JP3676943B2 - Yeast mutant for alcoholic beverage production and method for producing alcoholic beverages using the yeast mutant - Google Patents

Description

【００３３】
（実施例２） ワインの製造試験
実施例１において取得した二重変異を有するMA2504株及びMA2601株並びにこれらの親株であるサッカロミセス・セレビシエOC-2を使用し、グラニュー糖にて糖度を23度に調整した甲州種ブドウ果汁を原料として常法によりワイン製造を実施した。製造されたワインの一般成分分析結果を表2に示す。MA2504株及びMA2601株の発酵速度は、親株に比較してやや遅くなったが、アルコール濃度、エキス分、総酸、pH、有機酸組成の分析値に大きな差は認められなかった。また、アミノ酸分析結果を表3に示す。親株により製造したワインはそのアミノ酸の大部分がプロリンであったが、MA2504株により製造したワインは、プロリン含有量が多く、さらには、アスパラギン酸、トレオニン、セリン、アスパラギン、グルタミン酸、グルタミン、アラニン、バリン、イソロイシン、ロイシン、チロシン、フェニルアラニン及びヒスチジンのアミノ酸も多く含まれ、総アミノ酸量は親株により製造したワインの約1.9倍であった。MA2601株により製造したワインは、プロリン含有量がやや少なくなったが、アルギニンが果汁中の含有量と同程度含まれており、総アミノ酸量は親株により製造したワインの約1.3倍であった。
【００３４】
【表２】

【００３５】
【表３】

【００３６】
上記の如くして製造されたワインについて、ワイナリーの熟練した7名のパネラーにより官能試験を行った。その結果、MA2504株及びMA2601株により製造したワインは親株により製造したワインに比較して味に厚みがあるとの評価が得られた。
【００３７】
【発明の効果】
本発明によれば、酵母によるアミノ酸の取込みを抑制して、安定的にアミノ酸含有量が改善された酒類、特にはワインを製造できる方法を提供することが可能となる。
【図面の簡単な説明】
【図１】図１は、本発明の酵母変異株を得る工程の好適な実施形態を示すフローチャートである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a yeast used for producing alcoholic beverages, and also relates to a method for producing alcoholic beverages using yeast.
[0002]
[Prior art]
In general, amino acids are one of the taste components of wine and other liquors, and are considered to be important factors affecting quality. Therefore, it is important in the production of alcoholic beverages to control the amino acid content in the produced alcoholic beverages.
[0003]
However, for example, in the production of wine, most of the amino acids in the wine produced are from the grape juice, but many of the amino acids in the grape juice are consumed during fermentation as a nutrient source for yeast. It is extremely difficult to control the amino acid content in wine.
[0004]
The wine produced from Koshu grape, which is a typical grape varieties produced in Japan, has a relatively flat taste and is thought to be caused by the low content of nitrogen compounds including amino acids. ing. Therefore, Koshu wines are mainly sweet wines that leave sugar to supplement the flat taste. When producing dry wines, the Shu-Lee method, that is, by contacting the wine with starch, The method of giving a body feeling to wine by eluting components such as amino acids from self-digestion is adopted (K. Ariizumi et al .: Am. J. Enol. Vitic., Vol.45, pp.312, 1994) . However, the Sur-Lee method has a problem that the contact period between wine and starch is long, and further, the increase in amino acids in wine due to yeast autolysis is not always large. In recent years, diversification of liquor quality has been demanded, and in order to meet this demand, development of a new liquor production method capable of controlling the amino acid content in the produced liquor is desired.
[0005]
By the way, in order for yeast to use the amino acid which exists outside a cell as a nutrient source, it is essential to take in an amino acid into a microbial cell. As an enzyme to perform the incorporation of such amino acids, many amino acids transmission enzymes having different substrate specificities, for example, many amino acids transmission enzymes, including arginine permease encoded by the general amino acid permease and CAN1 gene encoded the GAP1 gene (B. Andre: Yeast, vol.11, pp.1575, 1995, B. Nelissen et al .: FEBS Letters, vol.377, pp.232, 1995, M. Grenson : In De Pont, JJHHM (ed.), Molecular Aspects of Transport Proteins, Chapter 7, "Amino acid transporters in yeast; structure, function and regulation", Elsevier, Amsterdam, pp.219, 1992, etc.).
[0006]
To date, there have been many reports on the method of obtaining mutant strains in which mutations have been made in genes involved in the amino acid permease of yeast and the effect of the mutations on amino acid uptake. For example, gap1 mutant strains can be obtained from strains resistant to D-amino acids, and it is known that incorporation of many amino acids including citrulline is suppressed (J. Rytka: J. Bacteriol., vol.121, pp.562, 1975). The can1 mutant can be obtained from a strain resistant to L-canavanine, an analog of arginine, and is known to suppress arginine uptake (M. Grenson et al .: Biochim. Biophys. Acta, vol. 127, pp. 325, 1966, M. Grenson, Molecular Aspects of Transport Proteins, Chapter 7, “Amino acid transporters in yeast: structure, function and regulation, Elsevier Science Publishers, 1992, etc. ) and apf1 mutants Can be obtained from strains resistant to L-azetidine-2-carboxylate or 3,4-dihydro-DL-proline, which are proline analogs, and the inhibition of proline uptake and various amino acid permeases activity is known to be inhibited, further, in the yeast with a double mutation gap1 the APF 1, it is known to not grow a number of amino acids, including glutamic acid as the sole nitrogen source (M. Grenson and C. Hennaut: J. Bacteriol, vol.105, pp.477, 1971, PF Lasko and MC Brandriss:.. J. Bacteriol, vol.148, pp.241, 1981).
[0007]
The SHR3 gene encodes a protein essential for the processing and transport of amino acid permeases from the endoplasmic reticulum, and shr3 and apf1 mutations have been reported to be non-complementary alleles (PO Ljungdahl et al .: Cell , vol. 71, pp. 463, 1992, J. Horak and A. Kotyk: Biochemistry and Molecular Biology International, vol. 29, pp. 907, 1993).
[0008]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for improving the amino acid content, which is one of the important unexposed components of alcoholic beverages, in the production of alcoholic beverages, particularly wine.
[0009]
[Means for Solving the Problems]
The present invention relates to a yeast mutation for producing alcoholic beverages belonging to the genus Saccharomyces (preferably Saccharomyces cerevisiae) having double mutations in genes involved in amino acid incorporation, in particular gap1 and apf1 (shr3) or gap1 and can1. Regarding stocks.
[0010]
The present invention also relates to a yeast mutant MA2504 strain (FERM P-17407) belonging to Saccharomyces cerevisiae and having a mutation of gap1 and apf1 (shr3) as a mutation of a gene involved in amino acid incorporation.
[0011]
The present invention also relates to a yeast mutant MA2601 strain (FERM P-17408) belonging to Saccharomyces cerevisiae and having gap1 and can1 mutations as mutations of genes involved in amino acid incorporation.
[0012]
The present invention also relates to a method for producing a liquor {preferably wine or malt alcoholic beverage (for example, beer, happoshu)} using the above-mentioned yeast mutant strain for liquor production, and in particular, the amino acid content in the produced liquor. The present invention relates to a method for producing alcoholic beverages.
[0013]
The present invention also relates to a method for producing wine using the above-described liquor-producing yeast mutant, and more particularly to a method for producing wine with an improved amino acid content in the produced wine.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a yeast mutant strain belonging to the genus Saccharomyces and having a double mutation in a gene involved in amino acid uptake, and particularly to a yeast mutant strain having a double mutation of gap1 and apf1 or gap1 and can1 .
[0015]
The present invention also relates to a method for producing an alcoholic beverage with improved amino acid content, particularly a wine, characterized by inoculating the yeast mutant to which the double mutation has been added as a liquor and performing fermentation.
[0016]
The yeast used in the present invention is a yeast belonging to the genus Saccharomyces, for example, Saccharomyces cerevisiae classified according to The Yeasts A Taxonomic Study, 4th ed. CP Kurtzman and JW Fell (ed.), Elsewier (1998). ), Saccharomyces bayanus, Saccharomyces pastorianus, and the like, and wine yeast and / or beer yeast. Examples of yeast belonging to Saccharomyces cerevisiae include wine yeasts such as Saccharomyces cerevisiae OC-2, W3, and Prize de Mousse.
[0017]
As a method for mutating yeast, there are a physical method such as ultraviolet irradiation or a chemical method of suspending in a mutating agent (for example, a solution such as ethylmethane sulfonate), and a known method can be appropriately used.
[0018]
A method for selecting mutants will be described. The gap1 mutant strain can be obtained from strains that are resistant to D-amino acids such as D-histidine after mutagenesis in yeast. That is, from a mutant exhibiting D-histidine resistance, a minimal medium supplemented with citrulline as a single nitrogen source (glucose 0.5%, Difco Bacto yeast nitrogen base (without amino acids and ammonium sulfate) 0.17%, agar 2 %) (% = W / v%) and in a complementation test using a known gap1 mutant strain, the strain can be obtained by selecting a strain having no complementarity. The can1 mutant can be obtained from a strain exhibiting resistance to L-canavanine, which is an analog of arginine, and can be obtained by selecting a strain having no complementarity in a complementation test using a known can1 mutant. apf1 mutant strains can be obtained from strains resistant to proline analogs L-azetidine-2-carboxylate or 3,4-dihydro-DL-proline, and resistant to β-2-thienyl-DL-alanine In a complementation test using a known apf1 mutant strain, which is unable to grow in a medium supplemented with proline as a single nitrogen source, it can be obtained by selecting a strain having no complementarity.
[0019]
Next, in order to obtain a double mutant strain from the mutant strain thus obtained, a method of remutation or a cross strain between mutant strains is obtained, and a single spore strain derived from the cross strain is obtained. A method of selecting from among them can be used. The above-described method can be used when selecting a target mutant strain from the subsequent mutation treatment. For example, the mutant strain having gap1 and apf1 mutations may be subjected to the mutation treatment again using the strain already having the gap1 mutation, and the above-described method for obtaining the apf1 mutant strain may be performed. In the method of obtaining double mutant strains by hybridization, the hybrid strain can be obtained by spore-spore, spore-cell, or cell-cell junction as appropriate in consideration of yeast mating type and ploidy. Induction of ascospores in the culture medium (sodium acetate 0.82%, glucose 0.1%, yeast extract 0.25%, potassium chloride 0.18%, agar 2%) (% = w / v%) followed by spore isolation and colony formation Thus, a single spore strain is obtained, and the target mutant strain can be selected by combining the above-described methods suitable for selecting the target mutant strain.
[0020]
The mutant yeast of the present invention can be suitably used for the production of alcoholic beverages such as wine and malt alcoholic beverages (for example, beer and happoshu), and is particularly preferably used for the production of wine.
[0021]
In order to produce wine in the present invention, the obtained yeast mutant strain is used as a liquor mother, and a method similar to the usual wine production method, for example, the sake brewing method (edited by Yamanashi Prefectural Industrial Technology Center) You can make wine by the method.
[0022]
Further, in order to produce alcoholic beverages other than wine in the present invention, the obtained yeast mutant strain is used, and a method similar to the usual method for producing alcoholic beverages, for example, “Technology Brewing and Malting”, by Kunze, VLB , 1996; "Die Bierbrauerei, Band I, II", (7th edition), Narziss, Enke, 1992; "Brew brewing science" by Matsuyama Shigesuke, Toyo Keizai Shinposha, 1969. Can be manufactured.
[0023]
In the production of alcoholic beverages using the yeast mutant of the present invention, since the incorporation of amino acids is suppressed, it is possible to produce alcoholic beverages having a stable improved amino acid content. Further, in the production of wine using the yeast mutant of the present invention, since the incorporation of amino acids is suppressed, it is not necessary to contact wine and starch stably as in the conventional surreal method. In addition, it is possible to produce wine with improved amino acid content in a short period of time.
[0024]
【Example】
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
[0025]
(Example 1) Acquisition of a double yeast mutant As a wine yeast, Saccharomyces cerevisiae OC-2 (IAM4274), which is homothallic, diploid, and shows a high ascospore formation rate and germination rate, was used as a parent strain. McClary et al.'S medium (sodium acetate 0.82%, glucose 0.1%, yeast extract 0.25%, potassium chloride 0.18%, agar 2%) (% = w / v%) (% = w / v%) to induce the formation of ascospores, The sac wall lysing enzyme (trade name: Zymolyce 20T, manufactured by Seikagaku Corporation) was dissolved in a phosphate buffer solution (pH 7.5) containing 250 ppm, washed with sterilized water, and then washed with phosphate buffer (pH 8. 0) was added to obtain 4.6 ml of a spore suspension. To this, add 0.25 ml of 40 w / v% glucose solution and 0.15 ml of ethyl methanesulfonate and perform mutation treatment at 30 ° C. for 80 minutes, and add an equal amount of 10 w / v% sodium thiosulfate solution to carry out mutation treatment. After stopping, it was washed with sterilized water and suspended in sterilized water to obtain a mutation-treated spore suspension.
[0026]
Mutation-treated spore suspension was prepared using a minimal medium containing 10 mM D-histidine and 0.1 w / v% proline (glucose 0.5%, yeast nitrogen base (without amino acids and ammonium sulfate) 0.17%, agar 2%) (% = w / v%), a strain having no known gap1 mutation, such as 2512c, which does not grow on a minimal medium containing citrulline as a single nitrogen source, and grows after 3-7 days of culture -2A (a gap1 ) strain in a complementation test, showing D-histidine resistance and no growth in minimal medium with citrulline as a single nitrogen source, that is, a strain with no complementarity (MA1007 strain) Was selected as a gap1 mutant.
[0027]
Similarly, among colonies growing on minimal medium containing 0.5 mM L-azetidine-2-carboxylate and 0.1 w / v urea, β-2-thienyl-DL-alanine (100 mg / l) and urea 0.1 w In a complementary test using a strain having a known apf1 mutation, such as 30967c ( apf1ura3 ), which grows in a minimal medium containing / v%, does not grow in a minimal medium containing proline as a single nitrogen source, and L-azetidine-2-carboxylate resistance and the property that growth was not observed in a minimal medium containing proline as a single nitrogen source, that is, a strain having no complementarity (MA1325 strain) was selected as an apf1 mutant.
[0028]
Complementation using a strain having a known can1 mutation, for example, AH-22 (a leu2-3,112his4can1 ), from colonies growing on a minimal medium containing L-canavanine 40 mg / l and ammonium sulfate 0.5 w / v% In the test, a strain (MA1205 strain) exhibiting L-canavanine resistance, ie, having no complementarity, was selected as a can1 mutant.
[0029]
Next, each of the strains MA1007 and MA1325 was used to form ascospores in the medium of McClary et al., And after the ascomytes wall was lysed with zymolyce in the same manner as described above, the spores were used using a micromanipulator. -A sexual cross between spores was obtained, and a strain (MA2504 strain) having a double mutation of gap1 and apf1 was obtained from the phenotype and complementation test of the obtained single spore strain derived from the cross. This strain was deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology on June 1, 1999, and its deposit number is FERM P-17407. In addition, a strain (MA2601 strain) having a double mutation of gap1 and can1 was obtained from the MA1007 strain and the MA1205 strain by the same method. This strain has been deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology on June 1, 1999, and the deposit number is FERM P-17408.
[0030]
The procedure shown above is described as a flowchart in FIG. 1 for explanation.
[0031]
The mutant strains obtained as described above were examined for growth in a minimal medium supplemented with various amino acids as a single nitrogen source. The results are shown in Table 1. The gap1 mutant MA1007 strain did not grow on a minimal medium with citrulline as a single nitrogen source, and the apf1 mutant MA1325 strain did not grow on a minimal medium with proline as a single nitrogen source. Both were able to grow on a minimal medium with glutamic acid or alanine as the single nitrogen source. However, the gap1 and apf1 double mutant MA2504 strains did not grow in a minimal medium containing glutamate and alanine as a single nitrogen source as well as citrulline and proline. On the other hand, the can1 mutant MA1205 strain was able to grow on a minimal medium using arginine as a single nitrogen source, whereas the gap1 and can1 double mutant MA2601 strains could grow on a minimal medium using arginine as a single nitrogen source. It was not recognized, and it was shown that incorporation of these double mutations suppressed the uptake of specific amino acids and prevented growth.
[0032]
[Table 1]

[0033]
(Example 2) Wine production test MA2504 and MA2601 strains having double mutations obtained in Example 1 and their parent strain Saccharomyces cerevisiae OC-2 were used, and the sugar content was increased to 23 degrees with granulated sugar. Wine production was carried out in a conventional manner using the adjusted Koshu grape juice as a raw material. Table 2 shows the results of general component analysis of the wines produced. The fermentation rates of MA2504 and MA2601 strains were slightly slower than the parent strain, but no significant differences were found in the analytical values of alcohol concentration, extract content, total acid, pH, and organic acid composition. In addition, Table 3 shows the results of amino acid analysis. Most of the amino acids in the wine produced by the parent strain were proline, but the wine produced by the MA2504 strain has a high proline content, and further, aspartic acid, threonine, serine, asparagine, glutamic acid, glutamine, alanine, A lot of amino acids such as valine, isoleucine, leucine, tyrosine, phenylalanine and histidine were contained, and the total amino acid amount was about 1.9 times that of the wine produced by the parent strain. The wine produced with the MA2601 strain had a slightly lower proline content, but contained arginine as much as the content in the fruit juice, and the total amino acid content was about 1.3 times that of the wine produced with the parent strain.
[0034]
[Table 2]

[0035]
[Table 3]

[0036]
The wine produced as described above was subjected to a sensory test by seven panelists skilled in the winery. As a result, it was evaluated that the wine produced by the MA2504 strain and the MA2601 strain had a thicker taste than the wine produced by the parent strain.
[0037]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the method which can manufacture alcoholic beverages by which the uptake of the amino acid by yeast and the amino acid content were improved stably, especially wine.
[Brief description of the drawings]
FIG. 1 is a flowchart showing a preferred embodiment of a process for obtaining a yeast mutant of the present invention.

Claims (5)

Yeast mutant MA2504 (FERM P-17407) belonging to Saccharomyces cerevisiae and having a mutation of gap1 and apf1 (shr3) as a mutation of a gene involved in amino acid incorporation. A yeast mutant MA2601 (FERM P-17408) belonging to Saccharomyces cerevisiae and having a mutation of gap1 and can1 as a mutation of a gene involved in amino acid incorporation. A method for producing alcoholic beverages using the yeast mutant according to claim 1 or 2 in a method for producing alcoholic beverages using yeast. The method for producing an alcoholic beverage according to claim 3 , wherein the alcoholic beverage is a wine or a malt alcoholic beverage. A method for producing wine, wherein the yeast mutant according to claim 1 or 2 is used in a method for producing wine by inoculating a grape juice with a wine yeast strain as a liquor and fermenting it.

Images