JP2000041661A - Breeding of yeast with increased fermentation rate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently produce an alcoholic beverage of high alcohol content in a short period by breeding a yeast that have an increased fermentation rate through the treatment of a yeast in Saccharomyces cerevisiae under specific conditions. SOLUTION: A yeast in Saccharomyces cerevisiae is mutated or not mutated, then, a selection culture medium containing at least one sesquiterpene antibiotics selected from trichothecin, roridin A, verrucarin A or the like and anisomycin is used to select and isolate an multidrug-resistant yeasts. In addition, yeasts that causes no foam and has an increased fermentation rate are selected from the multidrug-resistance yeasts and a Sake yeast causing no foam with increased fermentation rate is bred to obtain, for example, Saccharomyces cerevisiae Ana 10 (FERM P-16888). In a preferred embodiment, this Sake yeast is used to brew Japanese Sake beverage.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to breeding of yeast, and more particularly to breeding of yeast having an increased fermentation rate. Furthermore, the present invention also relates to the breeding of sake yeast, which, especially with respect to sake yeast, has an increased fermentation rate and also has a non-foaming property. In addition, the present invention also aims to shorten the production period of alcoholic beverages, increase the amount of alcohol production, omit the defoaming step, etc. For efficient manufacturing processes.

[0002]

2. Description of the Related Art There have been breeding examples for the purpose of isolating yeast with an increased fermentation rate. However, since it is necessary to isolate a relatively fast-growing yeast, separation conditions must be set. However, at present, a satisfactory breeding method that can quickly and reliably select a target yeast has not yet been established.

[0003] On the other hand, anisomycins are known to be useful as antibiotics, but they have been successfully used in the selection medium of production yeast with an increased fermentation rate and have successfully produced brewed satisfactorily. No such example has been reported so far.

[0004]

SUMMARY OF THE INVENTION In brewing sake,
Obtaining more alcohol in a shorter period of time is a more important task than before.

[0005]

SUMMARY OF THE INVENTION As described above, the present invention has been made to achieve the acquisition of more alcohol in a shorter period of time than in the past.

Therefore, the inventors aimed to increase the amount of alcohol obtained in a shorter period of time, and set a new technical problem of breeding a production yeast with an increased fermentation rate in order to achieve the purpose. did. That is, the present inventors have aimed at the isolation and breeding of yeast with a newly increased fermentation rate from brewer's yeast, and as a result of the experiment, the fermentation rate has been reduced compared to conventional yeast using a selective medium. The present inventors have developed a method for isolating the increased yeast, and have succeeded in breeding Saccharomyces cerevisiae by this method.

That is, the present invention provides a method using a selective medium,
Saccharomyces cerevisiae (cer)
The basic technical idea is to isolate and breed yeast having an increased fermentation rate from yeasts belonging to evisiae). As the selection medium, any medium can be used as long as it can separate and breed the production yeast with an increased fermentation rate. For example, anisomycin-containing medium is one of suitable media.

[0008] In addition, the inventors proceeded with research on anisomycin-resistant yeast isolated as a yeast having an increased fermentation rate as described above, and focused on the resistance again. As a result, it has been found that the anisomycin-resistant yeast has anisomycin resistance. It has been discovered for the first time that it has a completely novel property of being resistant to antibiotics such as cycloheximide and other drugs structurally different from anisomycin.

That is, the present invention further discovered that the phenomenon of exhibiting resistance to a large number of drugs greatly contributed to the increase in fermentation ability, and based on these useful new findings, the results of further investigations A yeast that has been completed and has a multidrug resistance exhibiting resistance to two or more drugs exhibits an increased fermentation ability by utilizing a novel property of increasing the fermentation rate. It is also involved in the total system for brewing sake, including selection and creation of the same yeast. Hereinafter, the present invention will be described specifically.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In order to carry out the present invention, cerevisiae of the genus Saccharomyces is used.
Using the yeast belonging to e), anisomycin-resistant yeast is isolated using a selective medium such as a medium containing anisomycin. As a parent strain, Saccharomy
All yeasts can be used as long as they belong to the cerevisiae cerevisiae. For example, sake yeasts (Kyoto No. 7 yeast, Kyokai No. 9 yeast, etc.) and wine yeasts (Grape No. 1 yeast (Nihon Brewery) Saccharomyces cerevisiae, including practical yeasts such as the Association Wine No. 1 yeast, Wine No. 3 yeast, Wine No. 4 yeast), beer yeast, baker's yeast, and other yeasts commonly used for alcohol fermentation. All the yeasts belonging to the category (1) can be advantageously used.

In the present breeding method, the yeast used as the parent strain is:
The target yeast can be separated even if used without mutation treatment, but there is no problem if separation is performed after ordinary mutation treatment. In order to obtain a mutant yeast in the present invention, any method may be used as a mutation method. Examples of physical methods of mutation include ultraviolet irradiation and irradiation, and examples of chemical methods include mutagens such as ethyl methanesulfonate, N-methyl-N-nitrosoguanidine, nitrite, and acridine dyes. There is a method of suspending in a solution, and these methods can be appropriately used.

In the present invention, these mutation methods can be used as appropriate, but are characterized by selection of mutant yeast. That is, the growth medium of the mutant strain contains anisomycin. Although anisomycin is known as an antibiotic, there is no report that it was used as a selective medium for yeast for brewing sake, particularly for yeast having an increased fermentation rate. In the present invention, the target mutant yeast may be separated from a colony growing on a medium containing anisomycin (positive selection), so that the separation operation is easy and simple. In this respect, the breeding method of the present invention Are better.

[0013] Using the anisomycin-resistant yeast thus isolated, a fermentation test is performed to isolate those having an increased fermentation rate.

[0014]

Example 1 Method for Isolating Anisomycin-Resistant Yeasts Saccharomyces cerevisiae
e) Association No. 9 sake yeast (hereinafter abbreviated as K-9) belonging to e),
YPD medium (yeast extract 1%, peptone 2%, glucose 2%) was inoculated and grown, and then aseptically collected and washed, and 0.1 ml of the resultant was anisomycin (0.0034).
mg / ml), cultured at 30 ° C., and grown colonies were isolated on anisomycin (0.03 mg / ml).
(4 mg / ml) on a YPD agar medium. The anisomycin-resistant strain isolated in this way is
FERM P-1 was added to the National Institute of Advanced Industrial Science and Technology.
Deposited as 6888.

[0015]

Example 2: Isolation of yeast with increased fermentation rate from anisomycin-resistant yeast The anisomycin-resistant yeast strain isolated in Example 1 is shown in Table 1 below using 70% polished white rice. Three-stage charging was performed with the charging mixture. Yeast suspension was used for yeast (5 × 10 5
⁶ ), served at 15 ° C, medium at 12 ° C, and rest at 9 ° C,
From the next day, the product temperature was raised at 1 ° C./day until the temperature reached 15 ° C., and fermentation was continued at 15 ° C. Ethanol in the fermentation broth was measured using Alcomate (Woodson Co., Ltd.), and the acidity was 0.1 N NaOH and the amino acid content was 0.1
Analysis was carried out using N NaOH and formalin solution (50% aqueous solution) according to the analysis method specified by the NTA.

[0016]

[Table 1]

Table 2 below shows the progress of carbon dioxide gas reduction during the preparation of the anisomycin-resistant yeast. As is apparent from the results, the parent strain K-9 was fermented from the isolated anisomycin-resistant yeast. A strain with increased speed could be obtained.

[0018]

[Table 2]

The alcohol content, acidity, amino acid content, and yeast kill rate of the brewed sake brewed using the anisomycin-resistant yeast as described above were measured at the time of the upper tank. The results obtained are shown in Table 3 below.

[0020]

[Table 3]

The concentration of anisomycin in the selection medium is from 0.030 mg / ml to 0.050 mg / ml.
The above range may be used as a rough guide, but the concentration may be higher than the above concentration if there is no adverse effect of toxicity, or may be lower than the above range if effectiveness is recognized.

As is clear from the above description, the yeast isolated and bred by the present method has a characteristic that it has a high fermentation rate and produces high-quality brewed sake. High quality brews can be produced in a short time. In this example, K-9 was used as a parent strain as an example. However, anisomycin-resistant yeast can be easily and rapidly separated from other yeasts by the same method. By using it according to the usual method, it is possible to produce excellent brewed sake in a short time.

The yeast strain thus selectively isolated is
By using it in the same manner as ordinary yeast, excellent brewed sake can be efficiently produced in a short period of time. Further, the present method can be applied to yeast used for mash for brewing vinegar production or mash for brewing alcohol production to improve alcohol productivity.

Furthermore, the present invention provides a multi-drug resistant yeast, which has a high fermentation rate and exhibits multi-drug resistance, which is resistant to two or more drugs, and And its use.

For example, as described later, a yeast having a high fermentation rate (for example, Saccharomyce
s cerevisiae Ana 10: FERM P-16888)
Is a sesquiterpene antibiotic (tricosecin,
In addition to velcarin A, loridine A, etc.), it also shows resistance to cycloheximide, fluphenazine, cerulenin and the like (Table 4).

[0026]

[Table 4]

The drug is not limited to the above-mentioned drugs alone. In short, it is important to exhibit resistance to a large number of drugs, such as two or more drugs. For the first time, the point that the alcohol fermentation rate is closely related is clarified, and this phenomenon is characterized in that this phenomenon is utilized industrially.

The multidrug-resistant yeast having a high fermentation rate according to the present invention is selected on one or more drug-containing selective media without artificially mutating the parent strain or without artificial mutation (natural mutation). The target mutant may be screened according to a conventional method used for isolating strains or other resistant bacteria, and the method for isolation and breeding is not particularly limited. Alternatively, the target multidrug resistance may be imparted by genetic manipulation, and yeast having a high fermentation rate may be directly created.

The production of alcohol or the fermentation of alcohol using the multidrug-resistant yeast having a high fermentation rate isolated in this manner may be performed in the same manner as described above.

Further, the present inventors have found for the first time that, in the course of the above-mentioned research on the breeding of yeasts having an increased fermentation rate, multi-drug resistant yeasts also become foamless yeasts for sake yeasts. By selectively separating multidrug-resistant yeast, a method for breeding sake yeast that has an increased fermentation rate and also has no foaming properties has been completed.

Normally, sake yeast is covered with foam at the upper part of the moromi, particularly during the early to middle stages of fermentation, and eventually the foam overflows. For this reason, at present, the foam is crushed using a foam eraser in the course of the manufacturing process so that the foam does not overflow from the tank containing the moromi.

As for the sake yeast itself, a part of the yeast without foam is commercially available, which contributes to the rationalization of sake production. Therefore, the development of an excellent foamless yeast becomes important especially from the industrial aspect of sake production, and establishment of an efficient and accurate separation and breeding method is extremely important.

Not only has the invention been successful in this regard,
Sake yeast with an increased fermentation rate was also successfully screened at the same time. Heretofore, there has been no report that multidrug-resistant yeast is foamless (of course, there is no report of sake yeast with an increased fermentation rate),
The present invention, in which foamless yeast can be selected in a manner different from the conventional method for selecting foamless yeast, is extremely outstanding. Furthermore, according to the present invention, not only yeast without foam but also sake yeast with an increased fermentation rate can be selected at the same time, which is more excellent.

In fact, K-isolated by the method of the present invention
It was confirmed that the 9 Ana 10 strain (FERM P-16888) not only has a high alcohol fermentation rate but also has no foaming property.

[0035]

EFFECTS OF THE INVENTION Isolation of a mutant strain that has newly acquired useful properties from a practical yeast without deteriorating its excellent brewing properties is a difficult task that generally requires a lot of effort. The present invention is based on positive selection for isolating antibiotic-resistant yeast, and can efficiently isolate only the mutant strain of interest. Use of this separation method is extremely effective in breeding practical yeast. By using the yeast having a high alcohol fermentation rate obtained by separation and breeding in this manner, alcoholic beverages having a high alcohol content can be efficiently produced in a short period of time.

Further, the novel finding that the multidrug resistance phenomenon, which exhibits resistance to two or more drugs, closely contributes to the improvement of the fermentation rate, has led to a novel high fermentation utilizing multidrug resistance. An efficient screening system for rate yeast was also established. Furthermore, by imparting multidrug resistance, it has become possible to create a novel yeast having a high fermentation rate.

In addition, according to the present invention, with respect to sake yeast, yeast having a bubble-free property and an increased fermentation rate can also be separated and bred. Since the foaming step can be omitted, the production of sake can be greatly streamlined.

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[Procedure amendment]

[Submission date] July 23, 1999 (1999.7.2)
3)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Continuation of the front page (72) Inventor Yusho Ishikawa 3-7-1 Kagamiyama, Higashihiroshima City, Hiroshima Pref. National Tax Agency Brewing Research Institute F-term (reference) 4B065 AA80X AC10 AC20 BA16 BA23 CA42

Claims (11)

[Claims] 1. Saccharomyces cerevisiae (Saccha)
romyces cerevisiae), without or with a mutation treatment, and then selectively separating the yeasts with increased fermentation rates using a selective medium, wherein the yeasts with increased fermentation rates are characterized. Method. 2. Saccharomyces cerevisiae (Saccha)
romyces cerevisiae), without or after mutation treatment, using a selective medium to selectively isolate yeast having a bubble-free property and an increased fermentation rate. A method for breeding sake yeast having a property and an increased fermentation rate. 3. Saccharomyces cerevisiae (Saccha
A method for breeding yeast having an increased fermentation rate, characterized in that multi-drug-resistant yeast is selectively isolated without or after mutation treatment of yeast belonging to R. cerevisiae). 4. Saccharomyces cerevisiae (Saccha
romyces cerevisiae), without mutagenesis, or after mutagenesis, selective isolation of multidrug-resistant yeasts. Breeding method. 5. The drug to be contained in the selective medium or the drug-resistant drug is at least one selected from anisomycin, sesquiterpene antibiotics, cycloheximide, fluphenazine, and cerulenin. The method according to any one of claims 1 to 4. 6. The method according to claim 5, wherein the agent is anisomycin. 7. The sesquiterpene antibiotic is at least one selected from tricosecin, lorizin A, and velcarin A.
The method described in. 8. A yeast breeding and isolated by the method according to any one of claims 1 to 7, wherein the fermentation rate is increased. 9. A sake yeast breeding and separated by the method according to any one of claims 2, 4 to 7, which has a bubble-free property and has an increased fermentation rate. 10. A method for producing a brewed brew, comprising using the yeast according to claim 8 or 9. A brewed liquor produced by the method according to claim 10.