア法で一倍体化させ、ＦＰＡを含む選択培地（２５μｇ／ｍｌのＦＰＡを含むＳＧ寒天培地）で３０℃１週間培養して、増殖したａ型株を複数選抜した。さらに、このａ型株をきょうかい酵母９号由来の一倍体（α型）と接合させ二倍体化した。この二倍体化した株をランダムスポア法で一倍体化させ、ＦＰＡを含む選択培地（２５μｇ／ｍｌのＦＰＡを含むＳＧ寒天培地）で３０℃１週間培養して、増殖したα型株を複数選抜した。さらに、選抜したａ型株及びα型株を交雑させて二倍体化した（二倍体株のうち１株を「ＳＭＤ−３」と称する）。
Step of crossing with yeast-derived haploids belonging to the parent strain Saccharomyces cerevisiae (5)
Next, SM1 and SM3 were conjugated with a haploid (type a) derived from Kyokai Yeast No. 9 to form a diploid. This diploid strain was haploidized by the random spor method and cultured in a selective medium containing FPA (SG agar medium containing 25 μg / ml FPA) at 30 ° C. for 1 week to grow the a-type strain. Multiple selections were made. Furthermore, this type a strain was conjugated with a haploid (α type) derived from Kyokai Yeast No. 9 to form a diploid. This diploid strain was haploidized by the random spor method and cultured in a selective medium containing FPA (SG agar medium containing 25 μg / ml FPA) at 30 ° C. for 1 week to obtain a grown α-type strain. Multiple selections were made. Further, the selected a-type strain and α-type strain were crossed to form a diploid (one of the diploid strains is referred to as "SMD-3").
得られた二倍体株を、ＦＰＡを含む選択培地（２５μｇ／ｍｌのＦＰＡを含むＳＧ寒天培地）で３０℃１週間培養して、増殖することを確認した。 It was confirmed that the obtained diploid strain was cultured in a selective medium containing FPA (SG agar medium containing 25 μg / ml FPA) at 30 ° C. for 1 week to grow.
ＳＭＤ−３のＳＨＲ３遺伝子及びＧＡＰ１遺伝子の配列は、それぞれ配列番号７及び１９に示すとおりである。ＳＭＤ−３のＳＨＲ３遺伝子は、第３７６番グアニンをチミンにした変異を有し、ＧＡＰ１遺伝子に変異を有しない。つまり、ＳＭＤ−３は、ＳＨＲ３遺伝子がコードするタンパク質の第１２６番グルタミン酸に対応するコドンを終始コドンにした変異を有する。 The sequences of the SHR3 gene and the GAP1 gene of SMD-3 are as shown in SEQ ID NOs: 7 and 19, respectively. The SHR3 gene of SMD-3 has a mutation in which 376th guanine is thymine and does not have a mutation in the GAP1 gene. That is, SMD-3 has a mutation in which the codon corresponding to glutamic acid No. 126 of the protein encoded by the SHR3 gene is a stop codon from beginning to end.
＜実施例１＞
酵母としてＳＭ１、原料の米としてα米（精米歩合７７％、セブンライス工業製）、麹（精米歩合７５％）、及び水を配合して醪を仕込む仕込工程、前記醪を発酵させる工程、及び前記発酵させた醪から清酒を分離する工程、を含む方法にて、清酒を製造した。具体的には、表１に示す仕込配合（総米（α米及び麹米の合計）９７．５ｇ）で、初添処理、仲添処理、及び留添処理からなる三段仕込を行って、その後１３℃一定で醪を発酵させた。約２週間の発酵後、醪をこして清澄液分を回収（上槽）することにより清酒を得た。得られた清酒について、一般成分を測定し、アミノ酸合計及びオルニチンの含有量を測定し、アミノ酸合計の含有量におけるオルニチンの割合を算出した。結果は表２及び表３に示す。また、発酵中の炭酸ガス減量を図１に示す。
<Example 1>
SM1 as yeast, α-rice as raw material rice (rice polishing ratio 77%, manufactured by Seven Rice Industry), koji (rice polishing ratio 75%), and water to prepare mash, ferment the mash, and Sake was produced by a method including the step of separating sake from the fermented mash. Specifically, with the preparation composition shown in Table 1 (total rice (total of α-rice and koji rice) 97.5 g), three-stage preparation consisting of initial addition treatment, intermediate treatment, and addition treatment was performed. After that, the rice was fermented at a constant temperature of 13 ° C. After fermentation for about 2 weeks, sake was obtained by straining the mash and collecting the clarified liquid (upper tank). With respect to the obtained sake, the general components were measured, the total amino acid content and the ornithine content were measured, and the ratio of ornithine to the total amino acid content was calculated. The results are shown in Tables 2 and 3. In addition, the weight loss of carbon dioxide during fermentation is shown in FIG.
＜実施例２〜３＞
酵母としてＳＭ１に換えてＳＭ３（実施例２）又はＳＭＤ−３（実施例３）を用いた以外は、それぞれ実施例１と同様にして、清酒を製造した。得られた清酒について、一般成分を測定し、アミノ酸合計及びオルニチンの含有量を測定し、アミノ酸合計の含有量におけるオルニチンの割合を算出した。結果は表２及び表３に示す。また、発酵中の炭酸ガス減量を図１に示す。
<Examples 2 to 3>
Sake was produced in the same manner as in Example 1 except that SM3 (Example 2) or SMD-3 (Example 3) was used instead of SM1 as yeast. With respect to the obtained sake, the general components were measured, the total amino acid content and the ornithine content were measured, and the ratio of ornithine to the total amino acid content was calculated. The results are shown in Tables 2 and 3. In addition, the weight loss of carbon dioxide during fermentation is shown in FIG.
＜比較例１＞
酵母としてＳＭ１に換えてきょうかい酵母９号（Ｋ９）を用いた以外は、実施例１と同様にして、清酒を製造した。得られた清酒について、一般成分を測定し、アミノ酸合計及びオルニチンの含有量を測定し、アミノ酸合計の含有量におけるオルニチンの割合を算出した。結果は表２及び表３に示す。また、発酵中の炭酸ガス減量を図１に示す。
<Comparative example 1>
Sake was produced in the same manner as in Example 1 except that the yeast No. 9 (K9) was used instead of SM1. With respect to the obtained sake, the general components were measured, the total amino acid content and the ornithine content were measured, and the ratio of ornithine to the total amino acid content was calculated. The results are shown in Tables 2 and 3. In addition, the weight loss of carbon dioxide during fermentation is shown in FIG.
＜比較例２＞
特許文献１：特許第３８２２４１５号公報に記載される、Saccharomyces cerevisiaeに属する酵母であるＯＣ−２を用いて醸造したワインのアミノ酸の分析結果を表３に示す。
<Comparative example 2>
Table 3 shows the analysis results of amino acids in wine brewed using OC-2, which is a yeast belonging to Saccharomyces cerevisiae, described in Patent Document 1: Japanese Patent No. 3822415.
＜比較例３＞
特許文献１：特許第３８２２４１５号公報に記載される、Saccharomyces cerevisiaeに属する酵母であるＭＡ３９１１を用いて醸造したワインのアミノ酸の分析結果を表３に示す。
<Comparative example 3>
Table 3 shows the analysis results of amino acids in wine brewed using MA3911, which is a yeast belonging to Saccharomyces cerevisiae, described in Patent Document 1: Japanese Patent No. 3822415.
ＭＡ３９１１のＧＡＰ１遺伝子（配列番号２３）は、ＯＣ−２に対し、第３１４番グアニンをアデニンに、第４１５番グアニンをアデニンにした変異を有する。また、ＭＡ３９１１のＳＨＲ３遺伝子（配列番号１１）は、ＯＣ−２に対し、第２７１番シトシンをチミンにした変異を有する。 The GAP1 gene (SEQ ID NO: 23) of MA3911 has a mutation in OC-2 in which guanine No. 314 is adenine and guanine No. 415 is adenine. In addition, the SHR3 gene (SEQ ID NO: 11) of MA3911 has a mutation in OC-2 in which cytosine No. 271 is thymine.
表２に示すように、実施例１〜３のいずれもアルコール度数、日本酒度、酸度及びアミノ酸度は、従来多く使用されている清酒酵母であるきょうかい酵母９号（Ｋ９）と同等である。 As shown in Table 2, all of Examples 1 to 3 have the same alcohol content, sake content, acidity and amino acid content as Kyokai yeast No. 9 (K9), which is a sake yeast that has been widely used in the past.
図１に示すように、実施例１〜３のいずれも炭酸ガス減量は、従来多く使用されている清酒酵母であるきょうかい酵母９号（Ｋ９）と同等である。 As shown in FIG. 1, the carbon dioxide gas weight loss in each of Examples 1 to 3 is equivalent to that of Kyokai Yeast No. 9 (K9), which is a sake yeast that has been widely used in the past.
表３に示すように、ＳＨＲ３遺伝子及びＧＡＰ１遺伝子に変異を有しない酵母を用いた比較例１及び比較例２は、アミノ酸合計の含有量におけるオルニチンの割合が不十分である。また、ＳＨＲ３及びＧＡＰ１遺伝子に変異を有する酵母を用いた比較例３は、比較例１及び比較例２と比較し、アミノ酸合計の含有量におけるオルニチンの割合が特に低い。また、比較例に対し、ＳＨＲ３遺伝子に変異を有し、ＧＡＰ１遺伝子に変異を有しない酵母を用いた実施例１〜３は、オルニチンの含有量が高く、そしてアミノ酸合計の含有量におけるオルニチンの割合も高い。 As shown in Table 3, in Comparative Example 1 and Comparative Example 2 using yeast having no mutation in the SHR3 gene and the GAP1 gene, the ratio of ornithine in the total amino acid content is insufficient. In addition, Comparative Example 3 using yeast having mutations in the SHR3 and GAP1 genes has a particularly low proportion of ornithine in the total amino acid content as compared with Comparative Example 1 and Comparative Example 2. Further, as compared with the comparative example, Examples 1 to 3 using yeast having a mutation in the SHR3 gene and no mutation in the GAP1 gene had a high content of ornithine, and the ratio of ornithine to the total content of amino acids. Is also expensive.
以上より、実施例の酵母によればオルニチンの含有量が高い清酒が得られたことが分かる。 From the above, it can be seen that according to the yeast of the example, sake having a high content of ornithine was obtained.
Claims (6)
The production method according to claim 5, wherein the liquor is sake.
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