JPH067152A - High temperature-sensitive strain of yeast for practical brewing and breeding method therefor or production of liquors and other brewed foods with the same - Google Patents

Abstract

PURPOSE:To obtain the galactose-unassimilating yeast capable of being rapidly autodigested under a high temperature condition to give aged sake (rice wine) residues in a short time by screening a deoxy-galactose-resistant strain from practically used yeasts. CONSTITUTION:2-Deoxy-galactose (hereinafter referred to as 2-DG)-resistant stain is screened from yeasts used for practical brewings to provide the objective yeast. Concretely, a high temperature-sensitive autodigestive yeast gal 31 strain (FERN 12605) is obtained as follows: preliminarily culturing e.g. Saccharomyces cerevisae (No.7 strain of the Sake Yeast Association) in a YNBD medium, mainly culturing in a 2-DG flat plate medium, inoculating a generated large colony on a galactose medium and on a YNBD medium, obtaining a galactose- unassimilating stain, screening a stain not multiplying in the YNBD medium at 37 deg.C, and subsequently culturing the strain in a 2-DG flat plate medium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention has a higher temperature sensitivity than a 2-deoxy-galactose (hereinafter referred to as 2-DG) resistant strain of yeast (referred to as practical brewing yeast) used for brewing foods and drinks actually. The present invention relates to a practical brewing yeast, breeding method thereof and use thereof.

The engineered strain of the present invention exhibits the property of resistance to 2-DG, which is an analogue of galactose, and cannot utilize galactose as a carbon source.

By subjecting a practically brewed yeast to a very mild mutation treatment of 2-DG resistance, new properties such as high temperature sensitivity can be added without impairing practical characteristics. The acquired strain can be used as a labeled strain for cell fusion, transformation, etc. Therefore, the present invention can be used for breeding a practical brewer's yeast having unprecedented properties by utilizing high temperature sensitivity and the like, and greatly contributes to the production of alcoholic beverages and other brewed foods.

[0004]

2. Description of the Related Art It has been shown that a high temperature sensitive strain for aging sake lees, which is a by-product of sake brewing, is effective by using a haploid high temperature sensitive strain isolated from a commercial brewer's yeast. (Kitamura et al. (Jyokyo, 86, 605 (1991))

However, the practical brewer's yeast (Saccharomyces cerevisiae) usually used for the production of sake, shochu, fruit wine, beer, whiskey, etc. is a polyploid with diploid or higher polyploidy. , It was not possible to obtain a thermosensitive strain that is a recessive mutation without impairing its brewing characteristics.

[0006] On the other hand, in the laboratory strain yeast, it is known that mutant strains such as gal4 which is a galactose assimilation control gene show various phenotypes as well as regulation of galactose assimilation. Was known to be sensitive to high temperatures. In addition, 2-D, which is an analogue of galactose
Strains resistant to G are mainly genetically gal1
And gal4 mutant strains.
(T. Patt (Mol. Cell. Biol., 4,
994 (1984)))

[0007]

[Problems to be Solved by the Invention and Means for Solving the Problems]
From the above viewpoints, the present inventors obtained a strain in which galactose-assimilating ability was deleted from a practical brewer's yeast by positive selection of 2-DG resistance. A high temperature sensitive strain was selected from these mutants. The characteristics of this high temperature sensitive strain were examined and it was found that it causes autodigestion at high temperature. As a result of the growth test against temperature, the sake brewing test, and the aging test of sake lees using the thus obtained practically brewed yeast, it was confirmed that the yeast has useful properties, and the present invention has been completed.

[0008]

Example 1 Construction of galactose non-assimilating and high temperature sensitive strain by 2-DG resistance Yeast (Saccharomyces cerevisiae)
e) uses 2-DG, which is an analog of galactose,
A high temperature sensitive strain was selected because the resistant strain became high temperature sensitive. YNBD medium (yeast nitrogen source medium 0.67
%, Glucose 2%), the Sake Yeast Society No. 7 strain was pre-incubated with sterilized water, and then washed with 2-DG plate medium (2-DG0.02
%, Yeast nitrogen source medium 0.67%, glycerol 3%, agar 2%) and cultured at 25 ° C. for 5 days. After culturing, large colonies that appeared at a frequency of about 10 ⁻⁶ were inoculated into a galactose medium (yeast nitrogen source medium 0.67%, galactose 2%, agar 2%) and YNBD medium, and about 10
^The galactose non-assimilating strain was obtained at a frequency of ^-2 . Furthermore, from the acquired strains, those that did not grow in YNBD medium at 37 ° C could be acquired at a frequency of 1/2. However, since the mutant strain obtained here has a high reversion rate of several%, it was inoculated on a 2-DG plate medium and further cultivated at 25 ° C. 8 times to obtain a stable remutation rate of 0.1% or less. 4 high temperature sensitive strains were acquired. First, the nature of the acquired stock
Shown in the table. Of these, the gal31 strain was used as a high temperature sensitive autolyzed yeast (Microtechnology Research Institute No. 12605).

[0009]

Example 2 Properties of High Temperature Sensitive Autolyzed Yeast Gal31 was placed in a YNBD liquid medium at an allowable temperature (35
By pre-culturing at a temperature not higher than 0 ° C.) and transferring the cells in the logarithmic growth phase to a non-permissive temperature, the auto-digestion action starts immediately. The progress of this autodigestion action can be examined by leakage of alkaline phosphatase, which is one of the enzymes existing in vacuoles existing in cells, into the medium. Considering the leakage pattern of this enzyme (Fig. 1), the autolysis is almost completed within 10 hours after shifting to the non-permissive temperature. In addition,
Alkaline phosphatase activity is determined by A. Schurs et al. (J. Gen. Microbiol., 65, 2
91 (1971)), and can be measured by the decomposition activity of p-nitrophenyl phosphoric acid (hereinafter, pNPP).

[0010] Number of viable cells due to the autolysis represents a logarithmic decline, after 16 hours 10 ^- decreased to ³ or less.
(Fig. 2) Moreover, although protease activation is required for autolysis, J. H. Scott's method (J. Bac
Teriology, 142, 414 (1980)), and the activity was measured using azochol as a substrate. As a result, an increase in intracellular protease activity was confirmed under a non-permissive temperature. (Fig. 3)

A growth test of this gal31 at various temperatures was carried out using a biophoto recorder (TOYOKAGAKU).
TN-112D). The results are shown in FIG. This is a measurement of turbidity change at 660 nm in YPD liquid medium at each temperature. The strain did not grow at a non-permissive temperature of 37 ° C or higher, and the optimum growth temperature was 27 ° C. This temperature is 32 ° C of Association No. 7
The temperature was lower than that of the above, and the growth in the low temperature range of 20 ° C. or lower tended to decrease rapidly. K-7 at 18 ℃
Compared with, the growth was delayed by about 40 hours before reaching the maximum cell density.

The fermentation characteristics of this strain are shown in FIG. The fermentation liquid used was 10% glucose, 1% peptone, 0.5% yeast extract, the initial inoculum count was 10 ⁶ and 10 ⁸ / ml, and the fermentability was measured by reducing the amount of carbon dioxide at a constant 15 ° C. . Initial number of bacteria 1 that is hardly affected by bacterial growth
In 0 8 ^/ ml of test category showed parent equivalent fermentable, but the classification in 10 6 / ^ml out effect of inhibition of growth at a low temperature, slightly delayed tended.

[0013]

[Example 3 Sake small-brewing brewing test using gal31] Based on the mixing formulation shown in Table 2, the characteristics of this strain in sake-brewing were compared with those of the parent strain, and 70% polished white rice (variety Nihonbare) was used.
A small batch test was conducted using 150 g of total rice. Dance is 2
Yeast was added to the addition water so as to be 10 ⁷ / ml. However, as a result of the proliferation test, since the strain had the property of suppressing the proliferation at low temperature, the standard type with a constant temperature of 15 ° C and the first half of the fermentation was 20 ° C (up to the 5th day after the end of the second half).
FIG. 6 shows the fermentation process in two sections, that is, the warm type before and after 5 ° C. The strain showed the same fermentability as that of the parent strain in the pre-rapid and post-warm type, and there was almost no difference in the analytical values of the produced sake. (Table 3) However, in the standard course, the fermentation delay and the change in the produced liquor component, which are considered to be due to the reduced growth of the strain in the mash, were observed.

[0014]

[Example 4] Aging method of sake lees and changes in components About 150 g of the sake lees obtained in the warm type before and after the rapid heating of Example 3 were transferred to a beaker, and allowed temperature (25 ° C) and non-permissible temperature (37).
(° C), and 10 g of each sample was sampled over time to examine the state of autolysis of yeast. 50m sample
The suspension was suspended in 1 l of distilled water and the water-soluble components in the sake lees were extracted overnight at 4 ° C. After extraction, centrifuge (8,000 rpm
x 20 minutes), the supernatant was filtered through a 0.45 μm filter, and the filtrate was used as a sample for analysis. Chemo-copak Nucleosil 1 as an analytical column
S-by high performance liquid chromatography using OSA
The amount of adenosylmethionine was quantified. A large amount of S-adenosylmethionine, which is said to be accumulated in yeast vacuoles, was released in sake lees after 6 hours of storage at a non-permissive temperature. (FIG. 7) In addition, leakage of alkaline phosphatase and an increase tendency of protease activity were observed, and it was considered that aging of sake lees was in progress.

[0015]

EFFECTS OF THE INVENTION From the above results, the strain (gal31) of the present invention has a slightly reduced growth property under low temperature conditions, but has a fermentability equivalent to that of the parent strain due to the progress of warm type before and after the rapid heating, and has high temperature conditions. It had the property of completing self-digestion immediately below. Further, the time required for self-digestion is considered to be about 6 hours at the shortest, and the use of this yeast made it possible to ripen the sake lees in a short period of time.

Further, the effect of self-digestion of yeast on the fermentation and flavor of the brew is large, and fermentation is carried out by a high temperature sensitive strain, and non-permissive temperature treatment is carried out at the end of fermentation, whereby early ripening and components by self-digestion of yeast are carried out. It is possible to give a change or a change in flavor.

Furthermore, it was possible to obtain a galactose-requiring strain and a galactose-requiring and high temperature-sensitive strain as the present strain and the mutant strain obtained by this selection method. T. P
According to att et al., the former is a gal1 mutant strain, the latter is
It is highly likely that it is a 4 mutant strain. The strain thus obtained can be used as a host for molecular breeding as a labeled strain at the time of cell fusion.

[0018]

[Brief description of drawings]

Fig. 1 shows the progress of alkaline phosphatase activity that leaks into and out of the cells, Fig. 2 shows the change in the number of normal bacteria under permissive and non-permissive temperatures, and Fig. 3 shows both inside and outside the cells. Figure 4 shows the progress of leaking protease activity, Figure 4 shows the growth at various temperatures, Figure 5 shows the progress of fermentation, Figure 6 shows the progress of fermentation with sake, and Figure 7 shows the maturity in sake lees. -Figures seen from the course of adenosylmethionine leakage.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tetsuya Hoshino 889 Kasori-cho, Chiba Prefecture Chiba Prefectural Industrial Research Institute

Claims (2)

[Claims] 1. A method for producing a galactose non-assimilating and high temperature-sensitive practical brewer's yeast from a 2-deoxy-galactose resistant strain. 2. A method for producing alcoholic beverages and other brewed foods, which comprises using the high-temperature-sensitive practical brewer's yeast of claim 1.