JP3057557B2 - New yeasts and their uses - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hybrid yeast having a novel ginjo aroma and a method for producing a beer having a ginjo aroma using the yeast.

[0002]

2. Description of the Related Art In general, yeast used for beer brewing has a particularly high ability to take in and ferment maltose, which is a main component of wort sugar, as compared with other brewing yeasts such as sake yeast and wine yeast. Is characterized by low production of higher alcohols and esters (Food Industry February 1987 6
0-68). As a method of increasing the aroma component in beer, adjustment of wort aeration, change of the sugar composition of wort, change of fermentation temperature, and the like are known, but none of these methods specifically increase the ginjo aroma. .

[0003] As applications for obtaining novel yeasts having a high aromaticity by hybridizing haploid yeasts, Japanese Patent Application Laid-Open Nos.
There is 7-95877. However, the former aims at crossing a haploid strain of sake yeast to obtain a hybrid yeast that can be used for sake production. On the other hand, the latter aims to obtain a hybrid yeast which can be used for wine production by crossing a wine yeast with a yeast called Saccharomyces bayanus. On the other hand, in order to improve the aroma of beer, sake yeast and beer yeast are crossed, and there is no report on obtaining yeast having high aromaticity and fermentation characteristics and flavor as beer.

[0004]

Generally, isoamyl acetate and ethyl caproate, which are the main components of ginjo aroma contained in sake, have a fruity aroma and a gorgeous aroma, and give a mild taste when drunk. However, sake yeast has low fermentability of maltose, and no satisfactory beer can be obtained in a fermentation test using wort. Therefore, an object of the present invention was to brew a beer having a ginjo aroma utilizing the characteristics of sake yeast.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to obtain a yeast which imparts ginjo scent to beer. The present invention relates to a sake yeast, a monospore isolate of Association 9 (IFO 2377) and a brewer's yeast. is a cross yeast having sake aroma by crossing the single spore isolates of X yeast is one.

[0006] The present invention is also a method for producing a beer having a ginjo aroma using the obtained hybrid yeast. Sake yeast, Saccharomyces cerevisiae, which is a sake yeast, can be purchased from the Japan Brewing Association or the Fermentation Research Institute. The Association No. 9 yeast is a yeast widely used in brewing Ginjo sake as a yeast that produces a large amount of Ginjo aroma.

[0007] Among the hybrid yeast obtained by the exchange miscellaneous, useful strain Saccharomyces cerevisiae (Saccharomyces ce
revisiae) KS7 and Saccharomyces cerevisiae KS11, which have been deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (Accession No .: FERM P-15799, Accession No .: FERM P-1580).
0).

In the present invention, crossing is carried out on the basis of the monospore isolate of the No. 9 yeast and the monospore isolate of the yeast X, which is a kind of brewer's yeast. The hybrid yeast having the following is selected. In addition, the selected hybrid yeast having ginjo aroma was used for beer brewing and tested for ginjo aroma and sensory characteristics, and it was confirmed that the yeast was actually effective for beer brewing.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION A monospore isolate of Kyokai No. 9 yeast is obtained by culturing Kyokai No. 9 yeast in a normal sporulation medium and then using the method of Tani et al. (J. Ferment. Bioengineering, 69 (3)). , 1
84 (1990)). X yeast, which is one type of brewer's yeast, can be obtained by the same method for a monospore isolate.

As a hybridization method, both monospore isolates are crossed and inoculated on a solid medium such as a YPD agar medium.
After culturing, a method can be used in which cells grown at the intersections are obtained, and a single colony isolated from the obtained cells is selected as a diploid from those capable of forming spores. Selection of the excellent hybrid strains obtained can be performed by measuring fermentability, flavor components, sensory evaluation, and the like. The fermentability can be determined, for example, by measuring the weight difference between the residual extract at the end of fermentation and the fermented liquid before and after fermentation. For the fragrance component, for example, at the end of fermentation, measure the fragrance component such as ethyl acetate, isobutanol, isoamyl acetate, isoamyl alcohol, β-phenethyl acetate, β-phenethyl alcohol, ethyl caproate, and ethyl caprylate in the fermentation broth. Can be determined. Further, as a sensory test, for example, it is possible to determine a superior strain by inspecting beer brewed on a trial basis for ginjo aroma, taste of aroma, and taste as a beer.

The hybrid yeast having a ginjo aroma obtained in the present invention can be used in a normal beer production method to produce a beer having a gorgeous aroma like sake.

[0012]

The present invention will be specifically described below with reference to examples. 1. Acquisition of single spore isolate of Association No. 9 yeast Association No. 9 yeast was transferred to YPD agar medium (2% Peptone, 1% Yeast
Extract, 2% glucose, 1.5% agar) for 2-3 days
After preculture at 25 ° C, Maclay sporulation agar medium (0.82
% Sodium acetate, 0.1% glucose, 0.25% Yeast Ext
ract, 0.37% potassium chloride, 0.146% magnesium sulfate heptahydrate, 1.5% agar) and allowed to stand at 25 ° C. for 7 days to form spores. For spore staining, yeast cells were smeared on a slide glass, air-dried, flame-fixed, and stained with a 7.6% (W / V) malachite green solution for 10 minutes. afterwards,
Counterstaining was performed with a 0.25% safranin solution, and observed with a microscope.

[0013] The random spore decomposition is performed by the method of Tani et al.
According to rment. Bioengineering, 69 (3), 184 (1990)), the spores were adjusted to a cell wall lysis buffer (0.6 mg / ml Zymolyase20T, 0.01 M 2-mercaptoethanol, 0.6 M chloride) at a concentration of 10 ⁷ to 10 ⁸ cells / ml. Potassium, 66.7 mM potassium phosphate buffer (pH 7.5)) and reacted at 37 ° C. for 1 hour. After centrifugation at 15,000 rpm for 5 minutes with a microcentrifuge, the supernatant was discarded and suspended in sterile water. After repeating this operation twice, heat treatment was performed at 60 ° C. for 20 minutes to selectively kill only vegetative cells. Apply to YPD agar medium,
From the colonies that grew at 25 ° C, those that were not stained by spore staining were selected as spore-degrading strains, and
Got the strain.

Next, Saccharomyces cerevi having a conjugating ability
siae α-type strain or Saccharomyces cerevisi with conjugating ability
The ae type a strain was tested for conjugation ability with 21 strains (α
(Type 17 strains, type a 4 strains). The resulting hybrid α-type strains were assigned strain numbers 1 to 17, and the a-type strains were assigned strain numbers 18 to 21.

2. Hybridization with monospore isolate of brewer's yeast 21 monospore isolates (α type 17 strain and a type 4 strain) of Association No. 9 yeast obtained and monospore of brewer's yeast X yeast having conjugating ability 7 isolates (type 4 strains, strain numbers A, B, C,
D and α type 3 strains, strain numbers E, F and G) were inoculated by crossing on a YPD agar medium, cultured at 25 ° C., and from the colonies grown at the intersection, those having a spore-forming ability were determined. As a result, 61 hybrids were obtained. Table 1 shows the status of obtaining crossed strains based on the result of crossing between the single spore isolate of brewer's yeast X yeast and the single spore isolate of Association No. 9 yeast. The crossed strain was obtained in the column where each yeast crossed. For those that could not be obtained, the stock numbers were shown, and for those that could not be obtained, they were shown by x.

[0016]

[Table 1]

3. Judgment of excellent strains by fermentation test in beer brewing In brewing beer, yeast used must have strong wort fermentation power. Therefore, for the purpose of selecting a strain excellent in wort fermentability from the obtained 61 hybrid strains,
The residual extract at the end of fermentation of the hybrid strain and the weight loss of the fermented liquor before and after fermentation were examined on a 100 ml scale. That is,
After the obtained hybrid strain was cultured in a 10 ml YPD liquid medium with shaking at 25 ° C. for 3 days, 0.5 ml of the culture was inoculated into 50 ml beer wort, allowed to stand at 12 ° C. for 5 days, and cultivated. The main culture was performed using 100 ml beer wort and the initial yeast number was 10 × 10 ⁶ cells /
The mixture was left to stand and cultured at 12 ° C. for 6 days. The strength of the fermentability was determined by measuring the weight difference between the residual extract at the end of fermentation and the fermented liquid before and after fermentation.

The results are shown in FIG. 1 with the weight loss before and after fermentation in each test on the horizontal axis and the residual extract at the end of fermentation on the vertical axis. During beer fermentation, sugars are converted by yeast into alcohol and carbon dioxide. Therefore, it can be said that the larger the weight loss of the fermentation liquor before and after fermentation, the stronger the maltose fermenting power. In addition, it is expected that the less residual extract at the end of fermentation, the stronger the maltose utilization ability.
As shown in FIG. 1, the Association No. 9 yeast was located at the upper left of the graph because maltose had low fermentative power and assimilation ability.
In addition, the brewer's yeast X yeast is located relatively lower right,
It was found that the hybrid strain obtained this time exhibited fermentative and assimilating ability of maltose equivalent to or higher than that of yeast X.

Most of the 61 hybrid strains tested showed a large decrease in extract during fermentation and a decrease in weight before and after fermentation as compared to Kyokai No. 9 yeast, and their properties were close to those of brewer's yeast X yeast. Became clear. From the above results, among the strains showing a decrease in the extract equivalent to or higher than that of the brewer's yeast X yeast and a decrease in weight before and after fermentation, 21 strains having a good aroma of the fermentation liquor (strain numbers: 103, 104, 105, 10)
7, 113, 114, 201, 202, 206, 20
7, 301, 302, 303, 308, 309, 31
0, 311, 312, 314, 401, 504).

4. Aroma and sensory characteristics of beer brewed using the hybrid strain Next, the selected 21 strains were subjected to a 1-L fermentation test to test the ginjo aroma and other sensory characteristics. 10 ml of hybrid strain, 100 m
l and 500 ml to scale up and grow sequentially to collect cells, and the initial yeast count in 1 L beer wort is 15 × 10 ⁶ cells / ml
And fermented at 15 ° C. for 6 days using a 1 L cylinder. The aroma component content (unit: ppm) of the fermentation liquor at the end of fermentation was measured. As a result, in all the hybrid strains, the decrease of the extract during the fermentation process was as good as that of the brewer's yeast X yeast, and it was confirmed that the wort fermentability of the Association No. 9 yeast was improved by the hybridization. Table 2 shows the aroma component content of the obtained fermentation liquor. In the table, a large number of hybrid strains that produce more aroma components than the brewer's yeast X yeast were found. Among them, ethyl caproate and isoamyl acetate, which are generally considered to have a high contribution to ginjo scent, are considered. The increase was significant.

[0021]

[Table 2]

Table 3 shows the results of the sensory evaluation of the hybrid strain beer.
It was shown to. In the sensory evaluation, 6 to 9 panelists evaluated ginjo aroma, taste of aroma, and taste as a beer on a five-point scale out of five. As is evident from the results, the score of Ginjoka of beer fermented using the hybrid strain is high,
Many comments from panelists were found to be attributed to an increase in the content of aroma components such as ester aroma.

[0023]

[Table 3]

From the above results, two strains, 114 and 207, were selected, which had a high score for Ginjo aroma, and also had good scores for aroma preference and beer preference. 5. Pilot plant test Next, two selected plants were subjected to test brewing at a pilot plant scale close to actual beer brewing.
The method uses 100 L of beer wort and the initial yeast count is 10 × 10 ⁶
cells / ml, and fermented at 15 ° C for 6 days. FIG. 2 shows the decrease in the extract with the progress of fermentation. FIG. 3 shows the change in the number of floating yeasts during the fermentation process.
The fermentation progress in terms of the remaining extracts and yeast counts of 114 and 207 showed the same tendency as that of the control brewer yeast X yeast, and no inferiority was observed even when compared with the fermentation state of the brewer yeast X yeast. Was.

Regarding the flavor components of the hybrid strain brewed beer,
FIG. 4 is a radar chart showing the amounts of the fragrance components produced by the hybrid strain when the respective fragrance component production amounts of the beer yeast were set to 1. FIG. 4 shows how many times the higher alcohols or esters, which are usually measured when evaluating the flavor component content of beer, produced the flavor component when compared with X yeast. In both cases, an increase in ethyl caproate (apple-like odor) and isoamyl acetate (banana-like odor), which are the main components of Ginjo scent, was observed. In particular, 114 strains produced a lot of isoamyl acetate and 207 strains produced a lot of ethyl caproate. I tended to.

The actual analysis values are shown in Table 4. As shown in Table 4, isoamyl acetate of 114 strains was 3.2 ppm, and ethyl caproate of 207 strains was 1.3 ppm. .

[0027]

[Table 4] *: Harrison et al. J. Inst. Brew., 76 (1970) 486

FIG. 5 shows the results of sensory tests on beer using the hybrid strains. As for the ginjo aroma, all strains have higher scores than the brewer's yeast X yeast, and the results of the aroma component content are lower. It was thought to reflect well. Regarding scent preference and beer preference, 114 strains and 2
In the 07 strain, a score equivalent to that of X yeast of brewer's yeast was obtained. When the fermentation process was repeated, the fermentation progress, the aroma component content, and the results of the sensory test showed the same tendency in all strains.
Strain and 207 strain obtained good results with good reproducibility, so both strains were finally selected, and a brewer's yeast hybrid strain having Ginjo scent, Saccharomyces cerevisiae, was selected.
visiae) KS7, Saccharomyces cerevisiae (Saccha
romyces cerevisiae) KS11.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the wort fermentability of a hybrid strain in an example, with the horizontal axis representing weight loss before and after fermentation in each test and the vertical axis representing residual extract at the end of fermentation.

FIG. 2 is a graph showing the number of extracts (days) on the abscissa and the extract (%) on the ordinate, regarding the decrease in extract due to the progress of fermentation in Examples.

FIG. 3 shows changes in the number of floating yeasts during the fermentation process in the examples, with the number of yeasts (× 10
⁶ cells / ml) on the vertical axis.

FIG. 4 is a radar chart showing the generation amount of the fragrance component of the hybrid strain when the generation amount of each fragrance component of the brewer's yeast is set to 1, with respect to the flavor component of the hybrid strain brewed beer in the example.

FIG. 5 is a graph showing a result of a sensory test of beer using a hybrid strain in an example.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) C12N 1/16-1/19 C12C 11/02

Claims (3)

(57) [Claims] 1. Saccharomyces cerevisiae , a hybrid yeast having a novel ginjo scent obtained by crossing a single spore isolate derived from sake yeast and a single spore isolate derived from brewer's yeast.
Visier (Saccharomyces cerevisiae) KS7 (Industrial technology
Institute of Biotechnology, Industrial Technology Research Institute accession number FERM P-15799). 2. A monospore isolate derived from sake yeast and brewer's yeast.
Obtained by crossing with a monospore isolate of origin
Saccharomyces cereal, a hybrid yeast with a new ginjo scent
Visier (Saccharomyces cerevisiae) KS11 (Industrial technology
Institute of Biotechnology, Industrial Technology Research Institute accession number FERM P-15800). 3. A novel ginjo scent according to claim 1 or 2.
Ginjo scent, characterized by the use of hybrid yeast
Beer production method.