JPH0653065B2 - β-phenethyl alcohol, β-phenethyl acetate high-producing yeast strain, breeding method thereof, and liquor production method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for breeding a yeast that produces a high β-phenethyl alcohol and β-phenethyl acetate at the same time and a separated yeast strain, and further uses the yeast. The present invention relates to a method for producing alcoholic beverages having a characteristic aroma.

The aroma component is one of the important factors that determine the quality of alcoholic beverages. Aroma components are mainly produced during the fermentation period by the yeast Saccharomyces cerevisiae used for brewing alcoholic beverages.

In the development of demand for alcoholic beverages, the development of products with characteristic quality is desired, and products with aroma are also one of them. The discrimination thresholds for β-phenethyl alcohol and β-phenethyl acetate, which have a rose-like sweet scent, are 125 ppm and 3 ppm in beer, respectively (Hashimoto: Fragrance No. 11
2, p23 (1975)). On the other hand, the content of β-phenethyl alcohol and β-phenethyl acetate in liquor is 10 to 100 ppm and 3 to 5 ppm for beer, 20 to 70 ppm and 5 to 8 ppm for sake, and 10 to 7 for wine.
5ppm and 1-2ppm, shochu 9-27ppm and 2-28ppm, whiskeys 15-30ppm and 1-5ppm (new edition brewing ingredients list Japan Brewing Association (1977)), below or close to the discrimination threshold. is there. Therefore, in order to obtain products characterized by these aromas, it is necessary to use yeasts having a higher ability to produce current yeasts. From the above, breeding of yeasts that highly produce β-phenethyl alcohol and β-phenethyl acetate is expected to contribute to the development of new products in the field of alcohol production.

(Prior Art) Up to now, there is no breeding example aiming at isolation of a yeast that highly produces β-phenethyl alcohol and β-phenethyl acetate.

(Problems to be Solved by the Invention and Means for Solving the Problems) In view of the above circumstances, the present invention is aimed at the isolation and breeding of a yeast that newly produces β-phenethyl alcohol and β-phenethyl acetate in high yield from brewer's yeast. As a result of repeated intensive experiments and studies, a method for separating yeasts that produce significant amounts of β-phenethyl alcohol and β-phenethyl acetate as compared with conventional yeasts was developed, and yeast Saccharomyces (S
accharomyces) cerevisiae Kyokai7-FPAR1
This is a breeding of No. 8 (Microtechnology Research Institute No. 10868). Furthermore, by using the breeding yeast, it has succeeded in producing alcoholic beverages having excellent aroma.

The production of higher alcohols including β-phenethyl alcohol by yeast is closely related to the metabolism of amino acids, and the amino acid taken up by yeast is deaminated and then decarboxylated and reduced to form an alcohol with one carbon atom less. And, there are two pathways of a system produced by a similar reaction from keto acid, which is an intermediate metabolite of a synthetic system of amino acids. The production of higher alcohols from the synthetic system is suppressed by feedback-inhibiting the enzyme of the synthetic system by the amino acid when a large amount of amino acids are produced through the system. In the mutant strain in which this feedback inhibition is incomplete, higher alcohol is always produced regardless of the presence or absence of amino acids. One of the methods to obtain mutants with incomplete feedback inhibition is to use an analog of an amino acid that itself has feedback-inhibitory properties and isolate from mutants that have acquired resistance to this analog. There is a way. In the present invention, p-fluorophenylalanine, which is an analog of phenylalanine, was mainly used for the isolation of a mutant strain that highly produces β-phenethyl alcohol. It is also possible to use fluorophenylalanine, glycylphenylalanine, β-2-thienylalanine. This is one of the enzymes that phenylalanine inhibits feedback 3
It is based on the fact that all of the above analogs have an inhibitory effect on -deoxy-D-arabino-heptulonate 7-phosphate synthase (TAKAHASI et al .: Can.JB.
iochem. , 49, 1016 (1971)).

High production of β-phenethyl acetate can be expected along with high production of β-phenethyl alcohol. This is because yeast alcohol acetyltransferase produces it by the condensation reaction of acetyl CoA and β-phenethyl alcohol, and β-phenethyl acetate, which is one of the substrates, also increases, and β-phenethyl acetate also becomes highly produced. Yes (Akita et al .: Jyokyo, 82 , 369 (1987)). Although the target analog-resistant yeast can be isolated even if the yeast used as a parent strain in this breeding method is used without being subjected to mutation treatment, there is no problem even if the yeast is used after being subjected to usual mutation treatment.

Fermentation test is performed using the isolated analog-resistant mutant strain, β-phenethyl alcohol and β-phenethyl acetate in the fermentation broth are quantified, and these are produced in a large amount, and the yeast strain that does not differ from the parent strain in fermentation is isolated as the target yeast strain. To do. Furthermore, yeasts are produced by using the isolated yeast, and yeasts having excellent aroma and harmonious flavor are selected.

(Effects of the Invention) Isolation of a mutant strain that has acquired new properties from a yeast for practical use without impairing its excellent brewing properties is generally a very labor-intensive and extremely difficult task.

The present invention is based on positive selection for separating an analog resistant strain, and can efficiently separate only a target mutant strain. Utilization of this separation method is extremely effective in breeding practical yeast. Further, the cultivated β-phenethyl alcohol and the yeast that highly produces β-phenethyl acetate are suitable for the production of alcoholic beverages having aroma.

(Example) Example 1 (isolation example 1 of p-fluorophenylalanine-resistant yeast) Saccharomyces sp.
The association No. 7 sake yeast belonging to siae) is inoculated in YCB medium (yeast carbon base 1.17%, casamino acid 0.5%) and grown, and then aseptically collected and washed, and 0.1 M containing 2% glucose, pH7. It was suspended in 10 ml of a phosphate buffer of 0.0. Add 0.3 ml of ethyl methane sulfonic acid to this and add 3
Mutagenesis was performed by slowly shaking at 0 ° C. The survival rate after mutation treatment was 57%. After the mutation treatment, the cells were washed once with 10 ml of sodium thiosulfate and twice with 10 ml of sterile water.
Suspended in 100 ml of sterile water, 400μ (7 x 10 ⁷ live yeast)
YNB containing 0.5 mg / ml of p-fluorophenylalanine
It was applied to several sheets of agar medium (0.67% yeast nitrogen base, 5% glucose, 2% agar). When cultured at 25 ° C, colonies were observed in about 1 week. This colony was further subcultured several times in the same medium and isolated as p-fluorophenylalanine-resistant yeast.

Example 2 (Separation of β-phenethyl alcohol and β-phenethyl acetate high-producing strains from p-fluorophenylalanine-resistant yeast) About 80 p-fluorophenylalanine-resistant mutants isolated in Example 1, YCB medium (yeast carbon base Fermentation test was carried out by using 1.17%, casamino acid 0.5%, glucose 15%) at 25 ° C. and stationary. Β- in the fermentation broth
Phenethyl alcohol and β-phenethyl acetate contents were quantified by capillary gas chromatography. The column is PEG 20M 15m x 0.53mm (id) (Gaskuro Industrial Co., Ltd.)
It was used.

As shown in Fig. 1, the parent strain association No. 7 has β of 100 ppm or less.
-Phenethyl alcohol and about 5 ppm β-phenethyl acetate were produced, whereas about 30% of the p-fluorophenylalanine-resistant mutants produced 300 ppm or more β-phenethyl alcohol and 10 ppm or more β-phenethyl acetate. By isolating the p-fluorophenylalanine-resistant mutant strain as described above, β-
A strain producing a large amount of phenethyl alcohol and β-phenethyl acetate was obtained. Table 1 shows the amounts of ethanol and aroma components produced by 9 representative strains. The production of ethanol was the same as that of the parent strain, and the amount of isoamyl alcohol and isoamyl acetate tended to be higher than that of the parent strain. Further, there is no correlation between the production of β-phenethyl alcohol and the consumption amount of phenylalanine, and it is considered that β-phenethyl alcohol was produced from the derepressed phenylalanine synthesis system.

Example 3 (Post-saccharification fermentation test using β-phenethyl alcohol and β-phenethyl acetate high-producing mutant strain) β-phenethyl alcohol and β acetate obtained in Example 2
-The post-saccharification fermentation we developed (Akita et al .: Jōkyo, 81 , 537 (1986)) was carried out using a phenethyl highly producing mutant strain. The saccharified solution was prepared by using white rice and koji with a polishing rate of 75% at a ratio of 6: 1, and water was used in an amount three times that of white rice. Japanese Patent Sho 61-518
64). The composition of the saccharified liquid is glucose 18.2%, amino acids 17.7
mM (amino nitrogen 327 mg /). Fermentation was carried out at a constant temperature of 15 ° C with stirring with a stirrer, adding 300 ml of saccharified solution to a 500 ml Erlenmeyer flask equipped with a fermentation stopper. ^The fermentation was terminated when glucose became 1% or less.The composition of the fermented liquor was shown in Table 2. As in the case of fermenting the synthetic medium, β was specifically used in the mutant strain. -The content of phenethyl alcohol and β-phenethyl acetate was significantly higher than that of the parent strain, and there was no significant difference in ethanol and acidity produced by the mutant strain from the parent strain, and there was no practical problem.

Example 4 The method of Nanba et al. (Beta-phenethyl alcohol, Sake Brewing tests with acetic acid beta-phenethyl highly productive mutant) (醸協Journal, 73, 295 (1978)) Total US 150 by
g of sake brewing test was conducted and the results are shown in Table 3. 22
The fermentation was completed on the day, and the upper tank was prepared by centrifugation.

The ethanol production of the resistant strain was the same as that of the parent strain, and β-phenethyl alcohol and β-phenethyl acetate were remarkably large in the aroma components, and isoamyl alcohol and isoamyl acetate tended to be higher than those of the parent strain. The acidity and amino acid content were not significantly different from the parent strain. The low amino acid content is due to the use of pregelatinized rice. From the above, the isolation resistant strain has sufficient practicality even in the actual sake preparation,
Furthermore, β-phenethyl alcohol and β-phenethyl acetate high productivity, which are the characteristics of this strain, were exhibited, and it was possible to produce sake having aroma which was unprecedented.

Example 5 (separation example 2 of p-fluorophenylalanine-resistant yeast) Saccharomyces cerevisiae
A p-fluorophenylalanine-resistant strain was isolated from the haploid yeast A5-8-1 (aleul) belonging to siae) in the same manner as in Example 1. This yeast is a laboratory yeast and is a representative strain of the genus Saccharomyces cerevisiae. It was confirmed that the isolated resistant strain showed leucine auxotrophy and was a mutant strain of the parent strain. YC by isolate
B medium (yeast carbon base 1.17%, casamino acid 0.
Production rate of β-phenethyl alcohol and β-phenethyl acetate in the fermentation test using 5%, glucose 15%)
Shown in the table.

From the resistant strain, a strain capable of producing significantly higher amounts of β-phenethyl alcohol and β-phenethyl acetate than the parent strain could be isolated.

Example 6 (isolation example 3 of p-fluorophenylalanine-resistant yeast) Saccharomyces cerevisiae
haploid yeast C4488-1A (α leu2 [KIL-
From k]), a p-fluorophenylalanine-resistant strain was isolated in the same manner as in Example 1. This yeast is a laboratory yeast and is a representative strain of the genus Saccharomyces cerevisiae. It was confirmed that the resistant strain showed leucine auxotrophy and was a mutant strain of the parent strain. YCB medium (Yeast carbon base 1.17%,
Fermentation test using casamino acid 0.5%, glucose 15%) was carried out, and the production amounts of β-phenethyl alcohol and β-phenethyl acetate at that time are shown in Table 5.

From the resistant strain, it was possible to isolate a strain producing a significantly higher amount of β-phenethyl alcohol and β-phenethyl acetate than the parent strain.

Β-phenethyl alcohol of the present invention from Examples 5 and 6,
It was shown that the method for isolating β-phenethyl acetate high-producing yeast strain can be widely applied to Saccharomyces cerevisiae.

[Brief description of drawings]

FIG. 1 is a diagram showing the production of β-phenethyl alcohol and β-phenethyl acetate by a parent strain Association No. 7 sake yeast and a p-fluorophenylalanine-resistant strain isolated from the parent strain in Example 2.

Claims (4)

[Claims] 1. An analog-resistant mutant strain capable of growing in a medium containing a concentration of an analog of phenylalanine that cannot be normally grown without mutating or mutating yeast belonging to the genus Saccharomyces cerevisiae. And further separating and breeding β-phenethyl alcohol and β-phenethyl acetate high-producing yeast from the resistant mutant. 2. A strain of sake yeast belonging to the genus Saccharomyces cerevisiae, which is mutated or is not mutated and has a concentration at which it cannot grow normally.
A method of isolating a resistant mutant strain that has become able to grow in a medium containing fluorophenylalanine, and further separating and breeding β-phenethyl alcohol and β-phenethyl acetate high-producing yeast from the resistant mutant strain. 3. A cerevisiae K genus of the genus Saccharomyces isolated by the method of (2) above.
β displayed by yokai7-FPAR18 (Microtechnology Research Institute, No. 10868)
-Phenethyl alcohol and β-phenethyl acetate high-producing yeast. 4. A cerevisiae of the genus Saccharomyces isolated by the method of (1) above.
A method for producing alcoholic beverages, which comprises using β-phenethyl alcohol belonging to the category of β-phenethyl alcohol and yeast highly producing β-phenethyl acetate.