JP2020178634A - Method for evaluating early flocculation of yeast - Google Patents
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Abstract
Description
本発明は、下面発酵によりビールを製造する際に使用されるビール酵母の早期凝集性を評価する方法に関する。 The present invention relates to a method for evaluating the early cohesiveness of brewer's yeast used in producing beer by bottom fermentation.
ビール酵母は、上面ビール酵母と下面ビール酵母とに大別される。上面ビール酵母は、発酵により麦汁の上面に酵母が浮上する、又は特段沈降性を有さないビール酵母であり、上面発酵に使用される。一方で、下面ビール酵母は、発酵終了時に酵母が沈降する酵母であり、下面発酵に使用される。下面発酵では、発酵が終了して沈降した下面ビール酵母を回収して次回の発酵に使用するため、充分な凝集性を備えるビール酵母を選択して使用することが好ましい。 Saccharomyces cerevisiae is roughly classified into top brewer's yeast and bottom brewer's yeast. The top brewer's yeast is a brewer's yeast in which yeast floats on the upper surface of wort by fermentation or does not have special sedimentation property, and is used for top fermentation. On the other hand, bottom brewer's yeast is yeast in which yeast precipitates at the end of fermentation and is used for bottom fermentation. In bottom fermentation, the bottom brewer's yeast that has settled after fermentation is recovered and used for the next fermentation. Therefore, it is preferable to select and use brewer's yeast having sufficient cohesiveness.
実験室酵母サッカロマイセス・セレビシエ（Saccharomyces cerevisiae）では全染色体について塩基配列が明らかになっており、凝集性に関与する遺伝子として、ＦＬＯ１遺伝子、ＦＬＯ５遺伝子、ＦＬＯ８遺伝子、ＦＬＯ９遺伝子、ＦＬＯ１０遺伝子、ＦＬＯ１１遺伝子等が確認されている。これらの凝集性に関与する遺伝子を利用して、酵母の凝集性を判定する方法が知られている。例えば、特許文献１には、ＦＬＯ５遺伝子のオープンリーディングフレーム（ＯＲＦ）配列を利用したＰＣＲによりＦＬＯ５遺伝子の有無を検出し、ＦＬＯ５遺伝子を有する酵母は凝集性酵母であると判定する方法が開示されている。また、特許文献２には、ＦＬＯ１遺伝子によく似たＬｇ−ＦＬＯ１遺伝子が凝集性に関与する遺伝子であること、及び、当該遺伝子を有する酵母は凝集性酵母であると判定する方法が開示されている。 In the laboratory yeast Saccharomyces cerevisiae, the base sequences of all chromosomes have been clarified, and the genes involved in aggregation include the FLO1 gene, FLO5 gene, FLO8 gene, FLO9 gene, FLO10 gene, FLO11 gene, etc. It has been confirmed. A method for determining yeast cohesiveness using genes involved in these cohesiveness is known. For example, Patent Document 1 discloses a method of detecting the presence or absence of the FLO5 gene by PCR using an open reading frame (ORF) sequence of the FLO5 gene and determining that the yeast having the FLO5 gene is an aggregated yeast. There is. Further, Patent Document 2 discloses that the Lg-FLO1 gene, which is similar to the FLO1 gene, is a gene involved in cohesiveness, and a method for determining that the yeast having the gene is a cohesive yeast. There is.
また、ゲノム解析により、ビール酵母は、S. cerevisiae型（以降、「Ｓｃ型」ということがある。）の染色体と、S. cerevisiae以外の酵母の型（以降、「非Ｓｃ型」ということがある。）の染色体のキメラ染色体を含むことが知られている。例えば、特許文献３には、ビール酵母の菌株によっては特定の染色体がＳｃ型染色体、サッカロマイセス・バヤナス（S. bayanus）型染色体、又はこれらのキメラ染色体（Ｓｃ型塩基配列とS. bayanus型塩基配列が同一染色体上で見られる染色体）となっていることを利用して、下面ビール酵母であるか否かを判定する方法が開示されている。また、特許文献４には、第VIII番染色体に座位するＬｇ−ＦＬＯ１遺伝子配列内の類似アミノ酸配列の繰り返しを含む領域が欠失すると、凝集性が低下することが記載されており、この繰り返しを含む領域の欠失の有無から酵母の凝集性を判定する方法も記載されている。 In addition, according to genome analysis, brewer's yeast has a chromosome of S. cerevisiae type (hereinafter sometimes referred to as "Sc type") and a yeast type other than S. cerevisiae (hereinafter referred to as "non-Sc type"). It is known to contain a chimeric chromosome of the chromosome of S. cerevisiae. For example, in Patent Document 3, depending on the strain of Saccharomyces cerevisiae, specific chromosomes are Sc-type chromosomes, Saccharomyces bayanus-type chromosomes, or their chimeric chromosomes (Sc-type base sequence and S. bayanus-type base sequence). Is disclosed as a method for determining whether or not it is a bottom brewer's yeast by utilizing the fact that is a chromosome found on the same chromosome. Further, Patent Document 4 describes that deletion of a region containing a repetition of a similar amino acid sequence in the Lg-FLO1 gene sequence located on chromosome VIII reduces aggregation, and this repetition is repeated. A method for determining yeast aggregation based on the presence or absence of deletion of the containing region is also described.
下面発酵では、発酵終了時点では酵母は凝集して沈降していることが要求されるものの、凝集した酵母では発酵が停止する。このため、下面発酵において酵母の凝集が早期に開始すると、発酵不良（発酵不順）となる。そこで、下面ビール酵母では、充分な凝集性を備えることに加えて、早期凝集性が低く、発酵液中の資化性糖が充分に消費されてから凝集するものが好ましい。しかし、酵母の早期凝集性は、実際に酵母を接種して発酵させなければ判断することができず、評価に長時間を要するという問題がある。 In bottom fermentation, yeast is required to aggregate and settle at the end of fermentation, but the agglomerated yeast stops fermentation. Therefore, if yeast agglutination starts early in bottom fermentation, fermentation becomes poor (fermentation irregular). Therefore, it is preferable that the bottom brewer's yeast has sufficient cohesiveness, low early cohesiveness, and aggregates after the assimilating sugar in the fermentation broth is sufficiently consumed. However, the early cohesiveness of yeast cannot be determined unless the yeast is actually inoculated and fermented, and there is a problem that it takes a long time to evaluate.
本発明は、酵母の早期凝集性を評価する方法、及び当該方法により評価された酵母集団を用いてビール様発泡性飲料を製造する方法を提供することを目的とする。 An object of the present invention is to provide a method for evaluating the early cohesiveness of yeast and a method for producing a beer-like effervescent beverage using the yeast population evaluated by the method.
本発明者らは、上記課題を解決すべく、各種下面ビール酵母のゲノムと早期凝集性について分析した結果、Ｓｃ型３番染色体の左腕側の領域が欠損したビール酵母は、早期凝集性が高まることを見出し、本発明を完成させた。 As a result of analyzing the genomes and early aggregation of various lower surface brewer's yeasts in order to solve the above problems, the present inventors have found that brewer's yeast lacking the region on the left arm side of Sc-type chromosome 3 has increased early aggregation. We found that and completed the present invention.
本発明は、下記［１］〜［１０］である。
［１］ S. cerevisiae型第III染色体を含有する酵母の早期凝集性を評価する方法であって、
評価対象の酵母細胞について、S. cerevisiae型第III染色体の左腕に欠損があるか否かを検出し、前記欠損がある場合には、前記酵母細胞は早期凝集性であると評価する、酵母の早期凝集性の評価方法。
［２］ 前記欠損が、S. cerevisiae型第III染色体の左腕の先端からＢＵＤ５遺伝子座を含むＭＡＴ座までの部分の欠損である、前記［１］の酵母の早期凝集性の評価方法。
［３］ S. cerevisiae型第III染色体を含有する酵母の早期凝集性を評価する方法であって、
評価対象の酵母集団について、S. cerevisiae型第III染色体の左腕に欠損があるか否かを検出し、前記酵母集団に対する前記欠損がある酵母細胞の割合が、所定の基準値未満である場合に、前記酵母集団は早期凝集性であると評価する、酵母の早期凝集性の評価方法。
［４］ 前記欠損が、S. cerevisiae型第III染色体の左腕の先端からＢＵＤ５遺伝子座を含むＭＡＴ座までの部分の欠損である、前記［３］の酵母の早期凝集性の評価方法。
［５］ 前記酵母集団に対する前記欠損がある酵母細胞の割合は、前記酵母集団を構成する酵母細胞の全量に含まれている、S. cerevisiae型第III染色体の全量に対する、左腕に欠損があるS. cerevisiae型第III染色体の量の割合である、前記［３］又は［４］の酵母の早期凝集性の評価方法。
［６］ 前記酵母集団に対する前記欠損がある酵母細胞の割合は、前記酵母集団を構成する酵母細胞から抽出されたＤＮＡにおける、S. cerevisiae型第III染色体のＢＵＤ５遺伝子座を含むＭＡＴ座よりも右腕側に存在する領域のＤＮＡ量に対する、S. cerevisiae型第III染色体の左腕の先端から前記ＭＡＴ座までに存在する領域のＤＮＡ量の割合を、１から差し引いた割合である、前記［４］の酵母の早期凝集性の評価方法。
［７］ 前記酵母集団が、少なくとも１度の発酵を行った後の酵母集団である、前記［３］〜［６］のいずれかの酵母の早期凝集性の評価方法。
［８］ 前記酵母集団が、下面ビール酵母の集団である、前記［３］〜［７］のいずれかの酵母の早期凝集性の評価方法。
［９］ 下面発酵を経て製造されるビール様発泡性飲料の製造方法であって、
発酵に使用する酵母集団を、予め、前記［３］〜［８］のいずれかの酵母の早期凝集性の評価方法により評価し、
早期凝集性であるとは評価されなかった酵母集団を用いて下面発酵を行う、ビール様発泡性飲料の製造方法。
［１０］ 下面発酵を経て製造されるビール様発泡性飲料の製造方法であって、
発酵に使用する酵母集団を、予め、前記［３］〜［８］のいずれかの酵母の早期凝集性の評価方法により評価し、
早期凝集性であると評価された酵母集団と、早期凝集性であるとは評価されなかった酵母集団とを、混合後の酵母集団に対する前記欠損がある酵母細胞の割合が前記基準値未満となるように混合した酵母集団を用いて下面発酵を行う、ビール様発泡性飲料の製造方法。
The present invention is the following [1] to [10].
[1] A method for evaluating the early aggregation of yeast containing S. cerevisiae type III chromosome.
For yeast cells to be evaluated, whether or not there is a defect in the left arm of S. cerevisiae type III chromosome is detected, and if there is such a defect, the yeast cell is evaluated to be prematurely aggregated. Evaluation method of early agglutination.
[2] The method for evaluating early cohesiveness of yeast according to the above [1], wherein the defect is a defect in the portion from the tip of the left arm of S. cerevisiae type III chromosome to the MAT locus including the BUD5 locus.
[3] A method for evaluating the early aggregation of yeast containing S. cerevisiae type III chromosome.
For the yeast population to be evaluated, whether or not there is a defect in the left arm of S. cerevisiae type III chromosome is detected, and the ratio of yeast cells having the defect to the yeast population is less than a predetermined reference value. , A method for evaluating the early aggregation of yeast, wherein the yeast population is evaluated to be early aggregating.
[4] The method for evaluating early aggregation of yeast according to the above [3], wherein the defect is a defect in the portion from the tip of the left arm of S. cerevisiae type III chromosome to the MAT locus containing the BUD5 locus.
[5] The ratio of yeast cells having the defect to the yeast population is S. cerevisiae type III chromosome having a defect in the left arm, which is contained in the total amount of yeast cells constituting the yeast population. The method for evaluating the early aggregation of yeast according to the above [3] or [4], which is the ratio of the amount of cerevisiae type III chromosome.
[6] The ratio of the yeast cell having the defect to the yeast population is the right arm of the DNA extracted from the yeast cells constituting the yeast population than the MAT locus containing the BUD5 locus of the S. cerevisiae type III chromosome. The ratio of the amount of DNA in the region existing from the tip of the left arm of the S. cerevisiae type III chromosome to the locus of MAT with respect to the amount of DNA in the region existing on the side is the ratio obtained by subtracting from 1. A method for evaluating early aggregation of yeast.
[7] The method for evaluating the early cohesiveness of the yeast according to any one of [3] to [6] above, wherein the yeast population is a yeast population after at least one fermentation.
[8] The method for evaluating the early aggregation of yeast according to any one of [3] to [7], wherein the yeast population is a population of bottom brewer's yeast.
[9] A method for producing a beer-like effervescent beverage produced through bottom fermentation.
The yeast population used for fermentation is evaluated in advance by the method for evaluating the early cohesiveness of yeast according to any one of [3] to [8].
A method for producing a beer-like effervescent beverage, in which bottom fermentation is performed using a yeast population that has not been evaluated as early cohesive.
[10] A method for producing a beer-like effervescent beverage produced through bottom fermentation.
The yeast population used for fermentation is evaluated in advance by the method for evaluating the early cohesiveness of yeast according to any one of [3] to [8].
The ratio of yeast cells having the deficiency to the yeast population after mixing the yeast population evaluated to be early-aggregating and the yeast population not evaluated to be early-aggregating is less than the reference value. A method for producing a beer-like effervescent beverage, in which bottom fermentation is performed using the yeast population mixed as described above.
本発明に係る酵母の早期凝集性の評価方法により、実際に発酵を行って発酵推移を確認しなくとも、酵母の早期凝集性を評価することができる。
また、下面発酵に使用されるビール酵母について、発酵前に予め当該評価方法を使用して早期凝集性を評価することにより、実際に発酵を行う前に発酵不良を引き起こす可能性の高い酵母を識別することができる。
According to the method for evaluating the early cohesiveness of yeast according to the present invention, the early cohesiveness of yeast can be evaluated without actually performing fermentation and confirming the fermentation transition.
In addition, for brewer's yeast used for bottom fermentation, early aggregation is evaluated using the evaluation method in advance before fermentation to identify yeasts that are likely to cause fermentation failure before actual fermentation. can do.
本発明及び本願明細書において、「ビール酵母」とは、一般にビール醸造に使用される酵母を指し、生物学的分類でいうと、例えばS. cerevisiae、サッカロマイセス・パストリアヌス(Saccharomyces pastorianus)が挙げられる。
歴史的には、「上面ビール酵母」と「下面ビール酵母」との区別は、ビール醸造過程で発酵後に見られるビール酵母の動態（沈降しない又は凝集して沈む）により区別されてきた。今現在、当業者間では、下面ビール酵母と上面ビール酵母とは生物学的分類において異なる種であり、上面ビール酵母は主としてサッカロマイセス・セレビシエに分類され、下面ビール酵母はサッカロマイセス・セレビシエとサッカロマイセス・ユーバヤナス(Saccharomyces eubayanus)との交雑体であってサッカロマイセス・パストリアヌス(S. pastorianus)に分類されるという見解が一般的となっている。
In the present invention and the present specification, "brewer's yeast" refers to yeast generally used for brewing beer, and in terms of biological classification, for example, S. cerevisiae and Saccharomyces pastorianus can be mentioned.
Historically, the distinction between "top brewer's yeast" and "bottom brewer's yeast" has been distinguished by the dynamics of brewer's yeast (not settling or agglomerating and sinking) seen after fermentation during the brewing process. Currently, among those skilled in the art, bottom brewer's yeast and top brewer's yeast are different species in biological classification, top brewer's yeast is mainly classified into Saccharomyces cerevisiae, and bottom brewer's yeast is Saccharomyces cerevisiae and Saccharomyces eubayanas. The general view is that it is a hybrid with (Saccharomyces eubayanus) and is classified as S. pastorianus.
本発明及び本願明細書において、「S.cerevisiae型（Ｓｃ型）染色体」とは、ＦＡＳＴＡ、ＢＬＡＳＴといった配列比較アルゴリズムを用いて、Saccharomyces Genome Database（ＳＧＤ）（http://www.yeastgenome.org/）に公開されているS.cerevisiaeのゲノム塩基配列と比較した場合に９５％以上の相同性（配列同一性）を示す塩基配列からなる染色体を指す。「非S.cerevisiae型（非Ｓｃ型）染色体」とは、同様にS.cerevisiaeのゲノム塩基配列と比較した場合に、９５％未満の相同性（配列同一性）を示す塩基配列からなる染色体を指す。 In the present invention and the present specification, the "S. cerevisiae type (Sc type) chromosome" is referred to as Saccharomyces Genome Database (SGD) (http://www.yeastgenome.org/) using a sequence comparison algorithm such as FASTA or BLAST. ) Refers to a chromosome consisting of a base sequence showing 95% or more homology (sequence identity) when compared with the genomic base sequence of S. cerevisiae. "Non-S. cerevisiae type (non-Sc type) chromosome" refers to a chromosome consisting of a base sequence showing less than 95% homology (sequence identity) when compared with the genomic base sequence of S. cerevisiae. Point.
本発明及び本願明細書において、「早期凝集性」とは、酵母が発酵開始から凝集して沈降するまでに要する時間が短い性質をいう。早期凝集性の酵母では、発酵開始から１２０時間経過時点以降の浮遊酵母数が、早期凝集性のない通常の酵母よりも低く推移する。ビール製造時の下面発酵では、一般的に、発酵開始から３〜４日目で酵母数が最大となり、５日目以降に酵母の凝集し、沈降し始め、６〜７日目で大部分の酵母が沈降し、全体の酵母量は、発酵開始時に比して約２倍に増える。これに対して、早期凝集性の酵母は、発酵開始から５日目にはその大部分の酵母が沈降する。例えば、発酵開始から１６８時間経過時点における浮遊酵母数に対する発酵開始から１２０時間経過時点における浮遊酵母数の割合は、早期凝集性の酵母では５倍以下であり、早期凝集性が高い酵母は、３倍以下、好ましくは２倍以下である。 In the present invention and the present specification, "early cohesiveness" refers to the property that the time required for yeast to aggregate and settle from the start of fermentation is short. In the early-aggregating yeast, the number of floating yeasts after 120 hours from the start of fermentation remains lower than that of the normal yeast having no early-aggregating property. In bottom fermentation during beer production, the number of yeasts generally reaches its maximum 3-4 days after the start of fermentation, yeasts begin to aggregate and settle after 5 days, and most of them begin to settle 6-7 days later. Yeast settles and the total amount of yeast increases about twice as much as at the start of fermentation. On the other hand, in the case of early-aggregating yeast, most of the yeast settles on the 5th day from the start of fermentation. For example, the ratio of the number of floating yeasts at 120 hours after the start of fermentation to the number of floating yeasts at 168 hours after the start of fermentation is 5 times or less for yeasts with early aggregation, and 3 for yeasts with high early aggregation. It is twice or less, preferably twice or less.
本発明に係る酵母の早期凝集性の評価方法（以下、「本発明に係る評価方法」ということがある。）は、酵母の早期凝集性を、Ｓｃ型第III染色体の左腕の欠損の有無で評価する方法である。Ｓｃ型第III染色体を含有する酵母は、Ｓｃ型第III染色体の左腕側のおよそ５０〜６０％程度の領域が欠損すると、早期凝集性が高くなる。本発明においては当該知見を利用し、評価対象である酵母が早期凝集性であるか否かを、ゲノム解析により評価する。酵母を実際に培養して沈降するまでに要する時間を測定する場合には、培養自体に通常５〜７日間を要するが、本発明に係る評価方法では、培養を必要とせず、ゲノム解析の結果に基づいて評価するため、より短時間で評価することができる。 The method for evaluating the early cohesiveness of yeast according to the present invention (hereinafter, may be referred to as "evaluation method according to the present invention") determines the early cohesiveness of yeast based on the presence or absence of a defect in the left arm of Sc-type III chromosome. It is a method of evaluation. Yeast containing Sc-type III chromosome becomes highly prematurely cohesive when a region of about 50 to 60% on the left arm side of Sc-type III chromosome is deleted. In the present invention, the findings are used to evaluate whether or not the yeast to be evaluated has early cohesiveness by genome analysis. When measuring the time required to actually culture and settle yeast, the culture itself usually takes 5 to 7 days, but the evaluation method according to the present invention does not require culture, and the result of genome analysis. Since the evaluation is based on, the evaluation can be performed in a shorter time.
以降において、左腕側のおよそ５０〜６０％程度の領域が欠損しているＳｃ型第III染色体を「部分欠損Ｓｃ型第III染色体」ということがある。また、部分欠損Ｓｃ型第III染色体を含む酵母を、「部分欠損Ｓｃ３酵母」ということがある。本発明において、部分欠損Ｓｃ３酵母は、部分欠損Ｓｃ型第III染色体を少なくとも１本備えていればよい。例えば、部分欠損Ｓｃ型第III染色体と欠損のないＳｃ型第III染色体の両方を備える酵母も、部分欠損Ｓｃ３酵母に含まれ、早期凝集性を備える。 Hereinafter, the Sc-type III chromosome in which a region of about 50 to 60% on the left arm side is deleted may be referred to as a “partially defective Sc-type III chromosome”. In addition, yeast containing a partially deficient Sc-type III chromosome may be referred to as "partially deficient Sc3 yeast". In the present invention, the partially deficient Sc3 yeast may be provided with at least one partially deficient Sc type III chromosome. For example, yeast having both a partially deficient Sc-type III chromosome and a non-defective Sc-type III chromosome is also included in the partially deficient Sc3 yeast and has early cohesiveness.
本発明における部分欠損Ｓｃ型第III染色体において、欠損している左腕側領域は、当該欠損により酵母の早期凝集性が高められるものであれば特に限定されるものではなく、左腕の先端からＳｃ型第III染色体全体の５０〜６０％程度が欠損しているものが好ましく、通常は、Ｓｃ型第III染色体の左腕の先端からＢＵＤ５遺伝子座を含むＭＡＴ座（Mating-type locus）までの部分が欠損している。このＳｃ型第III染色体の左腕の先端から前記ＭＡＴ座までの領域内には、凝集性に関与することが報告されている遺伝子はなく、当該領域が欠落することで酵母の早期凝集性が高められる理由は明らかではない。 In the partially deficient Sc-type III chromosome in the present invention, the deficient left arm side region is not particularly limited as long as the deficiency enhances the early aggregation of yeast, and the Sc-type is formed from the tip of the left arm. It is preferable that about 50 to 60% of the entire chromosome III is deleted, and usually, the portion from the tip of the left arm of the Sc-type chromosome III to the MAT locus (Mating-type locus) containing the BUD5 locus is deleted. doing. There is no gene reported to be involved in cohesiveness in the region from the tip of the left arm of the Sc-type III chromosome to the MAT locus, and the lack of this region enhances the early cohesiveness of yeast. The reason for this is not clear.
左腕の先端からＢＵＤ５遺伝子座を含むＭＡＴ座までの部分欠損は、Ｓｃ型第III染色体において、よく観察される変異であり（例えば、非特許文献１及び２参照。）、当該部分領域は欠損しやすい領域である。早期凝集性が弱く、下面発酵に適している下面ビール酵母が、発酵を複数回繰り返すことにより早期凝集性を獲得する現象はよく観察されるが、これは、発酵を繰り返すうちに、Ｓｃ型第III染色体の左腕の先端からＢＵＤ５遺伝子座を含むＭＡＴ座までの部分の欠落が生じ、部分欠損Ｓｃ３酵母に変化してしまったためと推察される。 A partial defect from the tip of the left arm to the MAT locus containing the BUD5 locus is a commonly observed mutation in Sc-type chromosome III (see, for example, Non-Patent Documents 1 and 2), and the partial region is deleted. It is an easy area. It is often observed that bottom brewer's yeast, which has weak early aggregation and is suitable for bottom fermentation, acquires early aggregation by repeating fermentation multiple times, but this is due to the Sc type as the fermentation is repeated. It is presumed that the part from the tip of the left arm of chromosome III to the MAT locus containing the BUD5 locus was missing, and the yeast was changed to a partially defective Sc3 yeast.
本発明に係る評価方法において、評価対象が１個の酵母細胞である場合には、評価対象の酵母細胞について、Ｓｃ型第III染色体の左腕に欠損があるか否かを検出する。当該欠損が検出された場合には、当該酵母細胞は部分欠損Ｓｃ３酵母であり、早期凝集性であると評価する。 In the evaluation method according to the present invention, when the evaluation target is one yeast cell, it is detected whether or not the yeast cell to be evaluated has a defect in the left arm of the Sc type III chromosome. When the defect is detected, the yeast cell is a partially defective Sc3 yeast and is evaluated to be prematurely cohesive.
本発明に係る評価方法において、評価対象が酵母集団である場合には、当該酵母集団を構成する酵母細胞全体に対する部分欠損Ｓｃ３酵母の割合に基づいて、当該酵母集団の早期凝集性を評価する。酵母集団の早期凝集性は、部分欠損Ｓｃ３酵母の割合に依存しており、部分欠損Ｓｃ３酵母の割合が多くなるほど、当該酵母集団の早期凝集性は強くなる。本発明に係る評価方法では、評価対象の酵母集団について、Ｓｃ型第III染色体の左腕に欠損があるか否かを検出し、当該酵母集団に対する当該欠損がある酵母細胞（部分欠損Ｓｃ３酵母細胞）の割合が、所定の基準値超である場合に、当該酵母集団は早期凝集性であると評価する。 In the evaluation method according to the present invention, when the evaluation target is a yeast population, the early aggregation of the yeast population is evaluated based on the ratio of the partially deficient Sc3 yeast to the entire yeast cells constituting the yeast population. The early cohesiveness of the yeast population depends on the proportion of partially deficient Sc3 yeast, and the higher the proportion of partially deficient Sc3 yeast, the stronger the early cohesiveness of the yeast population. In the evaluation method according to the present invention, it is detected in the yeast population to be evaluated whether or not there is a defect in the left arm of the Sc type III chromosome, and the yeast cell having the defect in the yeast population (partially defective Sc3 yeast cell). The yeast population is evaluated to be prematurely aggregated when the proportion of yeast exceeds a predetermined reference value.
酵母集団に対する部分欠損Ｓｃ３酵母細胞の割合は、例えば、当該酵母集団を構成する酵母細胞の全量に含まれている、Ｓｃ型第III染色体の全量に対する、部分欠損Ｓｃ型第III染色体の量の割合で求められる。ここで、Ｓｃ型第III染色体の全量は、部分欠損Ｓｃ型第III染色体の全量と、左腕領域が欠損されていないＳｃ型第III染色体の全量の和である。このため、酵母集団に対する部分欠損Ｓｃ３酵母細胞の割合は、当該酵母集団を構成する酵母細胞の全量に含まれているＳｃ型第III染色体の全量に対する、左腕領域が欠損されていないＳｃ型第III染色体の量の割合を、１から差し引いた割合に相当する。 The ratio of the partially deficient Sc3 yeast cells to the yeast population is, for example, the ratio of the amount of the partially deficient Sc type III chromosome to the total amount of the Sc type III chromosome contained in the total amount of yeast cells constituting the yeast population. Is required by. Here, the total amount of Sc-type III chromosome is the sum of the total amount of partially defective Sc-type III chromosome and the total amount of Sc-type III chromosome in which the left arm region is not deleted. Therefore, the ratio of partially deficient Sc3 yeast cells to the yeast population is based on the total amount of Sc-type III chromosomes contained in the total amount of yeast cells constituting the yeast population, in which the left arm region is not deleted. It corresponds to the ratio of the amount of chromosomes subtracted from 1.
染色体の相対量は、当該染色体中の部分領域のＤＮＡ量に基づいて求めることができる。ここで、左腕領域が欠損されていないＳｃ型第III染色体の相対量は、Ｓｃ型第III染色体の左腕側の、部分欠損Ｓｃ型第III染色体において欠損した領域中の部分領域のＤＮＡ量を指標として求めることができる。一方で、左腕領域が欠損されていないＳｃ型第III染色体の右腕側は、部分欠損Ｓｃ型第III染色体と同じ核酸配列である。このため、Ｓｃ型第III染色体の相対量は、部分欠損Ｓｃ型第III染色体中の部分領域のＤＮＡ量を指標として求めることができる。例えば、酵母集団を構成する酵母細胞から抽出されたＤＮＡにおける、Ｓｃ型第III染色体のＢＵＤ５遺伝子座を含むＭＡＴ座よりも右腕側に存在する領域のＤＮＡ量に対する、Ｓｃ型第III染色体の左腕の先端から前記ＭＡＴ座までに存在する領域のＤＮＡ量の割合を、１から差し引いた割合を、酵母集団に対する部分欠損Ｓｃ３酵母細胞の割合とすることができる。 The relative amount of a chromosome can be determined based on the amount of DNA in a partial region of the chromosome. Here, the relative amount of the Sc-type III chromosome in which the left arm region is not deleted is an index of the DNA amount of the partial region in the partially defective Sc-type III chromosome on the left arm side of the Sc-type III chromosome. Can be obtained as. On the other hand, the right arm side of the Sc-type III chromosome in which the left arm region is not deleted has the same nucleic acid sequence as the partially deleted Sc-type III chromosome. Therefore, the relative amount of Sc-type III chromosome can be determined by using the amount of DNA in a partial region in the partially defective Sc-type III chromosome as an index. For example, in the DNA extracted from yeast cells constituting the yeast population, the amount of DNA in the region located on the right arm side of the MAT locus containing the BUD5 locus of the Sc type III chromosome is relative to the amount of DNA in the left arm of the Sc type III chromosome. The ratio of the amount of DNA in the region existing from the tip to the MAT locus, subtracted from 1, can be used as the ratio of partially defective Sc3 yeast cells to the yeast population.
以降において、ＤＮＡ量を測定する対象の「Ｓｃ型第III染色体の左腕側の、部分欠損Ｓｃ型第III染色体において欠損した領域中の部分領域」を「ＤＮＡ測定用標的領域Ａ」、ＤＮＡ量を測定する対象の「部分欠損Ｓｃ型第III染色体中の部分領域」を「ＤＮＡ測定用標的領域Ｂ」ということがある。酵母集団に対する部分欠損Ｓｃ３酵母細胞の割合（％）は、下記式（１）で求めることができる。 In the following, the "partial region in the region lacking in the partially defective Sc-type III chromosome on the left arm side of the Sc-type III chromosome" to be measured for the DNA amount is referred to as "target region A for DNA measurement", and the DNA amount is referred to as The "partial region in the partially defective Sc-type III chromosome" to be measured may be referred to as "target region B for DNA measurement". The ratio (%) of partially deficient Sc3 yeast cells to the yeast population can be calculated by the following formula (1).
式（１）・・・[酵母集団に対する部分欠損Ｓｃ３酵母細胞の割合（％）]＝〔１−[ＤＮＡ測定用標的領域ＡのＤＮＡ量（相対値）]／[ＤＮＡ測定用標的領域ＢのＤＮＡ量（相対値）]〕×１００（％） Formula (1) ... [Ratio of partially defective Sc3 yeast cells to yeast population (%)] = [1-[DNA amount (relative value) of target region A for DNA measurement] / [target region B for DNA measurement] Amount of DNA (relative value)]] x 100 (%)
なお、ＤＮＡ測定用標的領域Ａは、部分欠損Ｓｃ型第III染色体において欠損した領域中の部分領域であれば特に限定されるものではないが、Ｓｃ型第III染色体のＢＵＤ５遺伝子座を含むＭＡＴ座よりも右腕側に存在する領域であることが好ましい。同様に、ＤＮＡ測定用標的領域Ｂは、部分欠損Ｓｃ型第III染色体中の部分領域であれば特に限定されるものではないが、Ｓｃ型第III染色体の左腕の先端から前記ＭＡＴ座までに存在する領域であることが好ましい。 The target region A for DNA measurement is not particularly limited as long as it is a partial region in the region deleted in the partially defective Sc type III chromosome, but the MAT locus including the BUD5 locus of the Sc type III chromosome is not particularly limited. It is preferable that the region is located on the right arm side of the region. Similarly, the target region B for DNA measurement is not particularly limited as long as it is a partial region in the partially defective Sc-type III chromosome, but exists from the tip of the left arm of the Sc-type III chromosome to the MAT locus. It is preferable that the region is to be used.
染色体中の部分領域のＤＮＡ量の相対量は、ポリメラーゼを使用した核酸増幅法を利用した定量方法により定量することができる。ポリメラーゼを使用した核酸増幅法としては、ＰＣＲ（Polymerase Chain Reaction）を利用した方法が挙げられる。例えば、評価対象の酵母集団から抽出されたＤＮＡを鋳型として、ＤＮＡ測定用標的領域ＡとＤＮＡ測定用標的領域ＢをそれぞれＰＣＲ増幅し、得られた増幅産物の量を、それぞれの領域のＤＮＡ量の相対量とすることができる。ＰＣＲとその増幅産物の定量は、常法により行うことができる。例えば、超微量紫外可視分光光度計を用いてＤＮＡ量自体を測定することができる。また、増幅産物を電気泳動した後、エチジウムブロマイド等を用いてＤＮＡのバンドを染色し、染色強度に基づいて、ＤＮＡ量の相対値を求めることもできる。その他、評価対象の酵母集団から抽出されたＤＮＡを鋳型として、ＤＮＡ測定用標的領域ＡとＤＮＡ測定用標的領域ＢをそれぞれリアルタイムＰＣＲし、両領域のＤＮＡの相対量を求めることができる。また、評価対象の酵母集団から抽出されたＤＮＡを鋳型として、デジタルＰＣＲを行うことによっても、両領域のＤＮＡの相対量を求めることができる。リアルタイムＰＣＲやデジタルＰＣＲは常法により行うことができる。 The relative amount of DNA in a partial region of a chromosome can be quantified by a quantification method using a nucleic acid amplification method using a polymerase. Examples of the nucleic acid amplification method using a polymerase include a method using PCR (Polymerase Chain Reaction). For example, using the DNA extracted from the yeast population to be evaluated as a template, the target region A for DNA measurement and the target region B for DNA measurement are each PCR-amplified, and the amount of the obtained amplification product is the amount of DNA in each region. Can be a relative quantity of. PCR and its amplification products can be quantified by conventional methods. For example, the amount of DNA itself can be measured using an ultratrace ultraviolet-visible spectrophotometer. Further, after the amplification product is electrophoresed, the band of DNA can be stained with ethidium bromide or the like, and the relative value of the amount of DNA can be obtained based on the staining intensity. In addition, using the DNA extracted from the yeast population to be evaluated as a template, the target region A for DNA measurement and the target region B for DNA measurement can be PCRed in real time to determine the relative amount of DNA in both regions. In addition, the relative amount of DNA in both regions can be obtained by performing digital PCR using the DNA extracted from the yeast population to be evaluated as a template. Real-time PCR and digital PCR can be performed by a conventional method.
その他、ＤＮＡ測定用標的領域ＡとＤＮＡ測定用標的領域ＢのＤＮＡ量の相対量は、Ｓｃ型第III染色体以外の染色体中の部分領域のＤＮＡ量を内部標準とし、この内部標準領域のＤＮＡ量に対するＤＮＡ測定用標的領域ＡのＤＮＡ量と、内部標準領域のＤＮＡ量に対するＤＮＡ測定用標的領域ＢのＤＮＡ量とを相対比較することにより求めることもできる。内部標準領域のＤＮＡ量は、前記のＤＮＡ測定用標的領域ＡのＤＮＡ量の測定と同様にして測定することができる。 In addition, the relative amount of DNA in the target region A for DNA measurement and the target region B for DNA measurement is based on the amount of DNA in a partial region in the chromosome other than Sc-type III chromosome as an internal standard, and the amount of DNA in this internal standard region. It can also be obtained by making a relative comparison between the amount of DNA in the target region A for DNA measurement and the amount of DNA in the target region B for DNA measurement with respect to the amount of DNA in the internal standard region. The amount of DNA in the internal standard region can be measured in the same manner as the above-mentioned measurement of the amount of DNA in the target region A for DNA measurement.
酵母からのゲノムＤＮＡ抽出は、公知の方法のいずれを用いて行ってもよい。例えば、Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, P130 (1990)などに記載の方法が挙げられる。また、ＤＮＡ抽出用のキットやカラム等を用いて行うこともできる。 Genomic DNA extraction from yeast may be carried out by any known method. For example, the methods described in Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, P130 (1990). It can also be carried out using a kit or column for DNA extraction.
酵母は、染色体の交差や重複が生じやすく、また、近縁種同士の自然交雑も行われる（例えば、非特許文献３参照。）。例えば、ビール酵母は、サッカロマイセス・セレビシエがその近縁種と自然交雑して得られた株がさらに交差や重複を起こしながら進化した酵母である。このため、同じ属種の酵母であっても、株によってそのゲノム構造は様々である。さらに、酵母は、継代回数や培養条件、ストレスの有無などによっても染色体に変異が生じやすい。このため、単一株化された酵母を培養して得られた酵母集団であっても、遺伝的背景は必ずしも均一ではない場合がある。この酵母集団の遺伝的背景の多様性から、評価対象の酵母集団ごとに、早期凝集性を評価するための基準値を設定する。 Yeast is prone to chromosomal crossing and duplication, and natural crosses between closely related species are also carried out (see, for example, Non-Patent Document 3). For example, Saccharomyces cerevisiae is a yeast that has evolved while further crossing and duplicating strains obtained by natural crossing of Saccharomyces cerevisiae with its relatives. Therefore, even yeasts of the same genus have different genomic structures depending on the strain. Furthermore, yeast is prone to chromosomal mutations depending on the number of passages, culture conditions, and the presence or absence of stress. Therefore, even in a yeast population obtained by culturing a single strain of yeast, the genetic background may not always be uniform. Based on the diversity of the genetic background of this yeast population, a reference value for evaluating early cohesiveness is set for each yeast population to be evaluated.
当該基準値は、各酵母集団の部分欠損Ｓｃ３酵母の割合と早期凝集性の有無に基づいて、早期凝集性のない酵母集団と早期凝集性である酵母集団とを識別可能な値として、実験的に決定することができる。例えば、実際に培養して早期凝集性でないことが確認されている複数の酵母集団と、実際に培養して早期凝集性であることが確認されている複数の酵母集団について、各酵母集団の部分欠損Ｓｃ３酵母の割合を測定し、早期凝集性でない酵母集団と早期凝集性である酵母集団とを識別可能な閾値を統計的に設定することができる。 The reference value is experimentally set as a value capable of distinguishing between a yeast population without early cohesiveness and a yeast population with early cohesiveness based on the proportion of partially deficient Sc3 yeast in each yeast population and the presence or absence of early cohesiveness. Can be decided. For example, a portion of each yeast population for a plurality of yeast populations that have been actually cultured and confirmed not to be early agglutinating, and a plurality of yeast populations that have been actually cultured and confirmed to be not early aggregating. The proportion of deficient Sc3 yeast can be measured and a threshold can be statistically set to distinguish between a yeast population that is not prematurely aggregating and a yeast population that is prematurely aggregating.
また、実際に培養して早期凝集性を獲得していないことが確認されている特定の株の酵母を培養して得られる酵母集団の基準値は、例えば、以下の方法で設定することができる。まず、当該株が保有する全てのＳｃ型第III染色体について、その左腕の先端からＢＵＤ５遺伝子座を含むＭＡＴ座までの部分を欠損させた部分欠損Ｓｃ３酵母を作製する。この部分欠損Ｓｃ３酵母は、遺伝子組換え技術等を用いて常法により作製してもよく、継代の過程で自然に取得された部分欠損Ｓｃ３酵母を単離して用いてもよい。部分欠損Ｓｃ型第III染色体を含まない酵母に、含有する全てのＳｃ型第III染色体が部分欠損Ｓｃ型第III染色体である部分欠損Ｓｃ３酵母を混合して、両者の混合割合が様々である酵母集団の系列を作成し、各細胞集団について実際に培養して早期凝集性の有無を調べる。部分欠損Ｓｃ型第III染色体を含まない酵母からなる酵母集団は早期凝集性ではなく、部分欠損Ｓｃ３酵母からなる酵母集団は早期凝集性である。部分欠損Ｓｃ３酵母の含有割合が多くなるにつれ、酵母の凝集と沈降は早くなる。早期凝集性でない酵母集団のうち最も部分欠損Ｓｃ３酵母の割合が高かった酵母集団の部分欠損Ｓｃ３酵母の割合と、早期凝集性であった酵母集団のうち最も部分欠損Ｓｃ３酵母の割合が低かった酵母集団の部分欠損Ｓｃ３酵母の割合との間に、早期凝集性を評価するための基準値を設定することができる。 In addition, the reference value of the yeast population obtained by culturing yeast of a specific strain that has been confirmed not to acquire early cohesiveness by actually culturing can be set by, for example, the following method. .. First, a partially deficient Sc3 yeast is prepared by deleting the portion from the tip of the left arm to the MAT locus containing the BUD5 locus for all Sc-type III chromosomes possessed by the strain. This partially deficient Sc3 yeast may be prepared by a conventional method using a gene recombination technique or the like, or a partially deficient Sc3 yeast naturally acquired in the process of passage may be isolated and used. A yeast that does not contain a partially deficient Sc-type III chromosome is mixed with a partially deficient Sc3 yeast in which all the Sc-type III chromosomes contained are a partially deficient Sc-type III chromosome, and the mixing ratio of both is various. Create a population sequence and actually culture each cell population to check for premature aggregation. The yeast population consisting of yeast not containing the partially deficient Sc type III chromosome is not prematurely cohesive, and the yeast population consisting of partially deficient Sc3 yeast is prematurely cohesive. As the content of partially deficient Sc3 yeast increases, yeast aggregation and sedimentation become faster. The proportion of partially deficient Sc3 yeast in the yeast population that was not early-aggregating was the highest, and the proportion of partially deficient Sc3 yeast in the yeast population that was early-aggregating was the lowest. A reference value for assessing early aggregation can be set between the proportion of partially deficient Sc3 yeast in the population.
本発明に係る評価方法で評価対象とする酵母は、Ｓｃ型第III染色体を備える酵母であれば特に限定されるものではない。本発明に係る評価方法で評価対象とする酵母集団は、Ｓｃ型第III染色体を備える酵母細胞からなる集団であれば特に限定されるものではない。本発明に係る評価方法の評価対象としては、早期凝集性が問題となる下面ビール酵母であることが好ましい。下面発酵においては、早期凝集性の酵母を用いた場合には、エキス消費完了前に酵母が凝集して沈降してしまうため、エキス消費が遅延し、発酵完了までの時間が長くなり、製造効率が低下する。これに加えて、発酵不良により好ましくない香気（発酵不順臭）が生成したり、糖類の残留量が多くなり、香味が変化したりする。このように、下面発酵においては、発酵に使用する酵母集団が早期凝集性ではないことが重要であるが、従来は、早期凝集性の酵母を用いることに起因する発酵の不具合は、実際に１週間程度の時間をかけて発酵を行い、発酵推移を確認しなければ判断することができなかった。これに対して、本発明に係る評価方法は、発酵を行う必要がないため、１日程度という非常に短い時間で評価が可能である。 The yeast to be evaluated in the evaluation method according to the present invention is not particularly limited as long as it is a yeast having Sc type III chromosome. The yeast population to be evaluated by the evaluation method according to the present invention is not particularly limited as long as it is a population consisting of yeast cells having Sc-type III chromosome. The evaluation target of the evaluation method according to the present invention is preferably bottom brewer's yeast, which has a problem of early cohesiveness. In bottom fermentation, when early-aggregating yeast is used, the yeast aggregates and settles before the completion of extract consumption, which delays extract consumption and prolongs the time until fermentation is completed, resulting in production efficiency. Decreases. In addition to this, unfavorable aroma (unfermented fermentation odor) is generated due to poor fermentation, and the residual amount of sugars increases, resulting in a change in flavor. As described above, in bottom fermentation, it is important that the yeast population used for fermentation is not early-aggregating, but conventionally, the fermentation defect caused by using early-aggregating yeast is actually 1. Fermentation was carried out over a period of about a week, and it was not possible to make a judgment without confirming the fermentation transition. On the other hand, the evaluation method according to the present invention does not require fermentation, so evaluation can be performed in a very short time of about one day.
ビール様発泡性飲料の醸造における下面発酵では、発酵が終了して沈降した下面ビール酵母を回収して次回の発酵に使用する。発酵回数が多くなるほど、酵母集団の部分欠損Ｓｃ３酵母の割合が高くなり、早期凝集性を獲得する可能性が高くなるが、本発明に係る評価方法は、この少なくとも１度の発酵を行った後の酵母集団に対する早期凝集性の評価にも好適である。 In bottom fermentation in the brewing of beer-like sparkling beverages, bottom brewer's yeast that has settled after fermentation is collected and used for the next fermentation. As the number of fermentations increases, the proportion of partially deficient Sc3 yeast in the yeast population increases, and the possibility of acquiring early cohesiveness increases. However, in the evaluation method according to the present invention, after performing this at least one fermentation. It is also suitable for evaluation of early aggregation of yeast populations.
下面発酵を経て製造されるビール様発泡性飲料の製造において、発酵に使用する酵母集団を、予め、本発明に係る評価方法により評価し、早期凝集性であるとは評価されなかった酵母集団を用いて下面発酵を行う。発酵前に使用する酵母集団の早期凝集性を本発明に係る評価方法で評価することによって、下面発酵に不適当な酵母の使用を避けることができる。特に、早期凝集は染色体変化以外の要因で発生することもあり、発酵条件を変更すること等で対処できる場合もある。しかし、染色体変化に起因する早期凝集性の獲得の場合、酵母の状態が改善する可能性はない。本発明に係る評価方法により、下面発酵の繰り返し使用における酵母入れ替えの判断を早期に実施することが可能になる。 In the production of beer-like effervescent beverages produced through bottom fermentation, the yeast population used for fermentation was evaluated in advance by the evaluation method according to the present invention, and the yeast population that was not evaluated as early agglutination was evaluated. Use to perform bottom fermentation. By evaluating the early cohesiveness of the yeast population used before fermentation by the evaluation method according to the present invention, it is possible to avoid the use of yeast unsuitable for bottom fermentation. In particular, early aggregation may occur due to factors other than chromosomal changes, and may be dealt with by changing fermentation conditions. However, in the case of early acquisition of cohesiveness due to chromosomal alterations, yeast status is unlikely to improve. According to the evaluation method according to the present invention, it becomes possible to determine the yeast replacement in the repeated use of bottom fermentation at an early stage.
本発明に係る評価方法により早期凝集性であると評価された酵母集団については、早期凝集性であるとは評価されなかった酵母集団と混合し、混合後の酵母集団の部分欠損Ｓｃ３酵母の割合を低下させることにより、早期凝集性を低下させることができる。例えば、早期凝集性であると評価された酵母集団に、早期凝集性であるとは評価されなかった酵母集団を、混合後の酵母集団に対する部分欠損Ｓｃ３酵母の割合が所定の基準値未満となるように低下させる。この部分欠損Ｓｃ３酵母の割合を基準値未満とした酵母集団を用いることにより、早期凝集による発酵不良を避けて下面発酵を行うことができる。 The yeast population evaluated to be early-aggregating by the evaluation method according to the present invention was mixed with the yeast population not evaluated to be early-aggregating, and the proportion of partially defective Sc3 yeast in the mixed yeast population. By reducing the amount of yeast, the early cohesiveness can be reduced. For example, the ratio of the partially deficient Sc3 yeast to the yeast population after mixing the yeast population evaluated to be early-aggregating and the yeast population not evaluated to be early-aggregating becomes less than a predetermined reference value. To lower it. By using a yeast population in which the proportion of partially deficient Sc3 yeast is less than the reference value, bottom fermentation can be performed while avoiding fermentation defects due to early aggregation.
本発明に係る評価方法により早期凝集性を評価した酵母集団は、他の下面ビール酵母と同様に、下面発酵によるビール様発泡性飲料の製造に用いることができる。下面発酵のための発酵原料液（麦汁など）の調製や下面発酵の条件、発酵後の熟成等は、ビールをはじめとするビール様発泡性飲料の製造において一般的に行われている手法やその改良方法により行うことができる。 The yeast population evaluated for early aggregation by the evaluation method according to the present invention can be used for producing a beer-like effervescent beverage by bottom fermentation, like other bottom brewer's yeasts. Preparation of fermentation raw material liquid (wort, etc.) for bottom fermentation, conditions for bottom fermentation, aging after fermentation, etc. are methods commonly used in the production of beer-like effervescent beverages such as beer. It can be done by the improved method.
なお、本発明及び本願明細書において、「ビール様発泡性飲料」とは、アルコール含有量に関わらず、ビールと同等の又はそれと似た風味・味覚及びテクスチャーを有し、高い止渇感・ドリンカビリティーを有する発泡性飲料を意味する。すなわち、ビール様発泡性飲料は、アルコール飲料であってもよく、アルコール含量が１容量％未満であるいわゆるノンアルコール飲料又はローアルコール飲料であってもよい。ビール様発泡性飲料としては、具体的には、ビール、発泡酒、ローアルコールビール、ノンアルコールビール等が挙げられる。その他、麦芽を原料とし、発酵工程を経て製造された飲料を、アルコール含有蒸留液と混和して得られたリキュール類であってもよい。アルコール含有蒸留液とは、蒸留操作により得られたアルコールを含有する溶液であり、例えば、原料用アルコールであってもよく、スピリッツ、ウィスキー、ブランデー、ウオッカ、ラム、テキーラ、ジン、焼酎等の蒸留酒等を用いることができる。 In the present invention and the specification of the present application, the "beer-like sparkling beverage" has a flavor, taste and texture equivalent to or similar to beer regardless of the alcohol content, and has a high feeling of thirst and drin mold. It means an effervescent beverage with retaility. That is, the beer-like effervescent beverage may be an alcoholic beverage, or may be a so-called non-alcoholic beverage or a low-alcoholic beverage having an alcohol content of less than 1% by volume. Specific examples of the beer-like sparkling beverage include beer, low-malt beer, low-alcoholic beer, and non-alcoholic beer. In addition, liqueurs obtained by mixing a beverage produced from malt as a raw material through a fermentation step with an alcohol-containing distillate may be used. The alcohol-containing distillation solution is a solution containing alcohol obtained by a distillation operation, and may be, for example, alcohol for raw materials. Distillation of spirits, whiskey, brandy, wokka, lamb, tequila, gin, shochu, etc. Alcohol and the like can be used.
次に実施例及び参考例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例等に限定されるものではない。 Next, the present invention will be described in more detail with reference to Examples and Reference Examples, but the present invention is not limited to the following Examples and the like.
［実施例１］
同一の株を親株としているが、培養条件や継代回数等の培養履歴が異なる酵母のうち、早期凝集性の酵母と早期凝集性ではない酵母（健全酵母）について全ゲノム解析を行い、両酵母のゲノム上の違いを調べた。全ゲノム解析は、次世代シークエンサー（装置：Ｍｉｓｅｑ、解析ソフト：ＣＮＭｏｐｓ、いずれもｉｌｌｕｍｉｎａ社製）を用いて行った。
図１に、健全酵母の全ゲノム解析の結果（上段）と、早期凝集性の酵母の全ゲノム解析の結果（下段）を、それぞれ示す。早期凝集酵母は共通して、Ｓｃ型第III染色体の左端から約６０％の領域が欠損していた（部分欠損Ｓｃ３酵母）。この欠損領域の右腕側端はＢＵＤ５遺伝子座を含むＭＡＴ座であった。
[Example 1]
Among yeasts that have the same strain as the parent strain but have different culture histories such as culture conditions and number of passages, whole-genome analysis was performed on early-aggregating yeast and non-early-aggregating yeast (healthy yeast), and both yeasts were analyzed. We investigated the difference in the genome of yeast. Whole-genome analysis was performed using a next-generation sequencer (device: Miseq, analysis software: CNMops, both manufactured by Illumina).
FIG. 1 shows the results of whole-genome analysis of healthy yeast (upper row) and the results of whole-genome analysis of early-aggregating yeast (lower row). In common, early-aggregating yeast lacked about 60% of the region from the left end of Sc-type III chromosome (partially defective Sc3 yeast). The right arm lateral end of this defective region was the MAT locus containing the BUD5 locus.
麦芽粉砕物と原料水の混合物を加温して糖化処理し、穀皮を除去後、得られた麦汁にホップを添加して煮沸処理した後、ワールプールを用いてトルーブを除去して清澄な麦汁を得た。この麦汁に、全ゲノム解析に供した健全酵母又は早期凝集酵母を接種し、１０℃で１０日間発酵させた。その後、発酵液を貯酒槽へ移し、１０日間熟成させ、さらに０℃に冷却して１０日間安定化させた後、珪藻土濾過を実施し、製品ビールを得た。 A mixture of crushed malt and raw water is heated and saccharified to remove the grain, hops are added to the obtained wort and boiled, and then the trove is removed using a whirlpool to clarify. I got a nice wort. This wort was inoculated with healthy yeast or early agglutinating yeast used for whole genome analysis and fermented at 10 ° C. for 10 days. Then, the fermented liquid was transferred to a liquor tank, aged for 10 days, further cooled to 0 ° C. and stabilized for 10 days, and then filtered through diatomaceous earth to obtain a product beer.
発酵期間中、経時的にサンプリングし、浮遊酵母細胞数（ｃｅｌｌｓ／ｍＬ）と外観エキス濃度（％）を測定した。外観エキス濃度と浮遊酵母細胞数の測定は常法により行った。測定結果を図２に示す。この結果、どちらの酵母も、発酵開始から３〜４日目に酵母数が最大となり、５日目以降に酵母が凝集し、沈降し始めた。早期凝集酵母は、急激に凝集が進み、発酵から５日目にはそのほとんどが沈降してしまっていた。また、早期凝集酵母は、健全酵母よりもエキス消費が遅延していた。 During the fermentation period, sampling was performed over time, and the number of floating yeast cells (cells / mL) and the appearance extract concentration (%) were measured. The appearance extract concentration and the number of floating yeast cells were measured by a conventional method. The measurement results are shown in FIG. As a result, in both yeasts, the number of yeasts reached the maximum 3 to 4 days after the start of fermentation, and after the 5th day, the yeasts aggregated and began to settle. The early-aggregating yeast rapidly aggregated, and most of them had settled on the 5th day after fermentation. In addition, the early agglutinating yeast had a delayed extract consumption as compared with the healthy yeast.
＜増殖性の測定＞
早期凝集酵母と健全酵母を混合し、酵母集団に対する早期凝集酵母の含有割合が０、１０、２０、３０、４０、５０、６０、７０、８０、９０、又は１００％である酵母集団を作成した。この作成した酵母集団の系列について、増殖性を測定した。
具体的には、５ｍＬのＹＰＭ培地に、初発酵母数が１０×１０６ｃｅｌｌｓ／ｍＬとなるように接種し、２５℃で４８時間培養した。４８時間経過後の酵母数（×１０６ｃｅｌｌｓ／ｍＬ）を測定し、増殖性を評価した。
<Measurement of proliferation>
Early-aggregating yeast and healthy yeast were mixed to prepare a yeast population in which the content ratio of early-aggregating yeast to the yeast population was 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%. .. Proliferativeness was measured for the series of yeast populations prepared.
Specifically, 5 mL of YPM medium was inoculated so that the number of initial yeasts was 10 × 10 6 cells / mL, and the cells were cultured at 25 ° C. for 48 hours. 48 hours yeast counts after lapse of (× 10 6 cells / mL) was measured to evaluate the proliferative.
＜凝集性の測定＞
早期凝集酵母と健全酵母を混合して作成した酵母集団の系列について、凝集性を測定した。
具体的には、５ｍＬのＹＰＭ培地に、初発酵母数が１０×１０６ｃｅｌｌｓ／ｍＬとなるように接種し、２５℃で４８時間培養した。次いで、培養に得られた酵母を、５０ｍＬのＹＰＭ培地に、初発酵母数が１０×１０７ｃｅｌｌｓ／ｍＬとなるように接種し、１２℃で７日間発酵させた。７日間発酵した酵母について、マルトース存在下での凝集性をＨｅｌｍ’ｓ法により測定した。各酵母集団について、マルトース存在下で発酵させた後の相対凝集度（％：全く酵母が凝集しない場合を０％、全ての酵母が凝集する場合を１００％とする）を求めた。
<Measurement of cohesiveness>
Aggregation was measured for a series of yeast populations prepared by mixing early-aggregating yeast and healthy yeast.
Specifically, 5 mL of YPM medium was inoculated so that the number of initial yeasts was 10 × 10 6 cells / mL, and the cells were cultured at 25 ° C. for 48 hours. Then, the yeast obtained in the culture was inoculated into 50 mL of YPM medium so that the number of initial yeasts was 10 × 10 7 cells / mL, and fermented at 12 ° C. for 7 days. For yeast fermented for 7 days, the cohesiveness in the presence of maltose was measured by the Helm's method. For each yeast population, the relative agglutination degree after fermentation in the presence of maltose (%: 0% when no yeast aggregates at all and 100% when all yeast aggregate) was determined.
図３に、各酵母集団の４８時間培養後の酵母数（×１０６ｃｅｌｌｓ／ｍＬ）の測定結果を、図４に、各酵母集団の相対凝集度（％）の測定結果を、それぞれ示す。この結果、増殖性及び凝集性はいずれも、酵母集団に占める早期凝集酵母の割合と高い相関を示した。当該結果から、酵母集団に占める早期凝集酵母の割合が高くなるほど、早期凝集性が強くなることが確認された。 3, the measurement results of the yeast counts after 48 hours incubation of the yeast population (× 10 6 cells / mL) , 4, the measurement results of the relative cohesion of each yeast population (%), respectively. As a result, both proliferative and cohesiveness showed a high correlation with the proportion of early agglutinating yeast in the yeast population. From the results, it was confirmed that the higher the proportion of early-aggregating yeast in the yeast population, the stronger the early-aggregating property.
［実施例２］
同一の株を親株としているが培養履歴が異なる様々な酵母集団について、部分欠損Ｓｃ３酵母の割合と早期凝集性を調べた。Ａ工場で使用されている酵母集団のうち、早期凝集をしないことが発酵により確認されている酵母集団３種（Ａ−１−１、Ａ−１−２、Ａ−２−１）と早期凝集をすることが発酵により確認されている酵母集団４種（Ａ−３−１、Ａ−３−２、Ａ−３−２−１６、Ａ−３−３）、Ｂ工場で使用されている酵母集団のうち、早期凝集をしないことが発酵により確認されている酵母集団３種（Ｂ−１−１、Ｂ−１−２、Ｂ−１−３）、と早期凝集をすることが発酵により確認されている酵母集団４種（Ｂ−２−２、Ｂ−２−３、Ｂ−２−４、Ｂ−２−５）を用いた。
[Example 2]
The proportion of partially deficient Sc3 yeast and early cohesiveness were examined for various yeast populations having the same strain as the parent strain but having different culture histories. Among the yeast populations used in Factory A, three yeast populations (A-1-1, A-1-2, A-2-1) that have been confirmed by fermentation to not cause early aggregation and early aggregation 4 types of yeast populations (A-3-1, A-3-2, A-3-2-116, A-3-3) that have been confirmed to produce yeast by fermentation, yeast used in factory B Among the population, it was confirmed by fermentation that it did early aggregation with three yeast populations (B-1-1, B-1-2, B-1-3) that were confirmed not to aggregate early by fermentation. Four types of yeast populations (B-2-2, B-2-3, B-2-4, B-2-5) have been used.
＜部分欠損Ｓｃ３酵母の割合の測定＞
酵母集団から市販のＤＮＡ抽出キットを用いてＤＮＡを抽出した。ＤＮＡ抽出キットとしては、「Genomic-tip 100/G」（QIAGEN社製）又は「Genとるくん（酵母用）High Recovery」（タカラバイオ社製）を用いた。
<Measurement of the proportion of partially defective Sc3 yeast>
DNA was extracted from the yeast population using a commercially available DNA extraction kit. As the DNA extraction kit, "Genomic-tip 100 / G" (manufactured by QIAGEN) or "Gen Toru-kun (for yeast) High Recovery" (manufactured by Takara Bio Inc.) was used.
次いで、実施例１でゲノム解析した早期凝集酵母に共通していたＳｃ型第III染色体の左腕側の欠損領域中のＹＣＬ０４５Ｃ（ＥＭＣ１）遺伝子の遺伝子領域と、右腕側の非欠損領域中のＹＣＲ０７２Ｃ（ＲＳＡ４）遺伝子の遺伝子領域について、表１に記載のプライマーを用いてリアルタイムＰＣＲを行い、得られた核酸増幅量（相対量）を測定した。リアルタイムＰＣＲは、「TB Green Premix Ex Taq GC (Perfect Real Time)」（タカラバイオ社製）と「7500 Real-Time PCR System」（Applied Biosystems社製）を用いて行い、相対検量線法又はΔΔＣＴ法により相対定量を行った。 Next, the gene region of the YCL045C (EMC1) gene in the defective region on the left arm side of the Sc type III chromosome, which was common to the early-aggregated yeast genome-analyzed in Example 1, and the YCR072C (YCR072C) in the non-defective region on the right arm side For the gene region of the RSA4) gene, real-time PCR was performed using the primers shown in Table 1, and the obtained nucleic acid amplification amount (relative amount) was measured. Real-time PCR is performed using "TB Green Premix Ex Taq GC (Perfect Real Time)" (manufactured by Takara Bio Inc.) and "7500 Real-Time PCR System" (manufactured by Applied Biosystems), and the relative calibration curve method or ΔΔCT method. Relative quantification was performed.
表２に示す酵母集団について、ＹＣＲ０７２のＤＮＡ量に対するＹＣＬ０４５のＤＮＡ量の比（ＹＣＬ０４５／ＹＣＲ０７２比）を求めた。ＹＣＬ０４５／ＹＣＲ０７２比は、酵母集団を構成する全酵母細胞に占める、Ｓｃ型第III染色体の左腕側が欠損していない酵母（健全酵母）の割合を示す。当該酵母集団の酵母集団を構成する全酵母細胞に占める早期凝集酵母（部分欠損Ｓｃ３酵母）の割合は、１−ＹＣＬ０４５／ＹＣＲ０７２比で求められる。次いで、各酵母集団について、親株のＹＣＬ０４５／ＹＣＲ０７２比に対するＹＣＬ０４５／ＹＣＲ０７２相対比を算出した。結果を表２に示す。 For the yeast population shown in Table 2, the ratio of the amount of DNA of YCL045 to the amount of DNA of YCR072 (YCL045 / YCR072 ratio) was determined. The YCL045 / YCR072 ratio indicates the ratio of yeast (healthy yeast) in which the left arm side of the Sc type III chromosome is not deleted in all yeast cells constituting the yeast population. The ratio of early agglutinating yeast (partially defective Sc3 yeast) to all yeast cells constituting the yeast population of the yeast population is determined by the ratio of 1-YCL045 / YCR072. Then, for each yeast population, the relative ratio of YCL045 / YCR072 to the YCL045 / YCR072 ratio of the parent strain was calculated. The results are shown in Table 2.
早期凝集をしない酵母集団では、親株に対するＹＣＬ０４５／ＹＣＲ０７２相対比は、１近くであり、酵母集団に占める早期凝集酵母（部分欠損Ｓｃ３酵母）の割合は、親株と同程度であった。これに対して、早期凝集をする酵母集団では、親株に対するＹＣＬ０４５／ＹＣＲ０７２相対比は、いずれも小さく、酵母集団に占める早期凝集酵母（部分欠損Ｓｃ３酵母）の割合が、親株よりも明らかに増大していた。当該結果から、早期凝集の起こりやすさは、酵母集団に占める部分欠損Ｓｃ３酵母の割合に依存していることが確認された。 In the yeast population without early aggregation, the relative ratio of YCL045 / YCR072 to the parent strain was close to 1, and the ratio of early aggregation yeast (partially defective Sc3 yeast) to the yeast population was about the same as that of the parent strain. On the other hand, in the yeast population that aggregates early, the relative ratio of YCL045 / YCR072 to the parent strain is small, and the ratio of early aggregate yeast (partially defective Sc3 yeast) in the yeast population is clearly higher than that of the parent strain. Was there. From the results, it was confirmed that the susceptibility to early aggregation depends on the proportion of partially deficient Sc3 yeast in the yeast population.
各酵母集団について、実施例１と同様にして、増殖性と凝集性を調べた。図５に、各酵母集団の４８時間培養後の酵母数（×１０６ｃｅｌｌｓ／ｍＬ）の測定結果を、図６に、各酵母集団の相対凝集度（％）の測定結果を、それぞれ示す。この結果、早期凝集性である酵母集団は、早期凝集性を示さない酵母集団と比較して、増殖性と凝集性がいずれも高くなっていた。 Proliferativeness and cohesiveness were examined for each yeast population in the same manner as in Example 1. 5, the measurement results of the yeast counts after 48 hours incubation of the yeast population (× 10 6 cells / mL), in FIG. 6, the measurement results of the relative cohesion of each yeast population (%), respectively. As a result, the yeast population having early cohesiveness had higher proliferative and cohesiveness than the yeast population showing no early cohesiveness.
さらに、Ａ工場に由来する各酵母集団について、凝集性を調べた発酵液（熟成前のビール下し時の発酵液）のマルトトリオ―ス濃度（ｍｇ／ｍＬ）とビシナルジケトン（ＶＤＫ）（ｐｐｍ）を測定した。ＶＤＫは、発酵不良臭の一種である。発酵液のＶＤＫ値は、「ＢＣＯＪビール分析法（２００４．１１．１ 改訂版）８．１６ ダイアセチル ８．１６．２ ガスクロマトグラフィー法」の項に記載の方法に準じて測定した。また、発酵液のマルトトリオ―ス濃度は、ＨＰＬＣ分析により測定した。 Furthermore, for each yeast population derived from Factory A, the maltotriose concentration (mg / mL) and vicinal diketone (VDK) (ppm) of the fermented liquor (fermented liquor at the time of brewing beer before aging) whose aggregation was examined were determined. It was measured. VDK is a kind of poor fermentation odor. The VDK value of the fermented liquor was measured according to the method described in the section "BCOJ Beer Analysis Method (2004.11.1 Revised Edition) 8.16 Diacetyl 8.16.2 Gas Chromatography Method". The maltotriose concentration of the fermentation broth was measured by HPLC analysis.
図７に、各酵母集団の発酵液のマルトトリオ―ス濃度（ｍｇ／ｍＬ）の測定結果を、図８に、各酵母集団の発酵液のＶＤＫ濃度（ｐｐｍ）の測定結果を、それぞれ示す。この結果、早期凝集性である酵母集団は、早期凝集性を示さない酵母集団と比較して、発酵終了後の発酵液中のマルトトリオ―ス濃度が有意に高く、ＶＤＫ濃度も高かった。これらの結果から、部分欠損Ｓｃ３酵母の含有割合が高くて早期凝集性である酵母集団では、発酵不良が生じていることが確認された。 FIG. 7 shows the measurement results of the maltotriose concentration (mg / mL) of the fermentation broth of each yeast population, and FIG. 8 shows the measurement results of the VDK concentration (ppm) of the fermentation broth of each yeast population. As a result, the yeast population having early aggregation had a significantly higher maltotriose concentration and a higher VDK concentration in the fermentation broth after the completion of fermentation than the yeast population showing no early aggregation. From these results, it was confirmed that poor fermentation occurred in the yeast population having a high content ratio of the partially deficient Sc3 yeast and having early cohesiveness.
Claims (10)
評価対象の酵母細胞について、S. cerevisiae型第III染色体の左腕に欠損があるか否かを検出し、前記欠損がある場合には、前記酵母細胞は早期凝集性であると評価する、酵母の早期凝集性の評価方法。 A method for evaluating the early aggregation of yeast containing S. cerevisiae type III chromosome.
For yeast cells to be evaluated, whether or not there is a defect in the left arm of S. cerevisiae type III chromosome is detected, and if there is such a defect, the yeast cell is evaluated to be prematurely aggregated. Evaluation method of early agglutination.
評価対象の酵母集団について、S. cerevisiae型第III染色体の左腕に欠損があるか否かを検出し、前記酵母集団に対する前記欠損がある酵母細胞の割合が、所定の基準値超である場合に、前記酵母集団は早期凝集性であると評価する、酵母の早期凝集性の評価方法。 A method for evaluating the early aggregation of yeast containing S. cerevisiae type III chromosome.
For the yeast population to be evaluated, whether or not there is a defect in the left arm of S. cerevisiae type III chromosome is detected, and the ratio of yeast cells having the defect to the yeast population exceeds a predetermined reference value. , A method for evaluating the early aggregation of yeast, wherein the yeast population is evaluated to be early aggregating.
発酵に使用する酵母集団を、予め、請求項３〜８のいずれか一項に記載の酵母の早期凝集性の評価方法により評価し、
早期凝集性であるとは評価されなかった酵母集団を用いて下面発酵を行う、ビール様発泡性飲料の製造方法。 A method for producing a beer-like effervescent beverage produced through bottom fermentation.
The yeast population used for fermentation is evaluated in advance by the method for evaluating the early cohesiveness of yeast according to any one of claims 3 to 8.
A method for producing a beer-like effervescent beverage, in which bottom fermentation is performed using a yeast population that has not been evaluated to be early cohesive.
発酵に使用する酵母集団を、予め、請求項３〜８のいずれか一項に記載の酵母の早期凝集性の評価方法により評価し、
早期凝集性であると評価された酵母集団と、早期凝集性であるとは評価されなかった酵母集団とを、混合後の酵母集団に対する前記欠損がある酵母細胞の割合が前記基準値超となるように混合した酵母集団を用いて下面発酵を行う、ビール様発泡性飲料の製造方法。 A method for producing a beer-like effervescent beverage produced through bottom fermentation.
The yeast population used for fermentation is evaluated in advance by the method for evaluating the early cohesiveness of yeast according to any one of claims 3 to 8.
The ratio of yeast cells having the deficiency to the yeast population after mixing the yeast population evaluated to be early-aggregating and the yeast population not evaluated to be early-aggregating exceeds the reference value. A method for producing a beer-like effervescent beverage, in which bottom fermentation is performed using the yeast population mixed as described above.
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