JPH0675500B2 - Yeast that leaks physiologically active ingredient and method for producing alcoholic beverage using the yeast - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel yeast that leaks physiologically active components, particularly glutathione, out of the cells, and a novel method for producing alcoholic beverages using the yeast. More specifically, yeast that is produced in yeast cells and is normally stored and consumed inside the cells leaks out physiologically active components such as glutathione to the outside of the cells, and yeast that has brewing aptitude, and alcohol fermentation using the yeast. And then
The present invention relates to a method for producing alcoholic beverages having a high content of physiologically active components such as glutathione.

BACKGROUND ART Glutathione is a physiologically active peptide to which three kinds of amino acids, glutamic acid, cysteine and glycine are bound, is widely distributed in cells of animals and plants, and is involved in detoxification and redox equilibrium in vivo. Focusing on this physiologically active action, glutathione has been widely used as a therapeutic agent for liver diseases such as a therapeutic agent for alcoholic fatty liver.

On the other hand, in recent years, functional foods have come into the limelight from the viewpoint of health, and in the field of foods, foods containing glutathione have begun to attract attention due to the physiologically active action of glutathione. However, most alcoholic beverages do not contain glutathione at present, and even if they contain glutathione, the amount thereof is usually as low as 1 ppm or less.

This is due to the following circumstances.

That is, first, the yeast itself has a glutathione-producing ability, but the outer periphery of a yeast cell that maintains the so-called yeast type morphology of an oval type and an egg type is covered with a strong cell wall and a cell curtain, and is produced. Glutathione is restricted to flow out of the cells. Secondly, as a method for producing glutathione by fermentation in cells and continuously flowing it out of the cells, Saccharomyces glyoxalphilus (Saccharowyces) is used.
glyoxalphilus) (JP-A-53-94088), Candida
Candida tropicalis PK233 (JP-A-8)
-1462974) is proposed, but
All of these methods focus on the production of glutathione itself, and the yeasts used do not have an adequate fermentation ability for brewing. Furthermore, in brewing, these methods cannot be applied because the manufacturing method and additives are restricted by the regulation of the liquor tax law. The same applies to other physiologically active ingredients during this period.

Therefore, if you dare to produce a liquor containing a physiologically active ingredient such as glutathione, by adding a separately produced physiologically active ingredient to the liquor, or by self-digestion with alcohol, the yeast in the brewing process is artificially done in the brewing process. It had to be self-digested to leak glutathione out of the cells. However, the former method is not appropriate due to restrictions imposed by the liquor tax law, and the latter method remarkably deteriorates the quality of alcoholic beverages due to self-digesting odor.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention Under such circumstances, brewing of alcoholic beverages containing a large amount of useful physiologically active ingredients such as glutathione has not been realized.

Therefore, there is a new manufacturing method that can be applied to normal liquor charging methods without problems under the liquor tax law, and that contains a lot of physiologically active ingredients such as glutathione, and that is similar in quality to regular liquors. It has been requested.

The present inventors have diligently studied a method for producing a liquor containing a physiologically active ingredient in order to meet such a demand.

Means for Solving the Problems In conventional yeasts, the components produced inside the cells are difficult to be released to the outside of the cells by ordinary culture. Further, a specific component such as glutathione is produced and stored in the microbial cells, and decomposed and utilized in the microbial cells as needed. Therefore, firstly, in order to release the target component to the outside of the microbial cell in a sufficient amount, it is necessary to suppress the decomposition of the component within the microbial cell. Secondly, it is necessary to develop a yeast that releases the target component to the outside of the cell and has alcoholic fermentability at the same time, and also has aptitude for brewing alcoholic beverages. Yeast that can be applied to the liquor manufacturing process is desirable.

Therefore, the present inventors first focus on glutathione, which is one of the physiologically active components in yeast cells, and the presence of γ-glutamyl transpeptidase, which is a glutathione-degrading yeast, and the organ in which glutathione is stored and degraded. Breeding of a strain that does not form vacuoles, which is one of the above. The vacuolar-deficient strain was selected because it is expected that glutathione will leak out of the cells due to an abnormality in the substance transport system within the cells due to the vacuole defect, and that vacuoles are not formed. This is because it was considered that the cells became sensitive to the osmotic pressure inside and outside the cells, and the cells collapsed during the growth process to release other useful bioactive components.

As the vacuole-deficient strain, the method of Kitamoto et al., Which is one of the present inventors [Journal of Bacteriology (J.
Bateriol.), 170, 2687 (1988)], Saccharomuces cerevisiae.
KL-197-12B was used as the starting strain. Upon examination, this KL-197-12B was found to have extremely low growth rate and fermentative power as compared with the conventional yeast for brewing, as shown in Reference Example 1 described later, and was found to have no brewing aptitude for alcoholic beverages. Therefore, brewing yeast (Kyoto Yeast No. 7, hereinafter referred to as K-7) was used as a recurrent parent, and an attempt was made to backcross to enhance brewing aptitude.

As a result of such backcrossing, it was found that a yeast which is satisfactory in terms of proliferation ability, fermentation ability, leakage of intracellular physiologically active components such as glutathione, and quality of sake can be obtained, and the present invention was completed. Came to do.

That is, the present invention provides a novel yeast which is deficient in vacuolar formation ability, has a substance transport mutation or osmotic sensitivity, and has brewing aptitude such as fermentation ability and growth ability. In addition, alcohol fermentation is performed using such yeast,
Also provided is a method for producing alcoholic beverages, which comprises obtaining alcoholic beverages having an increased content of physiologically active ingredients.

Thus, the yeast of the present invention is backcrossed with a vacuole-forming ability deficient strain of Saccharomyces cerevisiae and a brewing yeast according to a known method, and the obtained strain is screened for fermentation ability, growth ability and glutathione leakage ability. Obtained by selecting strains.

The vacuolar formation-deficient strain used is KL-197-12B from the previous period.
However, the present invention is not limited to this, and for example, a known vacuolar formation-deficient strain of Saccharomyces cerevisiae strain may be used according to the method of Kitamoto et al.

Moreover, the yeast strain for brewing is not limited to K-7, but other sake yeasts, wine yeasts, brewer's yeasts, etc.
Known brewing yeast strains can be used.

The obtained yeast can maintain its characteristics stably by passage, and using this, alcohol can be obtained by a normal brewing process. For example, the yeast can be used for alcoholic fermentation of rice or millet with koji or enzyme. For example, liquor containing glutathione in an amount of 15 to 20 ppm, which is significantly higher than that of conventional liquor, can be obtained.

EXAMPLES The present invention will be described in more detail with reference to Reference Examples and Examples below.

Reference Example 1 Properties of KL-197-12B that leaks glutathione to the outside of the bacterial cells To a rice malt that has been brewed in the field, water with a pumping ratio of 250% was added, and the mixture was heated at 56 ° C for 16
The supernatant obtained by saccharification for a period of time and then centrifugation was used as a koji juice medium (Be5.0), and 2 × 10 ⁸ Saccharomyces cerevisiae KL-197-12B and Saccharomyces cerevisiae K-7 were added to 100 ml of this medium. The cells were inoculated and statically cultivated at 13 ° C., and changes in the amount of glutathione leaked with time were measured.

The amount of glutathione was measured by the Tietze method (hereinafter, the amount of glutathione is measured by this method), and the amounts of other components were measured by the National Tax Agency prescribed analysis method. The mortality was determined by a methylene blue staining method under a microscope.

As shown in Table 1, in the KL-197-12B, unlike K-7, the extracellular amount of glutathione leaked out increased with the lapse of time in culture. In addition, the mortality of the bacterium was not high, and glutathione was leaked from the early stage of culture, suggesting that the leakage of glutathione was not due to autolysis of the cells, but due to abnormal substance transport in the cells. .

However, as is clear from Table 1, KL-197-1
2B is inferior to K-7 in the growth of bacteria and the ability to produce alcohol, and is not suitable for brewing. Therefore, as described above, when backcrossing with K-7 was performed, KL-197-12B was given brewing aptitude while retaining the glutathione leaking ability.

Example 1 According to the procedure shown below, a vacuolar formation-deficient strain KL-197-12B
The back crossing was repeated using S. cerevisiae as the donor parent and Sake yeast K-7 as the recurrent parent.

1) The monophasic strains (K-7-H-5, mating type a and K-7-H-6, mating type α) are separated from K-7 and used as recurrent parents.

2) KL-197-12B (zygote type α) is crossed with K-7-H-6 to obtain a first-generation hybrid.

3) Isolate the monophasic body from the first-generation hybrid, and select a strain having no vacuole forming ability and good fermentation ability from the hybrid.

4) Determine the mating type of the selected strain.

5) Backcrossing the selected strain with a K-7 monophasic strain compatible with the mating type to obtain a second-generation hybrid.

6) Thereafter, backcrossing is repeated in the same manner.

As a result of backcrossing, an excellent strain lacking vacuolar formation ability and fermentability was selected, and Saccharomyces cerevisiae No. 133 (hereinafter referred to as No. 133) was obtained.

This No. 133 strain was placed on February 8, 1990 under the accession number FERM P-
Deposited under 11269 to the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology.

The bacterial characteristics of this No. 133 strain are described below.

Growth rate of No.133 strain Strain No.133 and amount Parent strain were planted in 7 ml of YPD liquid medium (1% Bacto-ist extract, 2% Bacto-peptone, 2% glucose, the same below) 7 × 10 ⁵ each. Fungus and 25
The growth rate was measured by shaking culture and static culture at 0 ° C. and measuring the turbidity (OD ⁶⁶⁰ ) of the culture broth with a turbidimeter over time. The results are shown in Tables 2 and 3.

From Tables 2 and 3, it was revealed that the yeast strain No. 133 of the present invention had a growth rate intermediate between those of the parent strains.

Effect of glucose concentration on alcohol production No.133 and parent strains were inoculated into ⁷ ml of YPD liquid medium containing 2 to 20% glucose in an amount of 1 x 10 ⁷ each, and then static culture was performed at 25 ° C for 7 days. The effect of glucose concentration on production was investigated. The growth was observed by the amount of CO ₂ gas generated, and the amount of alcohol produced was measured by gas chromatography using the filtrate after the culture as a sample.

From Table 4, it was revealed that the yeast No. 133 of the present invention has properties almost similar to those of the yeast for brewing (K-7) in terms of growth ability and fermentation ability.

Leakability of glitathione Next, the properties of the yeast of the present invention were examined in a saccharified solution using white rice bran before brewing with white rice. Shiranuka saccharified solution (6th
(See the table), the yeast cells were cultivated in the same numerical value, and static culture was performed at 13 ° C., and the fermentation ability and the leakage ability of glutathione as one of physiologically active components in the cells were measured with time.

The saccharified rice solution uses crushed rice with a rice polishing rate of 70%, and the saccharified rice bran solution uses bran produced between 80% to 75% of the rice polishing rate. It was prepared using Protease M Amano, manufactured by Amano Pharmaceutical Co., Ltd.

Despite the low content of sulfur-containing amino acids, which can serve as a substrate for glutathione biosynthesis, in the saccharified rice syrup (see Table 6), No. 133 leaks glutathione, and its glutathione leakage ability is , Which had a capacity comparable to that of the parent strain, a vacuole-deficient yeast (KI-197-12B) (Table 5). Further, the fermenting ability was different from KL-197-12B and had the same ability as that of the other parent strain K-7 (Table 5).

Furthermore, when estimated from the substrate amino acid content in the raw material,
It could be expected that the concentration of glutathione would be further increased by using white rice as a raw material.

From the above measurement, it was confirmed that the yeast of the present invention has aptitude for brewing alcoholic beverages and further has a novel property of leaking glutathione produced and stored in the yeast cells outside the cells. Therefore, it was considered that glutathione-containing sake can be produced by using this yeast for brewing sake.

Therefore, it was decided to carry out a small preparation test using this new yeast.

Example 2 Small preparation test In this example, sake production was carried out by a two-stage preparation according to a conventional method. Tables 7 and 8 show the charge composition and the fermentation process.

From Table 8, it can be seen that the yeast of the present invention has a glutathione content 10 times or more higher than that of liquor charged with conventionally used brewing yeast (K-7). In addition, the amount of glutathione leaked out was also higher than that obtained when the saccharified bran solution was used (see Table 5). on the other hand,
KL-197-12B, which is a parent strain of the vacuole-forming ability-deficient strain, had poor growth and fermentation ability, and leakage of a sufficient amount of glutathione and production of alcohol were not observed.

Furthermore, the yeast of the present invention (No. 133) can be obtained by using Kikishu after the upper tank.
The liquor produced according to the present invention is not inferior to the liquor quality of liquor produced according to the conventional brewing yeast (K-7), and
It was revealed that no offensive odor such as self-digesting odor or off taste was felt.

As described above, it is understood that by using the yeast of the present invention, alcoholic beverages containing glutathione in a higher concentration than conventional alcoholic beverages can be produced.

Therefore, in the following examples, production was tried by scaling up.

Example 3 Two-stage preparation In this example, two-stage preparation of 400 g of total rice was performed according to ordinary conditions. Table 9 and 10 show the mixing recipe and the mixing result.
Shown in the table.

Even in the two-stage preparation of 400 g of total rice, the sake prepared with the yeast of the present invention (No. 133) showed a satisfactory fermentation process and the amount of alcohol produced was sufficient, as was the case with 200 g of total rice (Table 8). . Even in the amount of glutathione leaked out, the liquor of the present invention contained a larger amount of glutathione than the liquor charged with the conventional brewing yeast (K-7), and the odor of bonito sake, such as self-digesting odor, was also observed in the quality evaluation. I didn't feel any strangeness.

Thus, the desired alcoholic beverage was obtained when the yeast of the present invention was used in the two-stage charging.

Example 4 Three-stage preparation In this example, a three-stage preparation used for ordinary preparation of sake was performed.

As a result of carrying out three-stage preparation using the yeast of the present invention, the same as the result of the two-stage preparation (see Tables 8 and 10), K-
It was possible to produce a liquor containing more glutathione than that of No. 7. Similarly, the results of Kiki sake were not inferior to the sake produced by the conventional brewing yeast (K-7), and no offensive odor such as self-digesting odor or off taste was observed at all.

As described above, it was found that the desired glutathione-containing sake can be obtained by using the yeast of the present invention even in the brewing method by the usual preparation of sake.

Example 5 Ladder history and physiologically active ingredients In this Example, 40 g of total rice of the yeast of the present invention was charged in two stages, and the history and the content of physiologically active ingredients other than glutathione are also described.

As a result of charging with the yeast of the present invention, it was confirmed that fermentation of mash was successful, and that glutathione was leaked into sake after the middle of the mash with the production of alcohol (Table 13). on the other hand,
With conventional yeast (K-7), the leakage of glutathione into sake is extremely low (Table 14).

This time, it was found that the liquor produced by the present invention contained more physiologically active components in the liquor than the conventional liquor, which is shown by the following measurement results.

First, the ultraviolet absorption values (OD ²⁶⁰ , OD ²⁸⁰⁾ and nucleic acid purity (OD ²⁶⁰ / OD ²⁸⁰ ) during fermentation of mash are shown.

OD showing absorption of nucleic acid in mash when charged to this yeast
²⁶⁰ value, and increase in the value of OD ²⁶⁰ / OD ²⁸⁰ indicating the purity of nucleic acids was observed (Table 15). It is considered that the cause of the increase of these nucleic acids is due to the autolysis of bacterial cells or the destruction of bacterial cells. However, since no offensive odor normally associated with yeast autolysis was not observed at all, it is considered that the leakage is not due to autolysis but to destruction of bacterial cells.

Therefore, in the yeast of the present invention, the extracellular leakage of intracellular components caused by the abnormal substance transport in the yeast cells described in Reference Example 1, and further the change in morbidity that can be considered from Table 15 of this Example. It is considered that the useful physiologically active ingredient in the yeast cell is leaked out due to both destruction of the bacterium itself due to the osmotic pressure change outside the yeast cell which occurs together with.

As examples of other physiologically active ingredients, the results of measurement of amino acids and vitamins are shown below.

1) Amino acid measurement method: The filtrate on the 18th day of the second-stage charging was analyzed using a Hitachi amino acid analyzer.

As shown in Table 16, the sake of the present invention contains about 3 times as many amino acids as the conventional sake. Moreover, the content ratio of each amino acid is well balanced with almost no difference from the conventional one.

2) Vitamin measurement method: Thiamine in the upper tank sake in the two-stage preparation was measured by the thiochrome method.

As shown in Table 17, the liquor produced by the method of the present invention has a higher thiamin content than K-7 liquor. Since this thiamine is originally produced and stored only in yeast cells and is not leaked outside the yeast cells, it was confirmed that the yeast of the present invention has an excellent leaking ability to leak even such physiologically active components. To be done.

EFFECTS OF THE INVENTION The present invention provides a yeast having fermentability comparable to that of yeasts used for ordinary brewing of alcohol and having a large leakage amount of glutathione and other physiologically active components. In addition, by using this yeast, it is not possible to produce by the usual preparation method of sake and the conventional production method, which is produced in the cells,
Provided is a method for producing alcoholic beverages containing a large amount of various physiologically active ingredients to be stored and having excellent quality.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Akinori Kanda 2-70, Suimeidai, Kawanishi City, Hyogo Prefecture (72) Inventor Masaaki Hamachi 4-3-5 Chikushigaoka, Kita-ku, Kobe City, Hyogo Prefecture (72) Inventor Takemitsu Motoma 1-226 Mitsugaoka, Takarazuka City, Hyogo Prefecture (72) Inventor Yataro Nunokawa 2-26 Hirata Town, Ashiya City, Hyogo Prefecture

Claims (2)

[Claims] 1. A yeast which is deficient in vacuolar formation ability, has a substance transport mutation or osmotic sensitivity, and has brewing aptitude. 2. A method for producing alcoholic beverages, which comprises alcoholic fermentation using the yeast according to claim 1 to obtain alcoholic beverages having an increased content of physiologically active ingredients.