JPH0394670A - Yeast strain having high productivity of beta-phenetyl alcohol and beta-phenetyl acetate, its breeding method, and production of liquors using the same yeast - Google Patents

Abstract

PURPOSE:To efficiently separate the title yeast useful for producing liquors having excellent sweet smell from a specific analog resistant strain by separating the analog resistant strain from a yeast belonging to Saccharomyces cerevisiae. CONSTITUTION:A yeast belonging to Saccharomyces cerevisiae is, as necessary, treated with a variant-inducing treatment and then an analog resistant variant which became proliferative even in a culture medium containing an analog of phenylalanine of a high concentration incapable of normally growing is separated from the yeast. Then a strain capable of producing beta-phenetyl alcohol and beta-phenetyl acetate at remarkable amounts is separated from the resist variant to provide the objective yeast [e.g. Saccharomyces cerevisiae Kyokai 7-FPAR 18 (FERM P-10868)].

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is a method for breeding yeast that simultaneously produces high amounts of β-phenethyl alcohol and β-phenethyl acetate.

Aroma components are one of the important factors that determine the quality of alcoholic beverages. The aroma components are mainly produced during the fermentation period by yeasts of the genus Saccharomyces cerevisiae used in alcoholic beverage brewing.

When developing demand for alcoholic beverages, it is desirable to develop products with distinctive quality, and products with distinctive aromas are one example of this. The discrimination thresholds for β-phenethyl alcohol and β-phenethyl acetate, which have a sweet rose-like aroma, are said to be 125 ppm and 3 ppm, respectively, in beer (Hashimoto: Fragrance No. 112. I) 23 (1975)).

On the other hand, the contents of β-phenethyl alcohol and β-phenethyl acetate in alcoholic beverages are 10 to 100 ppm and 3 to 5 p in beer.
pm, 20-70 ppm and 5-8 ppm for sake. 1O~75ppm and 1~2ppm for wine, 9~27ppm for shochu
pm and 2-28 ppm, whiskey 15-30 ppm
m and 1 to 5 ppm (New Edition of Jozo Orubun List, edited by Japan Brewing Association (1977)), and it is necessary to use yeast that has a further enhanced ability to produce discrimination threshold cells. Based on the above, breeding of yeast that can produce high amounts of β-phenethyl alcohol and β-phenethyl acetate is expected to contribute to the development of new products in the field of alcoholic beverage manufacturing.

(Prior Art) Until now, there has been no example of breeding for the purpose of isolating a yeast strain that produces high amounts of 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-mentioned circumstances, the present invention aims to isolate and breed a yeast strain that can produce high amounts of β-phenethyl alcohol and β-phenethyl acetate from brewer's yeast. As a result of intensive experiments and research, we developed a method for isolating a yeast strain that produces significantly more β-phenethyl alcohol and β-phenethyl acetate than conventional yeast, and by using this method, we were able to isolate the yeast Satsuromyces (
Saccharo Ru. Furthermore, by using breeding yeast, they succeeded in producing alcoholic beverages with excellent aroma characteristics.

The production of higher alcohols, including β-phenethyl alcohol, by yeast is closely related to the metabolism of amino acids, and the amino acids taken into the yeast are deaminated, decarboxylated, and reduced to alcohols with one fewer carbon number. There are two pathways: the first and the other produced by a similar reaction from keto acids, which are intermediate metabolites in the amino acid synthesis system. When higher alcohols are produced from a synthetic system or a large amount of amino acids are produced through that system, the amino acids are suppressed by feedback inhibition of enzymes in the synthetic system. In mutant strain j in which this feedback inhibition is incomplete, higher alcohols are always produced regardless of the presence or absence of amino acids. One way to obtain mutant strains with incomplete feedback inhibition is to use amino acid analogs that themselves have the property of feedback inhibition, and to isolate mutant strains that produce high levels of acyl alcohol. Although p-fluorophenylalanine, an analog of phenylalanine, was mainly used, other analogues of phenylalanine, m-fluorophenylalanine,
- It is also possible to use fluorophenylalanine, glycylphenylalanine, β-2-chenylalanine. This is based on the fact that all of the above analogs have an inhibitory effect on 3-deoxy-D-arabinohepuronate 7-phosphate synthase, which is one of the enzymes that feedback inhibition is caused by phenylalanine (TAKAH
ASI et al. :Can. J. Bioche
m. , 49.1016 (1971).

High production of β-phenethyl acetate can be expected along with high production of β-phenethyl alcohol. This is converted into acetyl-CoA by yeast alcohol acetyltransferase.
Since it is produced by the condensation reaction of β-phenethyl alcohol and β-phenethyl alcohol, it is one of the substrates.
987)). Although it is possible to isolate the target analog-resistant yeast even if the yeast used as the parent strain in this breeding method is used without mutation treatment, there is no problem even if isolation is performed after the usual mutation treatment.

We conducted fermentation tests using the isolated analog-resistant mutant strain, quantified β-phenethyl alcohol and β-phenethyl acetate in the fermentation liquid, produced these in significant amounts, and isolated those that fermented similarly to the parent strain as the target yeast strain. do. Furthermore, alcoholic beverages are produced using isolated yeast, and a yeast that has excellent aroma characteristics and can produce alcoholic beverages with a harmonious flavor is selected.

(Effects of the Invention) Isolating a mutant strain that has acquired new properties without impairing its excellent brewing properties from a practical yeast is generally an extremely difficult task that requires a lot of effort.

The present invention is extremely effective in breeding positive practical yeasts to isolate analog-resistant strains. Further bred β-
Yeasts that produce high amounts of phenethyl alcohol and β-phenethyl acetate are suitable for producing alcoholic beverages with distinctive aromas.

(Example) Example 1 (Example 1 of isolation of p-full lophenylalanine resistant yeast) Sake yeast No. 7 belonging to the genus Saccharomyces cerevisiae was cultured in YCB medium (yeast carbon base l.
17%, casamino acid 0.5%) and grown,
Aseptically washed to collect bacteria, 0.1M containing 2% glucose,
I) Suspended in 10 ml of H7.0 phosphate buffer.

To this, 0.3 ml of ethyl methanesulfonic acid was added and the mixture was slowly shaken at 30°C to carry out mutation treatment. The survival rate after mutation treatment was 57%. 1 bacterial cell after mutation treatment
After washing once with 0 ml of sodium thiosulfate and twice with 10 ml of sterile water, suspend in 10 ml of sterile water and 400 μl of the same.
(nitrogen base 0.67%, glucose 5%, agar 2%) was applied to several sheets. When cultured at 25°C, colonies were observed in about one week. This colony was further subcultured several times in the same medium and was isolated as a p-fluorophenylalanine-resistant yeast.

Example 2 (Isolation of β-phenethyl alcohol and β-phenethyl acetate high-producing strains from p-fluorophenylalanine-resistant yeast) Regarding the 80 p-fluorophenylalanine-resistant mutant strains isolated in Example 1, YCB medium (Yeast Carbon Base 1 .17%, casamino acids 0.5%, glucose l
A fermentation test was conducted using 5%) at 25°C and left standing. The contents of β-phenethyl alcohol and β-phenethyl acetate in the fermentation broth were determined by capillary gas chromatography. The column is PEG20M15mx0.53mm (i
．． d. ) (Gas Kuro Kogyo) was used.

As shown in Figure 1, the parent strain Kyokai No. 7 is 100 ppm, and about 30% of the orophenylalanine resistant mutants are 300 ppm.
m or more of β-phenethyl alcohol and 10 ppm or more of β-phenethyl acetate were produced. By isolating p-fluorophenylalanine-resistant mutant strains as described above, strains that frequently produce large amounts of β-phenethyl alcohol and β-phenethyl acetate were obtained. Furthermore, the amounts of ethanol and aroma produced by nine representative strains among these strains are shown in Table 1. Ethanol production was the same as the parent strain, and isoamyl alcohol and isoamyl acetate tended to be higher than the parent strain, but it is clear that this mutant strain specifically produces high levels of β-phenethyl alcohol. Furthermore, there is no correlation between the yield of β-phenethyl alcohol and the consumption amount of phenylalanine, and it is considered that β-phenethyl alcohol is produced from the deinhibited phenylalanine synthesis system.

Kyokai No. 7 (parent strain) 8.8 139 Kyokai No. 7 (parent strain) 4.9 Kyokai No. 7 (parent strain) 7enylalanine consumption (mM) 0.2 9 Example 3 (β-phenethyl alcohol, 0.34 Acetic acid Post-saccharification fermentation that we developed using a β-phenethyl acetate high-producing mutant strain (Akita et al.: Joukyo Journal, 8l, 537 (1986)
)) was done. The saccharification liquid is made of white rice with a polishing ratio of 75% and koji.
= 1, water was used in an amount three times that of polished rice, and was liquefied and saccharified using a heat-resistant liquefying enzyme and a saccharifying enzyme (Oba, Akita, Nakamura: Special Publication No. 61-51864).

The composition of the saccharified solution was 8.2% glucose and 17% amino acids.
7mM (amino nitrogen 327mg/1) Fermentation was carried out in a 500-mm Erlenmeyer flask equipped with a fermentation stopper.
Add the 00- saccharified liquid and stir with a stirrer.
The experiment was carried out at a constant temperature of ℃. The initial number of yeast was 5×10′/1.

Fermentation was terminated when glucose became 1% or less.

Table 2 shows the chemical composition of fermented sake. As in the case of fermentation using a mixed medium, the contents of β-phenethyl alcohol and β-phenethyl acetate were significantly higher in the mutant strain than in the parent strain. There was no noticeable difference in ethanol and acidity produced by the mutant strain from that of the parent strain, and there were no practical problems.

Association No. 7 (Parent Stock) Association No. 7 (R) Association No. 7 (Parent Stock) Ecl. 3 1.4 β-phenethyl alcohol β-7 enethyl acetate 0.8 Phenethyl-K7 ranin consumption 0.45 3.0 - Sake brewing test with high phenethyl-producing mutant strain) Method of Namba et al. (Jokyo Journal, U, 295) (1978) 'I conducted a sake brewing test using 150 g of total rice, and the results are shown in Table 3. Fermentation was completed on the 22nd day, and the sake was poured into a tank by centrifugation.

The ethanol production of the resistant strain was no different from that of the parent strain, and among the aroma components, β-phenethyl alcohol and β-phenethyl acetate were significantly more abundant, and isoamyl alcohol and isoamyl acetate were also more abundant than the parent strain. There was no significant difference in acidity and amino acid content from the parent strain. The reason why the amino acid content is low in all of them is because pregelatinized rice is used. From the above, the isolated resistant strain has sufficient practicality in actual sake brewing.
Furthermore, the high productivity of β-phenethyl alcohol and β-phenethyl acetate, which is a characteristic of this strain, was exhibited, and it was possible to produce sake with unprecedented aromatic characteristics.

Example 4 (β-phenethyl alcohol, β acetic acid Japan Sake Acidity and Amino Acidity Association No. 7 <yi strain) -13 3.2 Sensory test * Aroma Total Quality Association No. 7 (parent strain) 2,00 1.862.4 Ethanol Isoamyl alcohol (%)
μ g/m/ 18.1 169 Isoamyl acetate β-7 enethyl alcohol β-7 enethyl acetate Association No. 7 (parent strain) 3.2 135 3.6 Example 5 (Isolation of p-fluorodenylalanine resistant yeast Example 2) Saccharomyces
A p-fluorophenylalanine resistant strain was isolated from the lploid yeast A5-8-1 (Aleul) belonging to the genus Cerevisiae using the same technique as in Example 1. This yeast is a laboratory yeast, Satsurikuchi Myces (
Saccharomyces genus cerevisiae
evisiae). The isolated resistant strain showed leucine auxotrophy and was confirmed to be a mutant of the parent strain. YCB medium by isolate (yeast carbon base l. 17%, casamino acids 0.5%, glucose 15%
Table 4 shows the production amounts of β-phenethyl alcohol and β-phenethyl acetate in the fermentation test using ().

A strain that produced significantly more β-phenethyl alcohol and β-phenethyl acetate than the parent strain was isolated from the resistant strain.

*Judging using 3a method (1. Good, 2: Average, 3. Bad)
Average values of 7 panel members Table 4 p-Full-sized lophenyara ≧ Otsu-resistant yeast aromatic β-
7 enethyl alcohol Acetic acid/3 -7 enethyl Table 5 Aroma of p-fluorophenylalanine resistant yeast or β-7 enethyl alcohol β-F L-unthyl acetate 43 309
248 312
254 3
58 2 Example 6 (Example 3 of isolation of p-ferrophenylalanine resistant yeast) Saccharomyces
The haploid yeast C4488-IA (α leu2 (KIL-k
]) in the same manner as in Example 1 to isolate p-fluorophenylalanine-resistant strains. This yeast is a laboratory yeast, Saccharomyces (Saccharomyces).
s) is a representative strain of the genus Cerevisiae. The resistant strain showed leucine auxotrophy and was confirmed to be a mutant of the parent strain. isolate in YCB medium (yeast carbon base 1.17%, casamino acids 0
．． Table 5 shows the amounts of β-phenethyl alcohol and β-phenethyl acetate produced.

25 287
From the 3-resistant strains, we were able to isolate a strain that produced significantly more β-phenethyl alcohol and β-phenethyl acetate than the parent strain.

β-phenethyl alcohol of the present invention from Examples 5 and 6,
It has been shown that the method for isolating a yeast strain that produces high β-phenethyl acetate can be widely used for the genus Saccharomyces cerevisiae.

[Brief explanation of drawings]

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

Claims (4)

[Claims] (1) Saccharomyces
We isolated analog-resistant mutant strains of yeast belonging to the genus cerevisiae that can grow in a medium containing a phenylalanine analog at a concentration that would normally make it impossible to grow without mutagenic treatment or mutagenic treatment, and further developed resistant mutant strains. A method for isolating and breeding yeast with high productivity of β-phenethyl alcohol and β-phenethyl acetate. (2) Saccharomyces
) A resistant mutant strain of sake yeast belonging to the genus cerevisiae that can grow in a medium containing p-fluorophenylalanine at a concentration that would normally be impossible to grow was isolated without mutation treatment or mutagenic treatment. A method for isolating and breeding yeast with high productivity of β-phenethyl alcohol and β-phenethyl acetate from mutant strains. (3) The genus Saccharomyces cerevisiae (c
erevisiae) Kyokai7-FPAR18 (
A yeast that is highly productive of β-phenethyl alcohol and β-phenethyl acetate, which is displayed in the Microtechnical Research Institute No. 10868). (4) The genus Saccharomyces cerevisiae (c
1. A method for producing alcoholic beverages, characterized by using a yeast that is highly productive of β-phenethyl alcohol and β-phenethyl acetate belonging to the family A. erevisiae.