JP2663095B2 - New brewer's yeast - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to Saccharomyces cerevisiae, which has a high growth rate in barley shochu mash and has alcohol tolerance.
revivale).

[0002]

2. Description of the Related Art Generally, conditions required for yeast for shochu are as follows.
It is mentioned that it has sufficient ability to produce alcohol even in the presence of a large amount of citric acid and at a high temperature of 25 to 35 ° C. Sake yeast, beer yeast, wine yeast, and the like cannot sufficiently satisfy these conditions, and thus yeasts specially selected for shochu are used. At present, as yeasts for shochu, Kagoshima yeast and Miyazaki yeast are common and used in most breweries. On the other hand, after the shochu boom, shochu using various raw materials was commercialized, but now barley shochu,
Potato shochu, buckwheat shochu, rice shochu and the like make up the majority, and barley shochu in particular has increased sharply, reaching about 50% of the share of shochu type B. Shochu can be roughly divided into two types depending on the raw material used. That is, shochu using barley koji, ie, barley shochu, and shochu using rice koji, ie, rice shochu, potato shochu, and buckwheat shochu can be roughly classified. However, white koji mold is used for barley koji and rice koji as well.

[0003]

Recently, Kagoshima yeast and Miyazaki yeast are also commonly used for barley shochu, whose share has been expanded. It is low compared. This has been attributed to the raw material properties of barley having poor solubility. However, as a result of the research of the present inventors, it has been found that yeast is also a factor. That is,
Rice koji soup with white yeast mold as seed yeast,
As a result of a fermentation test using barley koji juice, it was found that the currently used yeast has a lower raw material utilization rate in barley koji than in rice koji. As mentioned above,
Despite the demand for yeasts suitable for barley shochu, satisfactory strains have not yet been obtained.

[0004]

Means for Solving the Problems The present inventors conducted a search to isolate a high alcohol-producing bacterium having a high growth rate in barley koji juice and having alcohol tolerance, and obtained a yeast obtained by subjecting Kagoshima yeast to a mutagenesis treatment. Saccharomyces cerevisiae (S. cerevisiae), which grows rapidly even in barley koji soup and has excellent growth ability even at low temperatures, has alcohol tolerance.
iae).

[0005] It is an object of the present invention to provide a novel useful microorganism having a high growth rate in barley shochu mash and having alcohol tolerance.

[0006]

The novel microorganisms provided by the present invention, which have a high growth rate in barley shochu mash and are highly resistant to alcohol, are strains belonging to Saccharomyces cerevisiae described below. .

[0007] (i) TTC staining test according to the method of Furukawa, Akiyama (Toshio Furukawa, Yuichi Akiyama: 37, 398 (1963)), that is, the cells were appropriately diluted (about 200 per plate). ) On the colonies plated on the lower medium at 30 ° C for 2 days.
After dissolving the agar to about 45 ° C., gently overlay it. After hardening, leave it at 30 ° C. for 2 to 3 hours to observe the staining of the colonies. When the colony is stained, it shows pink color and (ii) the method of Mizoguchi and Fujita. (Haruhiko Mizoguchi, Eishin Fujita: Fermentation, 59, 185 (19
81)). C. Staining test, that is, the cells were appropriately diluted (to approximately 200 per plate), dissolved on a colony which was cultured in a TTC lower layer medium at 30 ° C for 2 days, and dissolved on a soft agar for upper layer at approximately 45 ° C. And then gently layer, and after hardening, leave at room temperature for 30 minutes.
It is a new strain belonging to Saccharomyces cerevisiae, which is characterized by showing white color when the staining of the colony is observed.

The new strain provided by the present invention has the following mycological properties.

(A) Morphology of bacteria when cultured at 30 ° C. for 2 days using YM medium Size of vegetative cells: 4 to 9 μ Shape of vegetative cells: egg-shaped Growth form: budding

(B) Morphology of colony when cultured on YM agar plate medium at 30 ° C. for 2 days: Morphology: Circular protuberance: On convex circle Perimeter: Smooth Size (diameter): 2-3 mm Color tone: White Opaque surface: smooth and shiny

(C) Utilization of carbon source: Glucose, galactose, sucrose, D-maltose, trehalose, raffinose, inulin, mannose, ethanol, glycerol, lactic acid, citric acid, fructose and arabinose are used. Cellobiose, lactose, melibiose, melezitose, starch, α-methylglucoside, D-xylose, D-ribose, L-rhamnose, meso-erythritol, D-sorbitol,
D-mannitol, salicin, succinic acid and inositol do not assimilate.

(D) Vitamin requirement: Calcium pantothenate and biotin are required. It does not require pyridoxine, inositol or thiamine.

The following describes how the present inventors have found BAW-6, a new strain belonging to Saccharomyces cerevisiae, of the present invention.

The present inventors carried out a mutagenesis treatment on Kagoshima yeast as described below in order to isolate a high alcohol-producing bacterium having a high growth rate in barley koji juice and having alcohol tolerance. Among the yeasts obtained as a result of the treatment, it has excellent alcohol tolerance, its growth ability, especially at low temperatures, stands out, and its bacteriological properties are distinctly different from conventional Kagoshima yeast. The strain of the invention was obtained.

Hereinafter, the process of obtaining the novel strain of the present invention will be described.

1. Mutation treatment and acquisition of useful yeast

[Mutation treatment] One platinum loop of commercially available Kagoshima yeast (Ko) was inoculated into 2 ml of YPD medium and cultured with shaking at 30 ° C. overnight. The obtained cells were added to 10 ml of YPD medium.
Inoculate 00 μl, perform shaking culture at 30 ° C. overnight, collect cells in the logarithmic growth phase (4 to 10 × 10 ⁷ cells / ml) by centrifugation, and wash twice with the same amount of sterile water. did.
The washed cells were placed in a 0.1 M phosphate buffer (pH 7.
0) Suspended in 10 ml, EMS (ethyl meth
anesulfonate) 0.3 ml, add 30 ml
Mutation treatment was performed by gently shaking at a liquid temperature of 40 ° C. for 40 minutes.

[Acquisition of Useful Yeast] The mutated cells were centrifuged, and the collected cells were washed once with 10 ml of a 5% sodium thiosulfate solution and twice with sterile water, and then with 10 ml of sterile water. Suspended in water. 400 μl of the resulting suspension
[Including about 2 × 10 ⁷ viable cells. The number of the cells was 10 m of cells having a survival rate of 50% by the EMS treatment.
400 μl of l suspension (2-5 × 10 ⁸ cells)
(0.4 / 10). Was introduced into 10 ml of Bllg ° 8 10 ml barley koji containing 8% alcohol and cultured at 30 ° C. for 7 days. The obtained culture was diluted with sterile water to 10 ³ , 1 ml of the culture was embedded in a YPD agar medium, cultured at 30 ° C. for 3 to 4 days, and 43 colonies that appeared were picked on a YPD slant medium. Next, the obtained fishing strain was cultured in 10 ml of YPD medium at 30 ° C. for 2 days, and 100 μl of the suspension was added to Bllg containing 9% alcohol.
Cultivation was carried out at 30 ° C. for 7 days in 10 ml of barley koji juice at 9 ° C. The same procedure was repeated to isolate 28 strains of colonies, which were further repeated in the same manner as described above using 10 ml of barley koji juice containing 10% alcohol at Bllg ° 10. Ten strains of yeast having resistance were obtained. Furthermore, a fermentation ability test was performed using these strains, and three strains having the best growth, fermentation ability, and alcohol tolerance were obtained. The temperature suitability of these three strains was examined, and the strain (BA
W-6) was isolated.

2. Bacteriological properties The strain BAW-6 isolated in the above (1) was obtained from both the parent strain Kagoshima yeast (Ko), the Miyazaki yeast (MK) known as yeast for shochu, and the association shochu yeast SH-4. To determine if they are distinct,
Observations were made on mycological properties (morphological and physiological properties).

2- (1). Morphological properties Using YM medium, K o, MK, SH-4 and BAW-6
Of each of the four yeasts at 30 ° C. for 2 days
After cultivation, the obtained product was observed with an optical microscope. View
The findings are summarized in Table 1. As is clear from Table 1
In addition, all vegetative cells are 4-8 μm in size and oval
Met. And, on YM agar medium, any strain
A lustrous white colony was formed.

2- (2). Physiological properties

A. Carbon source utilization was tested by the replica method using a solid medium. sand
About 1 liter of medium for carbon source utilization test manufactured by Wachi Bact
One carbon compound each (glucose, galacto
Each yeast on agar plate with 10g dissolved
Cells were inoculated (stamped) and assimilation was observed. Table 2
The results of carbon source utilization were shown. Association Shochu yeast SH-4,
It did not change much between BAW-6. Kagoshima yeast
Mother (K In o), there was no assimilation of ethanol. Also deer
Kojima yeast (K o) and Miyazaki yeast (MK) assimilate citric acid
Did not.

B. Vitamin Requirement Nakata et al.'S method (Kubo Nakata, Ken Hosaka, Satoshi Sakai: Fermentation, 6)
3,509 (1985)). sand
Wachi Bacto's liquid culture medium for vitamin requirement test 11
About calcium pantothenate 2mg, pyridoxine 4
00 μg, biotin 200 μg, inositol 10 m
g and thiamine 400 μg of vitamin were removed respectively.
Omit method, K o, MK, SH-4 and BAW-6
A diluted suspension of the washed cells of the four yeasts was added to 8 × 10⁸cel
1 / ml, inoculated at 30 ° C for 7 days,
Vitamin requirements were observed. Table 3 shows the view on vitamin requirement.
The result of the observation was shown. Miyazaki yeast (MK) is the key to thiamine
Kagoshima yeast (K o), SH-4, BA
There was no for W-6. Other vitamin requirements
Was almost the same for all four yeasts.

C. TTC dyeing method Furukawa, Akiyama method (Toshio Furukawa, Yuichi Akiyama: Agriculture, 37,
398 (1963)). That is, K
o, four types of yeasts, MK, SH-4 and BAW-6
Dilute each cell appropriately (about 200 per plate)
Plate culture at 30 ° C for 2 days
After dissolving TTC agar on the colonies
And then gently layer, and after setting, release at 30 ° C for 2-3 hours.
The colony was observed for staining. Table 1 shows TTC dyeing
The results of observation of the color were shown. Clear from the results shown in Table 1
Like, Kagoshima yeast (K o), SH-4 and BAW-
6 was pink, but Miyazaki yeast (MK) was red.
Met.

D. D. C. Dyeability Mizoguchi, Fujita's method (Haruhiko Mizoguchi, Eishin Fujita: Fermentation, 59,
185 (1981)). That is, K
o, four types of yeasts, MK, SH-4 and BAW-6
Dilute each cell appropriately (about 200 per plate)
), Play on TTC underlayer medium at 30 ° C for 2 days
After dissolving the soft agar for the upper layer on the colony
Approximately, layer gently and allow to set to room temperature for 30 minutes
The colony was left to observe the state of staining of the colony. Table 1 shows D.
C. The results of observation of the dyeability were shown. From the results shown in Table 1
As is evident, Kagoshima yeast (K o) and SH-4 are tea
However, Miyazaki yeast (MK) and BAW-6 were white.
Was.

E. Formation of ascospores (Takeshi Hasegawa, ed .: Classification and identification of microorganisms (1), 170)
(1984) Academic Publishing Center)
K o, MK, SH-4 and BAW-6
For each, form ascospores on potassium acetate medium
After that, the cells were stained and observed under a microscope. Observed in Table 4
The results showed the formation rate of ascospores. Results shown in Table 4
As is evident from the above, Miyazaki yeast (MK) has poor sporulation.
In addition, the sporulation rate of SH-4 was slightly low at 74.1%.
Was. In addition, Kagoshima yeast (K o) and BAW-6
Their sporulation rates were 86.5% and 83.3%, respectively.
And it was high.

From the above observation results of mycological properties, the following
It turns out. That is, this bacterium, that is, BAW-6,
Kagoshima yeast (K o) is Ethanor
About Miyazaki Yeast (MK)
About sake yeast and wine yeast (Kubo Nakata, Hosaka
Ken, Sakai Tetsu: Fermentation, 63, 509 (1985)
This is different from meleptose and α-methylglucoside.
(B) Regarding vitamin requirement, Miyazaki yeast
(MK) means thiamine, sake yeast (Kubo Nakada,
Ken Hosaka, Satoshi Sakai: About Fermentation, 63, 509 (1985)
Different for pantothenic acid, biotin; (c)
TTC stainability and D.I. C. About the dyeability, Kagoshima yeast
(K o) and SH-4. C. Stainability, Miyazaki yeast
(MK) is different in TTC staining property. Further these genders
The quality is maintained even after 20 passages of subculture.
This is a unique property of bacteria, that is, BAW-6. Therefore, this fungus
That is, BAW-6 is objectively distinguished from conventional yeast.
It was found that these were new bacteria.
The strain was identified, and this strain was named BAW-6. This strain is
On March 12, 1992, the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology
Deposit No. 12871
Was.

3. Proliferation test in barley koji medium Medium similar to 100 ml of barley koji medium of Bllg ° 8
Kagoshima yeast (K o) and BA
2 ml each of W-6 was inoculated. Culture at 30 ° C after inoculation
OD every other hour₆₆₀It was measured. In this way,
Breeding test, the maximum specific growth rate μ_maxAre each
0.658 and 0.684. BAW-6 is the parent strain
Kagoshima yeast (K Proliferation in barley koji juice is better than in o)
It was quick.

4. Proliferation test at low temperature K o, MK, SH-4 and BAW-6
For each, a growth test at low temperature was performed as follows.
Test was carried out. That is, 2 ml of YPD medium at 30 ° C. for 2 days
Each yeast culture solution that had been cultured for 10 minutes was added to 10 ml of YPD medium.
Inoculate 0 μl and shake culture at 10 ° C after inoculation.
Photo Recorder TC-106 (Advantech Toyo Co., Ltd.)
OD at regular time intervals₆₆₀Was measured. this
Table 5 shows the results of the proliferation test. Table 5
From the results shown in Table 2, the maximum specific growth rate μ at 10 ° C.
_maxIndicates that BAW-6 is higher than other typical shochu yeasts.
I knew it.

5. Comparison of alcohol tolerance K o, MK, SH-4 and BAW-6
For each, adjust alcohol tolerance as follows:
Solid. That is, cultivation in 10 ml of YPD medium at 30 ° C. for 2 days.
Each of the washed yeast washed cells was dissolved in 8 to 14% ethanol,
Course 1%, 0.1 M acetate buffer (pH 4.3) 6 ml
Suspend in autoclave for 3 days at 30 ° C.
It was measured. Table 6 shows the results. Results shown in Table 6
From, the survival rate of BAW-6 at ethanol concentration of 8%,
Kagoshima yeast (K Approximately 15 times the ethanol concentration compared to o)
At 11%, it is about 5.5 times that of SH-4
understood.

6. Comparison of alcohol productivity K o, MK, SH-4 and BAW-6
For each, the alcohol productivity was increased as follows:
Examined. That is, in 10 ml of YPD medium at 30 ° C. for 2 days
Each cultured yeast-washed cell was suspended in 10 ml of sterilized water,
llg Inoculate 1 ml each to 100 ml of barley koji juice
After static culture at 0 ° C for 2 days, add 4g of glucose
After further culturing for 2 days, 4 g of glucose was added and 2 days
After the inter-culture, the alcohol concentration of the culture and the residual glucose
Was measured. Table 7 shows the results. Shown in Table 7
Based on the results, BAW-6 is more than any other shochu yeast.
It was also found that alcohol productivity was high.

[0032]

[Experimental Examples] Commercially available Kagoshima yeast (Ko) and yeast of the present invention
Barley shochu was produced using BAW-6. This preparation is
Table 8 shows the charging ratio. The product temperature course is as shown in Fig. 1.
Was. Aspergillus Kaw
achii) was used. In addition, yeast is Bllg ° 8
Refresh 10 mL of barley koji broth with 2 mL of the same medium.
Inoculate 100 μl of the washed yeast and culture at 30 ° C for 2 days
What was used was used.

The fermentation curve was as shown in FIG. From this figure, it was found that the fermentation state of the yeast BAW-6 of the present invention was better than that of Kagoshima yeast (Ko). Furthermore, Table 9 shows the alcohol yield of the obtained shochu.
From the results in Table 9, it was found that the yield was higher than that using the conventional shochu yeast, Kagoshima yeast.

As is apparent from the above description, the yeast BAW-6 of the present invention has a higher fermenting power and a higher alcoholicity in the production of barley shochu than the conventional shochu yeast, Kagoshima yeast (Ko). It was found that it could give a yield.

[0035]

[Table 1]

[0036]

[Table 2]

[0037]

[Table 3]

[0038]

[Table 4]

[0039]

[Table 5]

[0040]

[Table 6]

[0041]

[Table 7]

[0042]

[Table 8]

[0043]

[Table 9]

[0044]

SUMMARY OF THE INVENTION A novel microorganism having a high growth rate in barley shochu mash and having excellent alcohol tolerance and high utility.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing the temperature progress in fermentation.

FIG. 2 is a graph showing the history of fermentation.

Claims (3)

(57) [Claims] 1. (i) TTC staining test, that is, TTC agar was appropriately diluted on a plate (to about 200 per plate) and cultured on a lower medium at 30 ° C. for 2 days on a colony of TTC agar. After dissolution, the mixture was heated to about 45 ° C. and gently overlaid. After solidification, the mixture was allowed to stand at 30 ° C. for 2 to 3 hours.
i) D. C. Staining test, that is, the cells were appropriately diluted (to about 200 per plate), and cultured on a TTC underlayer medium at 30 ° C. for 2 days.
After melting the soft agar for the upper layer, the mixture was gently overlaid at about 45 ° C. after being melted, left to stand at room temperature for 30 minutes after hardening, and when the staining of the colonies was observed, the yeast for brewing was characterized by showing white color (Saccharomyces cerevisiae) ). 2. The brewer's yeast (Saccharomyces)
2. The strain according to claim 1, wherein S. cerevisiae has the following mycological properties. (A) Form of bacterium when cultured at 30 ° C. for 2 days using YM medium: size of vegetative cell: 4 to 9 μ form of vegetative cell: oval form of growth: budding (b) YM agar plate Colony morphology when used and cultured at 30 ° C. for 2 days: Morphology: Circular ridge: Convex circle Perimeter: Smooth Size (diameter): 2-3 mm Color tone: White and opaque Surface: Smooth and shiny (c) Carbon Source assimilation: glucose, galactose, sucrose, D-maltose, trehalose, raffinose, inulin, mannose, ethanol, glycerol, lactic acid, citric acid, fructose and arabinose. Cellobiose, lactose, melibiose, melezitose, starch, α-methylglucoside,
D-xylose, D-ribose, L-rhamnose, meso-erythritol, D-sorbitol, D-mannitol, salicin, succinic acid and inositol do not assimilate. (D) Vitamin requirement: calcium pantothenate and biotin are required. It does not require pyridoxine, inositol or thiamine. 3. The brewer's yeast (Saccharomyces)
Cerevisiae) BAW-6
The strain according to claim 1 or 2, wherein