JPH074214B2 - Sake lees maturation method - Google Patents

Description

Detailed Description of the Invention

(Field of Industrial Use) The present invention relates to a method for aging sake lees. The amount of sake lees produced as a by-product during the production of sake has tended to increase in recent years due to the high quality of sake, but its consumption has fallen considerably due to a decline in demand for food. Therefore, development of a new method of using sake lees is desired. The present invention has been made to meet the demands of the industry, and relates to a completely new type of sake lees aging method using a temperature-sensitive autolyzing yeast. (Prior art and problems) Conventional sake lees are mostly pickled in lees. When ripening for pickling, it requires long-term ripening to complete the autolysis of yeast. However, the conventional aging method causes an unpleasant scent or coloring during the aging period, which makes it unsuitable for modern food taste. On the other hand, in order to diversify the uses of sake lees, it is necessary to secrete useful components in the yeast cells to the outside of the cells in a short period of time before unfavorable changes in aroma and coloring occur. (Problems to be solved by the invention) Therefore, the inventors of the present invention ripened yeast cells containing about 20% in sake lees by self-digestion by the simplest possible method and ripening by finishing the ripening in a shorter time. The subject was to obtain an aged sake lees without unfavorable changes. (Means for solving the problem) As a method for autolyzing yeast in sake lees, there is a method of leaving it for a long time, but if left for a long time, the yeast will self-digest, but the quality of the sake lees deteriorates. However, further corruption will not be able to achieve the initial purpose. Therefore, in order to self-digest yeast in a short time, it is necessary to force or actively self-digest, and as a method therefor, a method using an organic solvent such as toluene or ethyl acetate and a cell wall lysing enzyme are used. Although there is a method of using an enzyme, the organic solvent method is not preferable in terms of food hygiene and consumer safety, and the enzyme method is not preferable in terms of cost and quality. Therefore, the present inventors have made a major shift in the idea, and instead of treating the yeast in this way, paying attention to the yeast itself, and in the same manner as ordinary sake yeast, it grows normally at 10 to 30 ° C. and undergoes alcohol fermentation. (This temperature range is an allowable temperature range because it is allowable for yeast from the viewpoint of growth and alcohol fermentation), but it is a yeast that self-digests above a certain temperature, that is, a temperature-sensitive self The digested yeast was screened. However, despite all efforts, it was unsuccessful in screening the yeast of interest. Therefore, when mutation processing was performed, the allowable temperature range (10
~ 30 ℃) grows normally and performs alcoholic fermentation, but when it reaches a certain temperature or higher, it die and self-digests (the temperature is a non-permissible temperature because it is unacceptable for yeast, The range may be higher than the temperature at which the yeast is killed, but from the viewpoint of energy cost, quality deterioration prevention of sake lees, etc., 35 to 40 ° C. is preferable, and the temperature range is the non-permissible temperature range). In other words, the present invention has succeeded in creating a yeast that is affected by the above-mentioned various kinds of temperature (that is, a target temperature-sensitive yeast), and finally completed the present invention. Yeast mutant strain of interest, by physically or chemically mutating Saccharomyces cerevisiae in accordance with a conventional method, select a colony that can grow in YPD medium etc. Colony
By culturing in YPD medium at 37 ° C. and screening for yellow halo, the desired temperature-sensitive autodigestive yeast belonging to the genus Saccharomyces is obtained. An example thereof is the Saccharomyces cerevisiae J504 K-ts8 strain, which was constructed by mutating Saccharomyces cerevisiae J504 as a parent strain with a chemical mutagen. This bacterium is a temperature-sensitive autodigesting strain with a permissible temperature range of 10 to 30 ° C and a nonpermissive temperature of 35 ° C or higher. As described above, the non-permissive temperature is the temperature at which the temperature-sensitive autodigesting yeast created by mutation treatment of yeast self-digests, and the permissive temperature means that the yeast normally grows. This is the temperature at which alcohol fermentation is performed. These temperatures are different within the above range depending on the temperature-sensitive autolyzing yeast used. In addition, the strains created as described above were transferred to FERM P-1
Deposited as 1736. In order to carry out the present invention, first, using a temperature-sensitive self-digesting yeast belonging to the genus Saccharomyces (hereinafter also referred to as the present yeast) thus produced, sake brewing is carried out according to a conventional method to obtain sake moromi mash. If sake is separated and sake lees are obtained as a residue and stored at a non-permissible temperature,
It is aged sufficiently in a short time. Therefore, according to the present invention, unlike ordinary yeast, since it has the characteristic of using a temperature-sensitive autodigestive yeast, autodigestion is completed at an extremely low temperature and in a short time, and therefore, a short time without quality deterioration. In addition, the remarkable effect that the sake lees that are significantly reduced in energy cost are aged is performed. For example, in the case of K-ts8 strain, autolysis is completed by keeping the temperature at 35 ° C or higher for only 3 hours,
Aging for a short time is achieved. Examples of the present invention will be described below.

[Example 1: Construction of temperature-sensitive autodigestive yeast] Alkaline phosphatase was localized in the vacuole of sake yeast, and a mutant strain was selected that leaks this enzyme by changing the environmental temperature. . Saccharomyces cerevici pre-cultured in YPD medium (1% yeast extract, 2% polypeptone, 2% glucose)
After washing 7-α-2063, which is a haploid of ae J504, with 0.1 M phosphate buffer (pH 8.0), it was adjusted to 10 ⁸ cells / ml, and ethyl methanesulfonic acid (EMS) was adjusted to 0.3%. Add to be 30
It was treated at 60 ° C. for 60 minutes. The EMS was neutralized with 5% sodium thiosulfate, diluted with sterile water, spread on a YPD plate medium, and cultured at 25 ° C for 3 to 4 days. After culturing, replica on 2 YPD plates and cultivate one at 25 ℃ and the other at 37 ℃ for 1 day.
Colonies that could grow at 25 ° C but not at 37 ° C were isolated. Next, the isolated colonies were inoculated on a YPD plate, and soft agar (agar 0.5%, 0.05 M glycine buffer (pH 9.7), 10 mM pNP containing p-nitrophenyl phosphate (pNPP) was placed on it.
P) was overlaid. The plate was incubated at 37 ° C for 1 hour to isolate yellow halo-forming colonies,
This is a temperature-sensitive autolyzed yeast (K-ts8, Microtechnology Research Institute
1736).

Example 2: Properties of temperature-sensitive autolysed yeast K-ts8 was pre-cultured in YPD liquid medium at permissive temperature,
When cells in the logarithmic growth phase are transferred to a non-permissive temperature, K-ts8 immediately begins to undergo autolysis. The progress of this autolysis can be examined by the leakage of alkaline phosphatase, which is one of the enzymes present in intracellular vacuoles, into the medium. That is, alkaline phosphatase is an enzyme originally present in the cells of yeast, but is leaked out of the cell, that is, in the medium with the autolysis of yeast cells. Therefore, by measuring this enzyme activity inside and outside the cell, the degree of autolysis of yeast can be known. Figure this leakage pattern
Shown in 1. As is clear from Fig. 1, temperature-sensitive autolyzed yeast K
In -ts8, alkaline phosphatase activity is maintained in the cell (left figure) because autolysis does not occur in the permissible temperature range (25 ° C), but in the non-permissive temperature range (37 ° C).
Due to self-digestion, most of this enzyme activity leaks out of the cell (right figure) as the culture time elapses. On the other hand, the parent strain of K-ts8, 2063, undergoes almost no autolysis in any temperature range, and thus this enzyme activity is maintained in the cell. Considering the leakage pattern (Fig. 1) of this enzyme illustrated here, it was recognized that the autolysis was almost completed within 6 hours after the temperature was changed to the non-permissive temperature. The alkaline phosphatase activity was measured by the method of A. Schuur et al. In addition, the viable cell count was measured and the change is shown in FIG. As is clear from Fig. 2, temperature-sensitive autolyzed yeast K
In -ts8, almost no autolysis occurs within the permissible temperature range (25 ° C), so that the cells normally grow and the viable cell count increases even after the culturing time as in the parent strain 2063. On the other hand, in the non-permissive temperature range (37 ℃), autolysis occurred, so the cells rapidly diminished with the lapse of the culture time, and the viable cell count decreased logarithmically. Furthermore, intracellular protease is essential for autolysis, but its activity was measured using Azocol as a substrate according to the method of JHScott, and it was confirmed that intracellular protease activity was increased when this strain was cultured at a non-permissive temperature. (Fig. 3). As is clear from the results shown in Fig. 3, the temperature-sensitive autodigestive yeast K-ts8 showed a rapid increase in intracellular protease activity (left figure) with incubation time in the non-permissive temperature range (37 ° C). In the temperature range (25 ° C), the increase was as small as that of the parent strain 2063, which is consistent with the tendency of autolysis. On the other hand, the extracellular (right) activity is hardly observed in any case. Proteins, nucleic acids, amino acids, etc. forming cells are increased in the medium as a product of yeast autolysis. Fig. 4
Shows the increase in amino acids. As is clear from the results shown in Fig. 4, in the temperature-sensitive autodigestive yeast K-ts8, autodigestion occurs in the non-permissive temperature range (37 ° C), so a large amount of amino acid is produced in the medium depending on the culture time. , Within the allowable temperature range (25 ℃)
Similar to 2063, there is little autodigestion, so amino production is low. As described above, it was proved that the K-ts8 strain markedly increased the amino acid content in the medium when treated at 37 ° C., reaching 1.5 times that of the control parent strain, and rapidly undergoing autolysis. Was done.

[Example 3: Sake small-capacity test using K-ts8 strain] Small-capacity test using 300 g of white rice (variety Nipponbare) of 65% polished white rice for the purpose of comparing the performance of this strain in sake brewing with that of the parent strain I went. Table 1 shows the formulation of rice, koji and water used. The course of mash was investigated by carbon dioxide reduction. That is,
The fermentation course in the allowable temperature range (15 ° C) was reduced by carbon dioxide by a small-scale sake-brewing test using temperature-sensitive autodigestive yeast K-ts8 and its parent strain 2063 (carbon dioxide is discharged as the fermentation progresses. The decrease in mash weight due to) was investigated.
The results are shown in Fig. 5. As is clear from the results shown in Fig. 5, carbon dioxide is discharged according to the number of days of fermentation (fermentation days), and fermentation proceeds smoothly. However, the temperature-sensitive self-digesting yeast K-ts8 is more carbonic than the parent strain 2063. The rate of gas loss is slow, indicating a relatively slow fermentation process.

[Example 4: Aging method of sake lees and changes in components] About 300 g of the sake lees obtained in Example 3 was put in a 500 ml plastic container and stored at permissible temperature and non-permissible temperature.
For the purpose of investigating the state of autolysis of yeast contained in sake lees, 10 g of each sample was sampled over time, suspended in 50 ml of distilled water, and water-soluble components in the sake lees were extracted overnight at 4 ° C. went. After completion of the extraction, centrifugation was performed (8000 rpm × 20 minutes), and the supernatant was filtered through a 0.45 μm filter to use the filtrate as a sample for analysis. The samples were analyzed for alkaline phosphatase activity and the amount of S-adenosylmethionine according to the method of Tatenuma et al. That is, the sake lees obtained in Example 3 were mixed with each other in the allowable temperature range (25
℃) and non-permissive temperature range (37 ℃), the changes in the activity of alkaline phosphatase during autolysis of yeast contained in each sake lees were investigated. Figure showing the obtained results
6 shows. As is clear from the results shown in Fig. 6, alkaline phosphatase is leaked out from the sake lees obtained by using the temperature-sensitive autodigestive yeast K-ts8 in the non-permissive temperature range (37 ° C), and the activity is rapidly increased. . However, as the storage time becomes longer, the alkaline phosphatase is decomposed by the action of the protease present in the sake lees, and the activity is reduced. On the other hand, since the degree of autolysis is low in storage in the permissible temperature range (25 ° C), the activity of alkaline phosphatase is at a low level as in the parent strain 2063. That is, when K-ts8 was stored at a non-permissive temperature, alkaline phosphatase activity increased linearly and autodigestion proceeded rapidly as in the case of using YPD medium (Fig.
6). However, almost no increase in alkaline phosphatase activity was observed in the parent strain stored as a control or in the sample stored at a non-permissive temperature of K-ts8. When K-ts8 is stored at a non-permissive temperature, the activity decreases after 12 hours, which is considered to be due to the inactivation of the malt-derived protease and sake in sake lees and the temperature. Furthermore, the content of S-adenosylmethionine, which is said to accumulate in the vacuoles of sake yeast, in the sake lees was measured, and the results shown in FIG. 7 were obtained. As is clear from the results shown in FIG. 7, the sake lees obtained in Example 3 were allowed to have an allowable temperature range (25 ° C.) and a non-allowable temperature range (37
By storing at (° C.), changes in S-adenosylmethionine content were examined as a measure of elution of components contained in each sake lees into the lees by autolysis of yeast. (As a measure of the elution of yeast components due to self-digestion, changes in amino acid content were examined in Figure 4; however, since sake lees already contain a large amount of amino acids, yeast-specific components are shown here. As a result, the S-adenosylmethionine content was examined for change.) In the sake lees obtained by using the temperature-sensitive self-digesting yeast K-ts8, self-digestion rapidly occurred due to storage at a non-permissive temperature (37 ° C). -The adenosylmethionine content rises sharply. That is, S-adenosylmethionine was released in large amounts in sake lees after storage for 3 hours at a non-permissive temperature. In contrast, the S-adenosylmethionine content is at a low level similar to that of the parent strain, 2063, due to the low degree of autolysis when stored in the permissible temperature range (25 ° C). (Effect of the Invention) From the above results, this strain (K-ts8) was capable of rapidly completing autolysis in a sake lees at a non-permissive temperature. Further, it is considered that the time required for self-digestion is about 3 hours at the shortest, and it became possible to ripen the sake lees in a very short time by using this yeast.

[Brief description of drawings]

Fig. 1 shows changes in alkaline phosphatase activity, Fig. 2
Shows changes in the number of viable bacteria, Fig. 3 shows changes in protease activity, Fig. 4 shows increases in amino acid content in YPD medium, Fig. 5 shows reduction of carbon dioxide in mash, Fig. 6 shows alkaline phosphatase activity during aging of sake lees Fig. 7 shows changes in S-adenosylmethionine content, respectively.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Jin Shimohan 2-6-30 Takinogawa, Kita-ku, Tokyo Inside the National Tax Agency Brewing Laboratory

Claims (5)

[Claims] 1. A method for aging sake lees derived from sake mash produced by a conventional method using a temperature-sensitive self-digesting yeast belonging to the genus Saccharomyces, which comprises performing a short-term aging by applying a temperature at which the yeast self-digests. A method for aging sake lees, which is characterized by being performed. 2. The method according to claim 1, wherein the temperature is 35 ° C. or higher. 3. A temperature-sensitive autolyzing yeast belonging to the genus Saccharomyces is Saccharomyces cerevisiae J504 K-ts8.
(Saccharomyces cereviciae J504 K-ts8), The method of Claim 1 or Claim 2 characterized by the above-mentioned. 4. A temperature-sensitive autolyzing yeast belonging to the genus Saccharomyces is Saccharomyces cerevisiae J504 (Saccha
The method according to claim 3, which is produced by subjecting romyces cereviciae J504) to a mutation treatment. 5. A temperature-sensitive autodigestive yeast belonging to the genus Saccharomyces, Saccharomyces cerevisiae J504 K-ts8 (Saccharo Saccharomyces cerevisiae J504 K-ts8 (Saccharo).
The method according to claim 4, which is myces cereviciae J504 K-ts8, FERM P-11736).