JP6270412B2 - Breeding method of sake yeast that reduces the production of ethyl-α-glucoside - Google Patents
Breeding method of sake yeast that reduces the production of ethyl-α-glucoside Download PDFInfo
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Description
本発明は、エチル−α−グルコシドの生成を低減する清酒酵母の育種方法、及び該育種方法で育種されたエチル−α−グルコシドの生成を低減する清酒酵母に関する。 The present invention relates to a method for breeding sake yeast that reduces the production of ethyl-α-glucoside, and a sake yeast that reduces the production of ethyl-α-glucoside bred by the breeding method.
清酒における消費者の嗜好は、時に大きく変わり、その嗜好に合った清酒を製造するには、様々な特徴を備える清酒酵母を育種する必要がある。消費者の嗜好に大きくかかわる清酒の要素として、芳香、甘味、酸味等が代表的であるが、その他に苦味も重要な要素に挙げられる。清酒における最近の消費者の嗜好としては、飲み易く、後味のすっきりした味が好まれ、甘味や苦味は敬遠される傾向にある。 Consumer preferences in sake change greatly, and it is necessary to breed sake yeast with various characteristics in order to produce sake that meets those preferences. Aroma, sweetness, sourness, etc. are typical as elements of sake that are greatly related to consumer preferences, but bitterness is also an important factor. The recent consumer preference for sake is that it is easy to drink and a refreshing aftertaste is preferred, and sweetness and bitterness tend to be avoided.
エチル−α−グルコシドは、清酒に含まれる糖質の中でも、グルコース等に次いで多量に含まれ、即効性の甘味と遅効性の苦味とを呈することが知られている。このエチル−α−グルコシドを多く含む清酒を飲酒すると、先ず即効性の甘味が口内に広がり、若干時間をおいて苦味を感じることになる。これは、清酒の飲みやすさと、後味のすっきり感とを低下させることになる。従来の清酒の製造方法では、淡麗な清酒を製造するために清酒中に含まれるグルコース等の糖質を低減させることを目的とする開発は行われてきたが、清酒中に含まれるエチル−α−グルコシド等の糖質に着目されてはいなかった。 It is known that ethyl-α-glucoside is contained in a large amount next to glucose and the like among sugars contained in sake, and exhibits fast-acting sweetness and slow-acting bitterness. If you drink sake that contains a lot of ethyl-α-glucoside, first you will have a quick-acting sweetness spread in your mouth and you will feel bitterness after some time. This reduces the ease of drinking sake and the refreshing aftertaste. In the conventional method for producing sake, development aimed at reducing carbohydrates such as glucose contained in sake has been performed in order to produce fresh sake, but ethyl- No attention has been paid to carbohydrates such as α-glucoside.
例えば、特許文献１では、醪を仕込む際にトランスグルコシダーゼ等の酵素を添加し、醪中のオリゴ糖やデンプン等を、酵母が容易に資化できるグルコースにまで分解することにより、醪中の酵母の発酵を促進させて清酒中に含まれる糖質を低減させる方法が開示されている。 For example, in Patent Document 1, an enzyme such as transglucosidase is added at the time of charging koji, and the oligosaccharide or starch in the koji is decomposed into glucose that can be easily assimilated by the yeast. Has been disclosed a method for reducing the sugars contained in sake by accelerating fermentation.
特許文献１に記載の清酒の醸造方法では、トランスグルコシダーゼ等の酵素によりグルコースに分解するオリゴ糖やデンプン等の糖質は低減できるものの、清酒中のエチル−α−グルコシドを考慮しておらず、製造条件によっては、飲み易く、後味のすっきりした清酒を製造できるとは限らない。 In the sake brewing method described in Patent Literature 1, although sugars such as oligosaccharides and starch that are decomposed into glucose by an enzyme such as transglucosidase can be reduced, ethyl-α-glucoside in sake is not considered, Depending on the production conditions, it is not always possible to produce sake that is easy to drink and has a clean aftertaste.
本発明は、上記問題点に鑑みてなされたものであり、飲み易く、後味のすっきりした清酒を製造するために、清酒中のエチル−α−グルコシドを低減する清酒酵母の育種方法を提供することを目的とする。さらに、当該育種方法で育種されたエチル−α−グルコシドの生成を低減する清酒酵母を提供することを目的とする。 The present invention has been made in view of the above problems, and provides a method for breeding sake yeast that reduces ethyl-α-glucoside in sake to produce a sake that is easy to drink and has a clean aftertaste. With the goal. Furthermore, it aims at providing the sake yeast which reduces the production | generation of the ethyl-alpha-glucoside bred by the said breeding method.
即ち、本発明は以下の発明を含む。
［発明１］
清酒酵母を変異処理してシクロヘキシミド耐性株を取得する耐性株取得工程と、
前記シクロヘキシミド耐性株の母集団からエチル−α−グルコシドの生成を低減する変異株を選択分離する変異株選択工程と、
を包含するエチル−α−グルコシドの生成を低減する清酒酵母の育種方法。
［発明２］
前記清酒酵母は、きょうかい酵母９０１号である発明１に記載のエチル−α−グルコシドの生成を低減する清酒酵母の育種方法。
［発明３］
前記変異処理は、メタンスルホン酸エチル処理である発明１又は２に記載のエチル−α−グルコシドの生成を低減する清酒酵母の育種方法。
［発明４］
発明１〜３の何れか一に記載のエチル−α−グルコシドの生成を低減する清酒酵母の育種方法で育種されたエチル−α−グルコシドの生成を低減する清酒酵母。
That is, the present invention includes the following inventions.
[Invention 1]
A resistant strain acquisition step of obtaining a cycloheximide resistant strain by mutating sake yeast,
A mutant selection step of selectively separating a mutant that reduces the production of ethyl-α-glucoside from the population of the cycloheximide-resistant strain;
A method for breeding sake yeast that reduces the production of ethyl-α-glucoside, comprising
[Invention 2]
The sake yeast is a method for breeding sake yeast that reduces production of ethyl-α-glucoside according to the first aspect of the present invention, which is Kyoto
[Invention 3]
The method for breeding sake yeast that reduces the production of ethyl-α-glucoside according to
[Invention 4]
The sake yeast which reduces the production | generation of the ethyl-alpha-glucoside bred by the breeding method of the sake yeast which reduces the production | generation of the ethyl-alpha-glucoside as described in any one of invention 1-3.
本構成のエチル−α−グルコシドの生成を低減する清酒酵母の育種方法は、上記構成を有することから、エチル−α−グルコシドの生成を低減する清酒酵母を高確率で取得することができる。その結果、当該育種方法を用いて取得したエチル−α−グルコシドの生成を低減する清酒酵母により、エチル−α−グルコシドを低減させた飲み易く、後味のすっきりした清酒を製造することができる。 Since the method for breeding sake yeast that reduces the production of ethyl-α-glucoside of the present configuration has the above-described configuration, it is possible to obtain a sake yeast that reduces the production of ethyl-α-glucoside with high probability. As a result, sake yeast with reduced ethyl-α-glucoside can be produced with a sake yeast that reduces the production of ethyl-α-glucoside obtained using the breeding method, and a clean sake with a clean aftertaste can be produced.
清酒中のエチル−α−グルコシドは、麹（米麹）由来の酵素等によって、糖類とエタノールとの糖転移反応により生成する。清酒酵母は、エチル−α−グルコシドの低減能力が低いことが知られている。そこで、本発明者らは、清酒中に生成するエチル−α−グルコシドを低減することができる清酒酵母の変異株を育種、取得することを試みた。抗生物質であるシクロヘキシミドに耐性を有するシクロヘキシミド耐性の変異株は、有機酸の代謝等（Ｊ．Ｂｒｅｗ．Ｓｏｃ．Ｊａｐａｎ．Ｖｏｌ．８８，Ｎｏ．８，ｐ．６４５〜６４７（１９９３））、様々な代謝に影響を受けることが知られている。本発明者らは、変異処理を行った清酒酵母の母集団からシクロヘキシミド耐性を指標として変異株を取得した。その結果、得られたシクロヘキシミド耐性変異株の形質を調べたところ、予期しないことに、その変異株の全菌株において、清酒中のエチル−α−グルコシドの生成量が、親株を使用したときよりも低減しているという新しい知見を得た。 Ethyl-α-glucoside in sake is produced by a transglycosylation reaction between saccharide and ethanol by an enzyme derived from koji (rice koji). Sake yeast is known to have a low ability to reduce ethyl-α-glucoside. Then, the present inventors tried to breed and acquire a mutant strain of sake yeast that can reduce ethyl-α-glucoside produced in sake. Cycloheximide-resistant mutants having resistance to the antibiotic cycloheximide include various organic acid metabolisms (J. Brew. Soc. Japan. Vol. 88, No. 8, p. 645-647 (1993)). It is known to be affected by metabolism. The inventors of the present invention obtained mutant strains using cycloheximide resistance as an index from a population of sake yeast that had undergone mutation treatment. As a result, when the traits of the obtained cycloheximide-resistant mutant strain were examined, unexpectedly, in all strains of the mutant strain, the amount of ethyl-α-glucoside produced in sake was higher than when the parent strain was used. I got new knowledge that it was decreasing.
本発明に係るエチル−α−グルコシドの生成を低減する清酒酵母の育種方法、及び該育種方法で育種されたエチル−α−グルコシドの生成を低減する清酒酵母に関する実施形態について、以下に具体的に説明する。ただし、本発明は、以下に説明する実施形態や図面に記載される構成に限定されることを意図しない。 Embodiments relating to a method for breeding sake yeast that reduces production of ethyl-α-glucoside according to the present invention and sake yeast that reduces production of ethyl-α-glucoside bred by the breeding method are specifically described below. explain. However, the present invention is not intended to be limited to the configurations described in the embodiments and drawings described below.
ここで、本明細書中に記載される「エチル−α−グルコシドの生成を低減する清酒酵母」とは、本発明の育種方法で用いられる清酒酵母である親株と比較して、清酒中のエチル−α−グルコシドの生成量を低減する清酒酵母を意味する。 Here, “sake yeast that reduces the production of ethyl-α-glucoside” described in the present specification refers to ethyl in sake compared to the parent strain that is the sake yeast used in the breeding method of the present invention. -It means sake yeast that reduces the amount of α-glucoside produced.
［エチル−α−グルコシドの生成を低減する清酒酵母の育種方法］
本発明に係る清酒酵母の育種方法は、清酒酵母を変異処理してシクロヘキシミド耐性株を取得する耐性株取得工程と、当該シクロヘキシミド耐性株の母集団からエチル−α−グルコシドの生成を低減する変異株を選択分離する変異株選択工程と、を有している。以下、本発明に係るエチル−α−グルコシド（以下、α−ＥＧと称す）の生成を低減する清酒酵母の育種方法について、詳細に説明する。
[Method of Breeding Sake Yeast to Reduce Production of Ethyl-α-Glucoside]
The method for breeding sake yeast according to the present invention includes a resistant strain obtaining step for obtaining a cycloheximide-resistant strain by mutating the sake yeast, and a mutant strain that reduces the production of ethyl-α-glucoside from the population of the cycloheximide-resistant strain. And a mutant strain selection step for selectively separating. Hereinafter, a method for breeding sake yeast that reduces the production of ethyl-α-glucoside (hereinafter referred to as α-EG) according to the present invention will be described in detail.
（育種に用いる親株）
本発明の清酒酵母の育種方法に用いる親株としては、サッカロマイセス・セレビジエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）に属する清酒酵母であればすべての酵母に利用することができる。親株として使用する清酒酵母は、実験室で利用する実験室酵母でもよいが、清酒の醸造で一般に利用される既存の清酒酵母が好ましい。既存の清酒酵母としては、例えば、公益社団法人日本醸造協会から頒布されている、きょうかい酵母６号、７号、９号、１０号、１１号、１４号、６０１号、７０１号、９０１号、１００１号、１４０１号、１５０１号、１６０１号、１７０１号、１８０１号及びＫＴ９０１号等；各県工業総合研究センターが開発した、まほろば華酵母、吟醸２号、宮城マイ酵母、愛美酵母、泡なし宮城マイ酵母、秋田流・花酵母、秋田純米酵母、こまち酵母、秋田流・雅酵母；学校法人東京農業大学が開発した、花酵母ＮＤ−４、ＡＢ−２、ＮＩ−２、ＨＮＧ−５、ＣＡＲ−１、ＫＳ−３、ＳＵＮＦ−５、ＢＫ−１、ＳＮ−３、ＭＲ−４、ＧＥ−１、ＫＡＦ−２及びＳＴ−４から容易に取得できる。また、秋田今野商店より販売されている、清酒用Ｎｏ．２４、清酒用Ｎｏ．２５、及び清酒用Ｎｏ．３５等を購入することにより取得することもできる。また、前記既存の清酒酵母に変異あるいは交配・育種したものを用いても良い。本発明の清酒酵母の育種方法は、これら清酒酵母を親株として用いることにより、親株の特徴を維持しながら、即効性の甘味及び遅効性の苦味の成分であるエチル−α−グルコシドの生成量を低減する清酒酵母を取得することができる。
(Parent strain used for breeding)
As the parent strain used in the method for breeding sake yeast of the present invention, any yeast can be used as long as it is a sake yeast belonging to Saccharomyces cerevisiae. The sake yeast used as the parent strain may be a laboratory yeast used in the laboratory, but an existing sake yeast generally used in sake brewing is preferred. As existing sake yeast, for example, Kyokai yeast 6, 7, 9, 10, 11, 14, 601, 701, 901 distributed by the Japan Brewing Association 1001, 1401, 1501, 1601, 1601, 1701, 1801 and KT901, etc .; Mahoroba flower yeast, Ginjo 2, Miyagi my yeast, Aimi yeast, no foam, developed by each industrial research center Miyagi My Yeast, Akita Ryu / Flower Yeast, Akita Junmai Yeast, Komachi Yeast, Akita Ryu / Masa Yeast; Flower Yeast ND-4, AB-2, NI-2, HNG-5 developed by Tokyo University of Agriculture , CAR-1, KS-3, SUNF-5, BK-1, SN-3, MR-4, GE-1, KAF-2 and ST-4. Also, No. for sake, sold by Akita Imano Shoten. 24, Sake No. 25, and sake no. It can also be obtained by purchasing 35 or the like. Moreover, you may use what was mutated or crossed and bred to the said existing sake yeast. The method for breeding sake yeast of the present invention uses these sake yeasts as parent strains, while maintaining the characteristics of the parent strain, while reducing the production amount of ethyl-α-glucoside, which is a component of immediate-effect sweetness and slow-acting bitterness. Sake yeast to be reduced can be obtained.
（シクロヘキシミド耐性株の取得）
シクロヘキシミドは、細菌の一種であるストレプトマイセス グリセウス（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｇｒｉｓｅｕｓ）によって生産される抗生物質であり、真核生物のタンパク質合成を選択的に阻害する。シクロヘキシミドはタンパク質合成の転位過程（リボソームに結合する二つのｔＲＮＡ分子とｍＲＮＡの移動）に干渉することでその効果を示し、タンパク質の翻訳を阻害している。このシクロヘキシミドに耐性を有する清酒酵母は、上述の既存の清酒酵母に対して交配、細胞融合、形質転換、及び変異処理等を行うことにより取得することができる。この中でも、変異処理によりシクロヘキシミド耐性株を分離することが好ましい。これにより、シクロヘキシミド耐性株を容易に取得することができる。
(Acquisition of cycloheximide resistant strain)
Cycloheximide is an antibiotic produced by Streptomyces griseus, a kind of bacteria, and selectively inhibits eukaryotic protein synthesis. Cycloheximide shows its effect by interfering with the translocation process of protein synthesis (two tRNA molecules that bind to ribosome and the movement of mRNA) and inhibits protein translation. The sake yeast having resistance to cycloheximide can be obtained by performing mating, cell fusion, transformation, mutation treatment and the like on the above-mentioned existing sake yeast. Among these, it is preferable to isolate a cycloheximide resistant strain by mutation treatment. Thereby, a cycloheximide resistant strain can be easily obtained.
清酒酵母を変異処理する方法としては、物理的変異処理及び化学的変異処理がある。物理的変異処理としては、紫外線、放射線、イオンビーム等による照射処理が挙げられ、化学的変異処理としては、亜硝酸、ナイトロジェンマスタード、アクリジン系色素、Ｎ−メチル−Ｎ’−ニトロ−Ｎ−ニトロソグアニジン（ＮＴＧ）、メタンスルホン酸エチル（以下、ＥＭＳと称す）等による化学処理が挙げられる。これらの変異処理は、使用する親株の清酒酵母に応じて適宜選択することができるが、好ましくはＥＭＳを用いた化学的変異処理である。これにより、清酒酵母のシクロヘキシミド耐性株を容易に取得することができる。 Methods for mutation treatment of sake yeast include physical mutation treatment and chemical mutation treatment. Examples of physical mutation treatment include irradiation treatment with ultraviolet rays, radiation, ion beams, etc., and chemical mutation treatment includes nitrous acid, nitrogen mustard, acridine dye, N-methyl-N′-nitro-N—. Examples include chemical treatment with nitrosoguanidine (NTG), ethyl methanesulfonate (hereinafter referred to as EMS), and the like. These mutation treatments can be appropriately selected depending on the parent sake sake yeast to be used, but are preferably chemical mutation treatments using EMS. Thereby, a cycloheximide resistant strain of sake yeast can be easily obtained.
ＥＭＳを用いて清酒酵母に変異処理を行う方法としては、例えば、先ず、親株である清酒酵母を、ＹＰＤ培地（２％グルコース、２％ポリペプトン、１％酵母エキス）を用いて、２５℃〜３７℃で、３０〜５０時間浸とう培養し、得られた培養液を遠心分離により集菌する。次いで、０．２Ｍリン酸緩衝液（ｐＨ６．０〜８．０）１０ｍｌに、ＥＭＳの濃度が０．３〜６．０％となるように混合し、当該混合液に上記集菌した菌体を再懸濁して、３０℃で、１５〜６０分間変異処理を行う。このとき、清酒酵母の生存率が０．０１〜９０％、好ましくは１０〜８０％、より好ましくは３０〜６０％となるように変異処理を行う。これにより、シクロヘキシミド耐性株の中から、効率的にα−ＥＧの生成を低減する清酒酵母を取得することができる。変異処理後、処理溶液に５％チオ硫酸ナトリウム溶液を添加してＥＭＳを中和し、０．２Ｍリン酸緩衝溶液を用いて変異処理菌体を２回洗浄する。洗浄した菌体を、シクロヘキシミド含有寒天培地上に塗布して、２５〜３７℃で、７〜１０日間培養し、増殖したコロニーを釣菌する。得られたシクロヘキシミド耐性株を、レプリカ法等を用いて、さらにシクロヘキシミド含有寒天培地で培養し、シクロヘキシミド耐性を再度確認することが好ましい。これにより、確実にシクロヘキシミド耐性株を取得することができる。使用するシクロヘキシミド含有寒天培地は、清酒酵母を培養できる寒天培地であればよいが、ＹＰＤ寒天培地（２％グルコース、２％ポリペプトン、１％酵母エキス、２％寒天）が好ましい。シクロヘキシミドの寒天培地への添加量は、０．５〜１．５μｇ／ｍｌであり、好ましくは０．５〜１．０μｇ／ｍｌである。培養条件は、使用する清酒酵母に応じて適宜選択される。 As a method for performing a mutation treatment on sake yeast using EMS, for example, first, a yeast yeast as a parent strain is treated at 25 ° C. to 37 ° C. using a YPD medium (2% glucose, 2% polypeptone, 1% yeast extract). Culturing is carried out at 30 ° C. for 30 to 50 hours, and the resulting culture is collected by centrifugation. Subsequently, 10 ml of 0.2 M phosphate buffer solution (pH 6.0 to 8.0) was mixed so that the concentration of EMS would be 0.3 to 6.0%, and the cells collected above in the mixture solution Is resuspended and subjected to mutation treatment at 30 ° C. for 15 to 60 minutes. At this time, the mutation treatment is performed so that the sake yeast has a survival rate of 0.01 to 90%, preferably 10 to 80%, more preferably 30 to 60%. Thereby, the sake yeast which reduces the production | generation of (alpha) -EG efficiently from a cycloheximide tolerance strain | stump | stock can be acquired. After the mutation treatment, the 5% sodium thiosulfate solution is added to the treatment solution to neutralize EMS, and the mutation-treated cells are washed twice using a 0.2 M phosphate buffer solution. The washed cells are spread on a cycloheximide-containing agar medium and cultured at 25 to 37 ° C. for 7 to 10 days to catch the grown colonies. The obtained cycloheximide-resistant strain is preferably further cultured on a cycloheximide-containing agar medium using a replica method or the like, and the cycloheximide resistance is confirmed again. Thereby, a cycloheximide resistant strain can be acquired reliably. The cycloheximide-containing agar medium to be used may be an agar medium capable of culturing sake yeast, but a YPD agar medium (2% glucose, 2% polypeptone, 1% yeast extract, 2% agar) is preferable. The amount of cycloheximide added to the agar medium is 0.5 to 1.5 μg / ml, preferably 0.5 to 1.0 μg / ml. The culture conditions are appropriately selected according to the sake yeast to be used.
（エチル−α−グルコシドの生成を低減する変異株の選択）
α−ＥＧの生成を低減する変異株の選抜方法として、例えば、一段仕込みの小仕込み試験を行い、上槽後、α−ＥＧの生成量を分析してα−ＥＧの生成量の少ない株を選択する。小仕込み試験は、例えば、１０〜１５℃で、１５〜２０日間培養を行う。小仕込み試験では、原料米を液化した融米を使用してもよい。液化した融米は、例えば、原料米又は原料米を粉砕した粉砕米に、汲水及び耐熱性酵素であるα−アミラーゼを添加し、６０〜９０℃で液化することにより得られる（特開昭５９−６６８７５号公報参照）。液化した融米を使用する場合、総米における融米の比率は、例えば、融米の調製に使用した原料米として換算することができる。α−ＥＧの分析方法としては、高速液体クロマトグラフィー（ＨＰＬＣ）による定量が好適に用いられる。
(Selection of mutant strains that reduce the production of ethyl-α-glucoside)
As a method for selecting mutant strains that reduce the production of α-EG, for example, a single preparation test is performed, and after the upper tank, the production amount of α-EG is analyzed to obtain a strain with a small production amount of α-EG. select. For example, the small preparation test is performed at 10 to 15 ° C. for 15 to 20 days. In the small preparation test, melted rice obtained by liquefying raw rice may be used. Liquefied melted rice is obtained, for example, by adding raw water or pulverized rice obtained by pulverizing raw rice rice and adding α-amylase, which is a heat-resistant enzyme, and liquefying at 60 to 90 ° C. 59-66875). When using the liquefied molten rice, the ratio of the molten rice in the total rice can be converted into, for example, raw material rice used for preparing the molten rice. As a method for analyzing α-EG, quantitative determination by high performance liquid chromatography (HPLC) is preferably used.
その他のα−ＥＧの生成を低減する清酒酵母の選抜方法として、例えば、得られたシクロヘキシミド耐性株を、麹汁培地、合成培地等の清酒酵母用の一般的な培地で１５℃〜３７℃で、１０〜２０日培養を行い、α−ＥＧの生成量を分析してα−ＥＧの生成量の少ない株を選択することも可能である。清酒酵母用の一般的な培地は、好ましくは麹汁培地である。麹汁培地は、例えば、米麹３００ｇに蒸留水１Ｌを加えて、５５〜６０℃で６時間糖化後、濾過した液を蒸留水でブリックス（Ｂｒｉｘ）を１０〜１５％程度に調整し、オートクレーブ滅菌（例えば、１２１℃、２０分）することで得ることができる。 As another method for selecting sake yeast that reduces the production of α-EG, for example, the obtained cycloheximide-resistant strain is a common medium for sake yeast such as a broth medium and a synthetic medium at 15 ° C. to 37 ° C. It is also possible to culture for 10 to 20 days, analyze the production amount of α-EG, and select a strain with a small production amount of α-EG. A common medium for sake yeast is preferably a broth medium. For example, 1 L of distilled water is added to 300 g of rice bran and saccharified at 55-60 ° C. for 6 hours, and the filtrate is adjusted to about 15% to 15% Brix with distilled water. It can be obtained by sterilization (eg, 121 ° C., 20 minutes).
［エチル−α−グルコシドの生成を低減する清酒酵母の育種方法の評価］
α−ＥＧの生成を低減する清酒酵母の育種方法に関して、α−ＥＧの生成を低減する清酒酵母の取得率を評価した。得られたシクロヘキシミド耐性株と、親株のα−ＥＧの生成量とを比較して、α−ＥＧの生成を低減する清酒酵母を評価した。
[Evaluation of Breeding Method of Sake Yeast to Reduce Production of Ethyl-α-Glucoside]
Regarding the method for breeding sake yeast that reduces the production of α-EG, the acquisition rate of sake yeast that reduces the production of α-EG was evaluated. The resulting cycloheximide-resistant strain was compared with the amount of α-EG produced by the parent strain to evaluate sake yeast that reduces α-EG production.
（シクロヘキシミド耐性株の取得）
親株は、きょうかい酵母９０１号（以下、Ｋ−９０１株と称す）を使用した。Ｋ−９０１株は、きょうかい酵母９号を親株とする泡なし酵母であり、低温でも醗酵力が旺盛で、酸が少なく、高い芳香を有する清酒を醸造するのに適した酵母である。Ｋ−９０１株で醸造する清酒中のα−ＥＧを低減できれば、飲み易く、すっきりとした清酒を製造することができる。
このＫ−９０１株を、ＹＰＤ培地（２％グルコース、２％ポリペプトン、１％酵母エキス）を用いて、３０℃で、２４時間培養後、ＥＭＳで変異処理を行った。変異処理条件としては、ＹＰＤ培地で培養した菌体を遠心分離にて集菌（１×１０８細胞／ｍｌ）し、０．２Ｍリン酸緩衝液（ｐＨ７．０）１０ｍｌに懸濁し、３％となるようにＥＭＳを添加して、３０℃で、４５分間変異処理を行った。５％チオ硫酸ナトリウム溶液でＥＭＳを中和し、０．２Ｍリン酸緩衝液（ｐＨ７．０）で、変異処理菌体を２回洗浄した。次いで、シクロヘキシミド含有ＹＰＤ寒天培地（１ｍｇ／Ｌシクロヘキシミド、２％グルコース、２％ポリペプトン、１％酵母エキス、２％寒天）１０枚に、洗浄した菌体（計１×１０８細胞）を塗布して、３０℃で、１０日間培養した。シクロヘキシミド含有ＹＰＤ寒天培地上で増殖したコロニーを同じシクロヘキシミド含有ＹＰＤ寒天培地にレプリカして、３０℃で、１０日間培養し、各菌株のシクロヘキシミド耐性を再度確認した。上記シクロヘキシミド耐性株の取得試験を２回（試験１及び試験２）実施し、シクロヘキシミド耐性株を、計８０菌株（各試験４０菌株）取得した。これらシクロヘキシミド耐性の８０菌株を、以下のα−ＥＧの生成量の評価試験に供した。
(Acquisition of cycloheximide resistant strain)
As a parent strain, Kyokai Yeast No. 901 (hereinafter referred to as K-901 strain) was used. The K-901 strain is a foam-free yeast whose parent strain is Kyokai Yeast No. 9, and is a yeast suitable for brewing sake having a high fermenting ability, low acidity and high aroma even at low temperatures. If α-EG in sake brewed with K-901 strain can be reduced, it is easy to drink and clean sake can be produced.
This K-901 strain was cultured at 30 ° C. for 24 hours using YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), and then subjected to mutation treatment with EMS. As mutation treatment conditions, cells cultured in YPD medium were collected by centrifugation (1 × 10 8 cells / ml), suspended in 10 ml of 0.2 M phosphate buffer (pH 7.0), and 3% EMS was added so that it might become, and the mutation process was performed for 45 minutes at 30 degreeC. The EMS was neutralized with a 5% sodium thiosulfate solution, and the mutation-treated cells were washed twice with 0.2 M phosphate buffer (pH 7.0). Next, 10 cells of cycloheximide-containing YPD agar (1 mg / L cycloheximide, 2% glucose, 2% polypeptone, 1% yeast extract, 2% agar) were applied with the washed cells (1 × 10 8 cells in total). The cells were cultured at 30 ° C. for 10 days. Colonies grown on a cycloheximide-containing YPD agar medium were replicated on the same cycloheximide-containing YPD agar medium and cultured at 30 ° C. for 10 days, and the cycloheximide resistance of each strain was confirmed again. The cycloheximide resistant strain acquisition test was performed twice (Test 1 and Test 2), and a total of 80 cycloheximide resistant strains (40 strains in each test) were acquired. The 80 strains resistant to cycloheximide were subjected to the following α-EG production evaluation test.
（シクロヘキシミド耐性株の発酵試験及びエチル−α−グルコシドの生成量の評価）
得られたシクロヘキシミド耐性株の８０菌株と、親株であるＫ−９０１株とを用いて発酵試験を行い、α−ＥＧの含有量を分析して、Ｋ−９０１株に対するシクロヘキシミド耐性株のα−ＥＧの生成量を評価した。また、各シクロヘキシミド耐性株の上槽酒の酒質についてもＫ−９０１株と比較した。発酵試験は、掛米の液化液１０．２ｍｌ、米麹の糖化液７．５ｍｌ、乳酸０．０３ｍｌに汲水を加え、最終酵母密度が１×１０７細胞／ｍｌとなるように清酒酵母を添加して、一段仕込みで実施した。発酵温度は、１５℃の一定で行い、１５日間の発酵を行った。得られた上槽酒を、α−ＥＧ及び酒質の分析試験に供した。掛米の液化には、液化酵素として、ＴＵ（商品名クライスターゼＳＤ−ＴＵ２０、酵素名α−アミラーゼ、天野エンザイム社製）とＹＣ（商品名コクゲンＳＤ−ＹＣ１５、酵素名α−アミラーゼ、天野エンザイム社製）とを重量比７：３で混合したものを使用し、酵素の総量と総米の総重量との比が、１：３，５００となるように調製した。米麹の糖化液は、米麹に液化液を加え、麹菌の酵素により麹米の澱粉を糖化したものを使用した。
(Fermentation test of cycloheximide-resistant strain and evaluation of production amount of ethyl-α-glucoside)
The obtained cycloheximide-resistant strain 80 and the parent K-901 strain were subjected to a fermentation test, the α-EG content was analyzed, and the cycloheximide-resistant strain α-EG against the K-901 strain was analyzed. The production amount of was evaluated. In addition, the quality of the upper tank liquor of each cycloheximide resistant strain was also compared with the K-901 strain. In the fermentation test, sake yeast was added so that the final yeast density would be 1 × 10 7 cells / ml by adding pumped water to 10.2 ml of liquefied rice cake, 7.5 ml of saccharified rice bran, and 0.03 ml of lactic acid. It was added and carried out in a single charge. The fermentation temperature was constant at 15 ° C., and the fermentation was performed for 15 days. The obtained upper tank liquor was subjected to α-EG and alcohol quality analysis tests. For liquefaction of rice cake, TU (trade name Christase SD-TU20, enzyme name α-amylase, manufactured by Amano Enzyme) and YC (trade name Kokugen SD-YC15, enzyme name α-amylase, Amano enzyme) are used as liquefaction enzymes. And a mixture of the total amount of enzyme and the total weight of total rice was 1: 3,500. As the saccharified solution of rice bran, a product obtained by adding a liquefied solution to rice bran and saccharifying the starch of glutinous rice with an enzyme of koji mold was used.
（酒質及びエチル−α−グルコシドの分析試験方法）
シクロヘキシミド耐性を有する８０菌株について、上記一段仕込みで得られた上槽酒について、酒質及びα−ＥＧの分析を行った。酒質の分析については、独立行政法人酒類総合研究所が定める「酒類総合研究所標準分析法」（平成２２年１１月４日、ｈｔｔｐ：//ｗｗｗ.ｎｒｉｂ.ｇｏ.ｊｐ/ｄａｔａ/ｎｒｉｂａｎａｌｙｓｉｓ.ｈｔｍ）に基づいて、アルコール度数及び酸度を分析した。具体的には、以下（１）及び（２）の方法である。
（１）アルコール：ガスクロマトグラフ分析法に従って、測定を行った。
（２）酸度：酸度は、清酒に含まれる、有機酸（乳酸、リンゴ酸、コハク酸等）の総量を示した値である。具体的には、１０ｍＬの清酒を中和するのに要する水酸化ナトリウム溶液の滴定量（ｍＬ）で表す。
α−ＥＧの分析については、高速液体クロマトグラフを用いて行った。具体的には、以下の分析条件で行った。
装置：高速液体クロマトグラフ「ＬＣ−２０型」（株式会社島津製作所製）
検出器：示差屈折率検出器（ＲＩ検出器）
カラム：「Ｓｈｏｄｅｘ Ａｓａｈｉｐａｋ ＮＨ２Ｐ−５０ ４Ｅ」（昭和電工株式会社製）
カラム温度：４０℃
移動相：アセトニトリル／水＝７０／３０（容積比）
流量：１ｍＬ／ｍｉｎ
(Analytical test method for alcohol and ethyl-α-glucoside)
For the 80 strains having cycloheximide resistance, the quality of sake and α-EG were analyzed for the upper tank sake obtained by the above-mentioned one-stage preparation. For the analysis of liquor quality, “Standard Analysis Method for Liquor Research Institute” established by the National Research Institute for Liquors (November 4, 2010, http://www.nrib.go.jp/data/nribanarisis. Based on htm), the alcohol content and acidity were analyzed. Specifically, the following methods (1) and (2) are used.
(1) Alcohol: Measurement was performed according to a gas chromatographic analysis method.
(2) Acidity: Acidity is a value indicating the total amount of organic acids (lactic acid, malic acid, succinic acid, etc.) contained in sake. Specifically, it is represented by a titration amount (mL) of a sodium hydroxide solution required to neutralize 10 mL of sake.
About the analysis of (alpha) -EG, it performed using the high performance liquid chromatograph. Specifically, the analysis was performed under the following analysis conditions.
Apparatus: High-performance liquid chromatograph “LC-20” (manufactured by Shimadzu Corporation)
Detector: Differential refractive index detector (RI detector)
Column: “Shodex Asahipak NH2P-50 4E” (manufactured by Showa Denko KK)
Column temperature: 40 ° C
Mobile phase: acetonitrile / water = 70/30 (volume ratio)
Flow rate: 1 mL / min
以下の表１（試験１で得られた菌株）及び表２（試験２で得られた菌株）に、シクロヘキシミド耐性を有する８０菌株のアルコール（％）、酸度（ｍｌ）、及びα−ＥＧ（ｇ／ｄＬ）に関する分析値を示した。これらの分析値は、２回のシクロヘキシミド耐性株の取得試験で得られた、シクロヘキシミド耐性の各４０菌株（計８０菌株）の酒質及びα−ＥＧの分析結果を示している。また、各シクロヘキシミド耐性株の親株に対するα−ＥＧの低減率については、親株であるＫ−９０１株のα−ＥＧの生成量を１００％とした場合におけるシクロヘキシミド耐性株のα−ＥＧの低減量の割合（％）で表している。
以下の表３には、得られたシクロヘキシミド耐性株中の親株Ｋ−９０１株に対するα−ＥＧの低減割合と、エチル−α−グルコシドの生成を低減する清酒酵母の取得率との関係を示した。表３では、取得した８０菌株のシクロヘキシミド耐性株を、親株であるＫ−９０１株のα−ＥＧの生成量を１００％とした場合における、シクロヘキシミド耐性株のα−ＥＧの低減量で分類分けしている。
In Table 1 below (the strain obtained in Test 1) and Table 2 (the strain obtained in Test 2), the alcohol (%), acidity (ml), and α-EG (g) of 80 strains having cycloheximide resistance. Analytical value for / dL) is shown. These analytical values show the analysis results of the alcohol quality and α-EG of 40 cycloheximide-resistant strains (total of 80 strains) obtained in two cycloheximide-resistant strain acquisition tests. Moreover, about the reduction | decrease rate of (alpha) -EG with respect to the parent strain of each cycloheximide resistant strain, when the production amount of (alpha) -EG of K-901 strain | stump | stock which is a parent strain is 100%, the reduction amount of (alpha) -EG of a cycloheximide resistant strain is Expressed as a percentage.
Table 3 below shows the relationship between the α-EG reduction ratio relative to the parent K-901 strain in the obtained cycloheximide-resistant strain and the acquisition rate of sake yeast that reduces the production of ethyl-α-glucoside. . In Table 3, the obtained cycloheximide-resistant strains of 80 strains are classified according to the α-EG reduction amount of the cycloheximide-resistant strain when the production amount of α-EG of the parent K-901 strain is 100%. ing.
表１及び表２より、２回のシクロヘキシミド耐性株の何れの試験においても、得られた全てのシクロヘキシミド耐性株は、親株であるＫ−９０１株と比較してα−ＥＧの生成量が低減する結果となった。また、これら得られた全てのシクロヘキシミド耐性株は、アルコールの生産性に関し、親株であるＫ−９０１株と同等の値を示していることから、何れのシクロヘキシミド耐性株も親株であるＫ−９０１株のアルコール生産能を維持していると考えられる。 From Tables 1 and 2, all the cycloheximide-resistant strains obtained in any of the two cycloheximide-resistant strains produced a reduced amount of α-EG compared to the parent strain K-901. As a result. Moreover, since all the obtained cycloheximide resistant strains showed the same value as the parent strain K-901 regarding the productivity of alcohol, any cycloheximide resistant strain was the parent strain K-901. It is thought that the alcohol-producing ability is maintained.
表３より、取得したシクロヘキシミド耐性株のうち、全ての菌株が親株であるＫ−９０１株よりα−ＥＧの生成量が低減しており、その内７８％のシクロヘキシミド耐性株（６２菌株）がＫ−９０１株と比較してα−ＥＧの低減量が４０％を超えて減少し、４３％のシクロヘキシミド耐性株（３４菌株）がＫ−９０１株と比較してα−ＥＧの低減量が６０％を超えて減少する結果となった。上記結果から、本発明のエチル−α−グルコシドの生成を低減する清酒酵母の育種方法では、エチル−α−グルコシドの生成を低減する清酒酵母を確実に取得することができ、さらに、α−ＥＧの生成量を大幅に低減する清酒酵母を高確率で取得できることが示された。また、試験結果には示していないが、これらシクロヘキシミド耐性株は、親株であるＫ−９０１株よりもα−ＥＧの生成を低減するため、全糖濃度においてもＫ−９０１株より低減していた。つまり、これらα−ＥＧの生成を低減する清酒酵母は、糖質濃度が低い清酒の製造にも好適に利用することができる。 From Table 3, among the obtained cycloheximide-resistant strains, all the strains have α-EG produced in a reduced amount from the parent strain K-901, of which 78% of the cycloheximide-resistant strains (62 strains) are K The reduction amount of α-EG is reduced by more than 40% compared to the -901 strain, and 43% of cycloheximide resistant strains (34 strains) are reduced by 60% of the α-EG compared to the K-901 strain. As a result, the result decreased. From the above results, in the method of breeding sake yeast for reducing the production of ethyl-α-glucoside of the present invention, sake yeast for reducing the production of ethyl-α-glucoside can be reliably obtained, and α-EG is further obtained. It was shown that sake yeast that greatly reduces the production amount of can be obtained with high probability. In addition, although not shown in the test results, these cycloheximide-resistant strains had a lower total sugar concentration than the K-901 strain in order to reduce α-EG production compared to the parent strain K-901. . That is, the sake yeast that reduces the production of α-EG can be suitably used for the production of sake having a low sugar concentration.
［小仕込み試験］
上記取得したα−ＥＧの生成を低減する清酒酵母の中から、Ｎｏ．２株、Ｎｏ．１９株、Ｎｏ．２１株、Ｎｏ３７株、Ｎｏ．５４株、Ｎｏ．５６株、Ｎｏ．６９株、Ｎｏ．７８株、及び親株であるＫ−９０１株を用いて清酒の小仕込み試験を行い、各菌株の発酵経過に伴う炭酸ガス減量を測定し、各菌株の発酵力を比較評価した。また、得られた上槽酒は、酒質分析及びα−ＥＧの分析に供し、親株であるＫ−９０１株と比較した。
[Small preparation test]
Among sake yeasts that reduce the production of the obtained α-EG, no. 2 strains, no. 19 strains, no. No. 21, No. 37 strain, No. 54, No. 56 strains, no. 69, No. A small amount of sake was prepared using 78 strains and the parent strain K-901, and the amount of carbon dioxide lost during fermentation of each strain was measured, and the fermentative power of each strain was compared and evaluated. Moreover, the obtained upper tank liquor was subjected to alcohol quality analysis and α-EG analysis, and compared with the parent strain K-901.
小仕込み試験は、掛米としてα化米（精米歩合７０％）３６ｇ、米麹として乾燥麹（精米歩合７０％）９ｇ、汲水９１ｍｌ、乳酸２００μｌを混合し、最終酵母密度が１×１０７細胞／ｍｌとなるように清酒酵母添加して、一段仕込みで実施した。発酵温度は、１５℃の一定で行い、１５日後に上槽した。上槽液の酒質分析及びα−ＥＧの分析結果を以下の表４に示す。 In the small preparation test, 36 g of α-rice (70% polished rice ratio) is used as the rice, 9 g of dried rice bran (70% polished rice ratio) is 9%, 91 ml of drawn water and 200 μl of lactic acid are mixed, and the final yeast density is 1 × 10 7. Sake yeast was added so that it might become a cell / ml, and it implemented by the one-step preparation. The fermentation temperature was kept constant at 15 ° C., and the upper tank was added after 15 days. Table 4 below shows the alcohol quality analysis of the upper tank liquid and the analysis results of α-EG.
図１は、本発明に係るエチル−α−グルコシドの生成を低減する清酒酵母の発酵経過に伴う炭酸ガス減少量を示すグラフである。図１から明らかなように、Ｋ−９０１株を親株とするα−ＥＧの生成を低減する株の炭酸ガス減少量の経過は、親株と比較して差異は殆ど認められなかった。従って、α−ＥＧの生成を低減する８菌株は、親株と同等の発酵力を有しており、清酒の製造に十分利用できる清酒酵母であることが確認された。また、α−ＥＧの生成を低減する８菌株は、表４に示すように、小仕込み試験でも安定してα−ＥＧを低減し、エタノールに関しても親株であるＫ−９０１株と同等の値のアルコール生産能を有していることが示された。 FIG. 1 is a graph showing the amount of carbon dioxide reduction with the progress of fermentation of sake yeast that reduces the production of ethyl-α-glucoside according to the present invention. As is clear from FIG. 1, the difference in the amount of carbon dioxide gas decreased in the strain that reduces the production of α-EG having the K-901 strain as the parent strain was hardly observed as compared with the parent strain. Therefore, it was confirmed that 8 strains that reduce the production of α-EG have a fermentation power equivalent to that of the parent strain, and are sake yeast that can be sufficiently used for the production of sake. Moreover, as shown in Table 4, 8 strains that reduce the production of α-EG stably reduce α-EG even in a small preparation test, and ethanol has a value equivalent to that of the parent strain K-901. It was shown to have alcohol production ability.
［官能試験］
酸度は、清酒の製造過程で酵母や麹、米から発生した乳酸・コハク酸・クエン酸・リンゴ酸等の有機酸の量を表したものである。酸度が高い場合、味に旨みと爽快感があり、酸度が低いと淡麗辛口のすっきり感がある。そこで、上記エチル−α−グルコシドの生成を低減する清酒酵母の中から、親株であるＫ−９０１株の酸度に近く、α−ＥＧの生成量が低いＮｏ．７８株を選択し、Ｎｏ．７８株の上槽酒と親株であるＫ−９０１株の上槽酒とを官能試験により比較した。官能試験は、熟練した８人のパネリストで、上槽酒の味わいについてフリーコメントを記載する形式で実施した。その結果、Ｎｏ．７８株の上槽酒は、Ｋ−９０１株の上槽酒と比較して、「シャープな味で、後味がすっきりしている」、「味が柔らかい」等のコメントが多く得られた。これは、α−ＥＧが減少したことにより、即効性の甘味と遅効性の苦味とが減少して、すっきりとした味わいになったと考えられる。
[Sensory test]
The acidity represents the amount of organic acids such as lactic acid, succinic acid, citric acid and malic acid generated from yeast, koji, and rice during the production of sake. When the acidity is high, the taste has a taste and refreshing feeling, and when the acidity is low, the taste is refreshing and dry. Therefore, among the sake yeasts that reduce the production of ethyl-α-glucoside, No. 1 is close to the acidity of the parent strain K-901 and has a low α-EG production. 78 strains were selected. The 78 tanks and the parent K-901 top tank were compared by a sensory test. The sensory test was carried out in the form of a free comment on the taste of the upper tank liquor by eight experienced panelists. As a result, no. Compared with the K-901 strain, the 78 tanks of the top tank liquor received many comments such as “sharp taste and clean aftertaste” and “soft taste”. This is thought to be due to the decrease in α-EG, which resulted in a decrease in immediate-effect sweetness and slow-acting bitterness, resulting in a refreshing taste.
本発明に係るエチル−α−グルコシドの生成を低減する清酒酵母の育種方法、及び該育種方法で育種されたエチル−α−グルコシドの生成を低減する清酒酵母は、清酒の製造において利用可能であり、本醸造、純米酒、吟醸酒、純米吟醸酒の製造に利用することができる。 The method for breeding sake yeast that reduces the production of ethyl-α-glucoside according to the present invention, and the sake yeast that reduces the production of ethyl-α-glucoside bred by the breeding method can be used in the production of sake. It can be used for the production of Honjozo, Junmaishu, Ginjoshu, Junmaiginjoshu.
Claims (4)
前記変異処理を施した親株をシクロヘキシミド含有寒天培地で培養し、シクロヘキシミドに耐性の変異株を選択分離することで、エチル−α−グルコシドの生成を低減する変異株を得る変異株選択工程と、
前記変異株によるエチル−α−グルコシドの生成量を分析する分析工程と、
を包含するエチル−α−グルコシドの生成を低減する清酒酵母の育種方法。 Mutation treatment step of subjecting sake yeast parent strain to ethyl methanesulfonate treatment ,
A mutant selection step of obtaining a mutant strain that reduces the production of ethyl-α-glucoside by culturing the parent strain subjected to the mutation treatment in an agar medium containing cycloheximide and selectively separating a mutant strain resistant to cycloheximide ;
An analysis step of analyzing the amount of ethyl-α-glucoside produced by the mutant strain;
A method for breeding sake yeast that reduces the production of ethyl-α-glucoside, comprising
前記シクロヘキシミド耐性株の母集団からエチル−α−グルコシドの生成を低減する変異株を選択分離する変異株選択工程と、A mutant selection step of selectively separating a mutant that reduces the production of ethyl-α-glucoside from the population of the cycloheximide-resistant strain;
を包含し、Including
前記変異株選択工程における前記選択分離は、エチル−α−グルコシドの低減率が前記親株に対して２０％を超える変異株を選択分離する操作であるエチル−α−グルコシドの生成を低減する清酒酵母の育種方法。The selective separation in the mutant selection step is a sake yeast that reduces the production of ethyl-α-glucoside, which is an operation for selectively separating mutant strains in which the reduction rate of ethyl-α-glucoside exceeds 20% relative to the parent strain. Breeding method.
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