KR101351431B1 - Saccharomyces cerevisiae jy218 with high concentration of ethyl caproate and process for preparing alcoholic liquors using the same - Google Patents

Abstract

The present invention relates to a serulinine resistant strain, Saccharomyces cerevisiae JY218 (KACC 91696P), which produces a high concentration of fatty acid ethyl ester, which is known as a main component of alcoholic yin-yang, and a method for preparing alcoholic beverage using the same. In addition, the mutant strain is not only resistant to cerulenin, but also produced in high concentrations of ethyl caprolate, ethyl caprate and ethyl myristicate, and thus can be usefully used for preparing flavorful liquor.

Description

Saccharomyces cerevisiae 213, produced in high concentrations of caprolate, and a method for producing alcoholic beverages using the same.

The present invention relates to a strain of Saccharomyces cerevisiae JY218 for producing fatty acid ethyl esters, preferably ethyl caprolate, ethyl caprate or ethyl myristicate in high concentration, and a liquor using the same. More specifically, the present invention relates to a mutant strain Saccharomyces cerevisiae JY218 strain obtained using JY174, which is an ethanol fermented yeast producing high caprolate ethyl as a parent strain, and a method for producing liquor using the same.

Recently, consumers' desire for alcoholic beverages has diversified, and the production and sales of distilled shochu with excellent flavor in addition to general distilled shochu is increasing, and mixed shochu with distilled shochu added to dilute shochu has become popular. In addition, in the case of makgeolli and Japanese sake, which have recently been increasing in consumption, the fragrance component plays a very important role in showing excellent quality.

Ingredients affecting the aroma of the liquor include higher alcohols such as butyl alcohol, isoamyl alcohol and 2-phenethyl alcohol and ethyl acetate, an ester compound thereof. acetates, isoamyl acetate, 2-phenethyl acetate, and other alcohol acetate esters. In addition, ethyl caproate, ethyl caprate, and And alcohol fatty acid esters such as ethyl caprylate. Among these fragrance components, fragrance components such as isoamyl acetate, ethyl caproate, and 2-phenethyl acetate are rich in Japanese sake, Yin-Yang liquor. Known as In particular, ethyl caprolate is a substance produced by esterification of capric acid, a fatty acid, and ethanol, the main ingredient of liquor. It is also known as an important ingredient in liquors such as shochu and Takju.

Ethyl caprolate is a cerulein-resistant mutant that is resistant to cerulenin, an antibiotic that inhibits the growth of yeast by specifically inhibiting Fatty Acid Synthase (FAS), which is responsible for fatty acid synthesis in yeast. It is known that in some of them, the amount of heavy chain fatty acid is increased and accordingly, the amount of ethyl caprolate is also increased (Eiji ICHIKAWA, et al ., Agric. , 1991] and [ARIKAWA YUKIHIKO, et al ., Journal of Bioscience and Bioengineering , 90 (6), 675-677, 2000).

Korean Patent No. 448515 discloses an ethanol fermentation yeast producing high caprolate ethyl, but the amount of ethyl caprolate produced by its mutants is not sufficient yet.

Therefore, the present inventors mutated by treating the ethanol fermentation strain Saccharomyces cerevise JY174 with a mutagen such as Ethyl Methanesulfonate (EMS) mutant Saccharomyces is a mutant that produces a high concentration of ethyl capronate component Celebrity JY218 was selected, and the present invention was completed by confirming that the mutant strain produced high concentrations of caprolate ethyl, ethyl caprylate and ethyl myristicate.

[Non-Patent Document 1] Eiji ICHIKAWA, et al ., Agric . Biol . Chem . , 55 (8), 2153-2154, 1991

[Non-Patent Document 2] ARIKAWA YUKIHIKO, et al ., Journal of Bioscience and Bioengineering , 90 (6), 675-677, 2000

[Patent Document 1] Republic of Korea Patent No. 10-0448515

Accordingly, it is an object of the present invention to provide a serulinine resistant strain Saccharomyces cerevisiae strain which produces a high concentration of fatty acid ethyl ester.

Another object of the present invention is to provide a method for producing liquor using the mutant strains.

In order to achieve the above object, the present invention provides a serulinine resistant strain Saccharomyces cerevisiae JY218 (KACC 91696P) for producing a high concentration of fatty acid ethyl ester.

In order to achieve the above another object, the present invention provides a method for producing liquor using the mutant strain.

In the production of alcoholic beverages when the fatty acid ethyl ester high-productivity cerulein-resistant mutant Saccharomyces cerevisiae JY218 is used, the main ingredient can be improved by producing high concentrations of caprolate ethyl as a fragrance component.

The present invention provides a serulenin-resistant strain Saccharomyces cerevisiae JY218 strain (KACC 91696P), which produces a high concentration of fatty acid ethyl ester. The fatty acid ethyl ester is characterized in that ethyl caproate, ethyl caprate or ethyl myristic acid.

Therefore, Saccharomyces cerevisiae JY218 strain of the present invention is characterized by high production of ethyl caprolate, ethyl caprate and ethyl myristic acid with fatty acid ethyl esters.

The strain is obtained by treating the mutagen such as ethyl methanesulfonate (EMS) with Saccharomyces cerevisiae JY174 as a parent strain. Specifically, the parent strain JY174 was treated with a mutagen to obtain a serulenin-resistant mutant strain first, and after obtaining a candidate strain producing a high concentration of fatty acid ethyl ester in the cerulein-resistant mutant strain, the ethanol fermentation ability with the parent strain, in particular The strain producing the highest concentration of ethyl caproate was selected as the JY218 strain of the present invention.

 Compared with the parent strain JY174, the fatty acid ethyl ester high-productivity serulinine resistant mutant Saccharomyces cerevisiae JY218 has similar bacteriological properties and ethanol fermentation capacity, but exhibits the following differences:

1) Cerulenin resistance: The parent strain JY174 is not resistant to 45 μM of cerulein, a type of fungal antibiotic, whereas JY218 of the present invention is resistant to 45 μM of cerulein;

2) Ethyl caprolate content: When preparing the liquor using the JY218 of the present invention, about 20 times more than the parent strain JY174, compared to JY-210 (KFCC-11287, Jinro Co., Ltd.), a high productivity patented strain of ethyl caprolate Increased the caprolate ethyl content by at least about two times (see Tables 4 and 8);

3) Fatty acid ethyl content: Ethyl Caprate and Ethyl Myristate content increased by about 80 times and 240 times, respectively, compared to the parent strain JY174 when preparing the liquor using JY218 of the present invention. (See Table 7).

As mentioned above, the JY218 mutant strain of the present invention has the same ethanol fermentation ability as the parent strain, but shows very good ethyl caprolate production ability compared to the parent strain, as well as ethyl caprate and ethyl myristic acid. The production ability of fatty acid ethyl esters is also superior to the parent strain. In addition, the JY218 mutant of the present invention is characterized by exhibiting resistance to cerulenin.

Therefore, the JY218 strain of the present invention can be usefully used in the liquor manufacturing method, the present invention in the production of liquor by ethanol fermentation of starch and sugar raw materials using Saccharomyces cerevises, saccharo of the present invention Provided is a method for preparing liquor using Meises Cervizer JY218. The alcohol production method refers to a conventional method well known to those skilled in the art, and means using the JY218 strain of the present invention in place of Saccharomyces cerevisiae, a yeast used in a conventional alcohol production method. On the other hand, the liquor is preferably alcohol, sake, Takju, fruit wine or distilled shochu, but is not limited thereto.

[Example]

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 Selection of Cerulenin-resistant Mutant

Saccharomyces cerevisiae JY174 (JINRO Co., Ltd.), a yeast for ethanol fermentation, was cultured in a 10 ml flask containing yeast malt broth (YMB) at 30 ° C. for 24 hours to obtain cells. Was washed three times with sterile water. Subsequently, the cells were suspended in sodium phosphate buffer (pH 7.0) to which ethyl methanesulfonate (EMS) was added at 5%, followed by shaking at 150 ° C. at 30 ° C. for 60 minutes. The mutated cells were washed with 5% sodium thiosulfate solution and suspended in sterile water, and then the suspension was plated 100 μl into agar medium for screening cerulenin-resistant mutants having the composition shown in Table 1 below. Colonies that grow by incubating for 1 week at ° C. were selected as the cerulenin resistant mutant strains.

 Medium composition for selection of cerulein resistant strains Yeast Nitrogen Base (without Amino Acids)
Glucose
Cerulenin
Baby 0.2%
2.0%
45 μM
2.0%

Cerulenin-resistant mutants that grow on agar medium for selection of cerulein-resistant mutant strains containing 45 μM of cerulein when mutated cultures of the parent strain JY174 yeast (10 ⁷ / ml) were treated according to the above-mentioned method. colony number as the average, about 27 check serul obtained probability of renin-resistant mutant strain is 2.7 × 10 ^- it was found to be ^six.

Example 2: Selection of high yielding mutant strain of ethyl caprolate

In order to determine the ethyl caprolate production capacity of 154 colonies of cerulein resistant mutants selected through a total of 12 mutations in the manner described in Example 1 were tested as follows.

Water was added to the white soup prepared using rice and Korean mold ( Aspergillus kawachii ), and the juice (20 Brix) was prepared by saccharifying for 48 hours at 50 ° C. The selected cerulein-resistant mutants were incubated for 24 hours at 30 ° C. using YMB medium (Difco) at 0.1 ° C. in the broth, respectively, and fermented at 25 ° C. for 7 days. The ethyl caprolate content of the fermentation broth obtained above was analyzed by gas chromatography (Gas Chromatography, GC) under the following conditions to select six mutant strains producing more than 5ppm ethyl caprolate and the results are shown in the following table. 2 is shown.

<GC analysis conditions>

1.Column: EC-WAX (0.53 mm × 30 m × 1.2 μm)

2. Oven temperature: 45 ℃ (3 minutes)-> 20 ℃ / min-> 180 ℃ (10 minutes)

3. Carrier gas: He (6.5 psig)

4. Split ratio: 10: 1

5. Detector: FID (300 ℃)

Ethyl caprolate content in cerulein-resistant mutant juice fermentation broth (unit: ppm)  JY174 (parent strain) 0.2  JY174CRN3008 5.1 JY174CRN6039 5.4 JY174CRN7022 6.5 JY174CRN9006 5.2 JY17CRN10010 5.7 JY174CRN12012 7.8

As shown in Table 2, JY174CRN12012 produced ethyl caprolate at about 39 times higher concentration (ie 7.8 ppm) than the parent strain JY174 among the mutant strains producing high concentration of caprolate ethyl in the juice fermentation broth. Accordingly, the present inventors named the serulinine resistant mutant JY174CRN12012, which produces the highest concentration of ethyl caproate, among the selected strains as Saccharomyces cerevisiae JY218 strain, to the National Agricultural Science Institute Agricultural Genetic Resource Center as of December 23, 2011. Deposited with Accession No. KACC 91696P.

Example 3 Preparation of Yakju Using Ethyl Caproate High Production Mutant

In order to check whether the Saccharomyces cerevisiae JY218 strain obtained in Example 2 produces high concentrations of caprolate ethyl at the time of producing the actual medicines, after preparing the medicines according to the conventional medicine preparation method using the strains, The content of ethyl caprolate in the chemical liquor was measured. At this time, as a control group was prepared by using the parent strain JY174.

First, rice wine was prepared by using a conventional method for preparing alcohol, as shown in Table 3 below.

Drunk and drunk ratio of Yakju
Drunk Drunkenness Primary Secondary Yeast (Raw Ingredients) 25% Capital increase (compared to raw material) 37.5% 37.5% Water (relative to raw material) 37.5% 61.25% 61.25% When to add 4th day of drinking 5th day of drinking

The content of ethyl caproate in the chemical liquor prepared by fermentation at 25 ° C. for 14 days after the addition of the second addition was measured using gas chromatography as described in Example 2. The measurement results are shown in Table 4 below.

Ethyl caproate content in Yakju prepared using JY174 and JY218 JY174 JY218 Ethyl caproate (mg / l) 0.5 10.0

As shown in Table 4, it was confirmed that the fermentation broth using the parent strain JY174 contained 0.5 mg / L ethyl caprolate, but fermented using the ethyl caprolate high productivity mutant JY218 according to the present invention. It was found that the content of ethyl caprolate in the medicinal herbs increased about 20 times larger than that of the parent strain.

Example 4: Determination of ethyl caprolate content in distilled liquor stock solution

In order to confirm the actual capronate ethyl content of distilled shochu prepared using the JY218 strain according to the present invention, distilled shochu was prepared by the conventional method from the medicinal liquor of Example 3 and the content of ethyl caprolate in the stock solution of soju Measured. At this time, the distillation shochu was prepared using the parent strain JY174 as a control.

The crude liquor prepared in Example 3 was distilled to a ethanol concentration of 45% at atmospheric pressure using a single distillation machine to prepare a stock solution of distilled soju. The ethyl caproate content in the distilled soju stock solution thus prepared was analyzed using gas chromatography. The measurement results are shown in Table 5 below.

Ethyl Capronate Content in Distilled Soju JY174 JY218 Ethyl caproate (mg / l) 1.8 27.3

As shown in Table 5, the ethyl caprolate content in the distilled soju stock solution produced using the high caprolate ethyl acetate high yielding cerulein resistant mutant JY218 increased about 15 times, compared with the case of using the parent strain JY174. Could know.

Example 5: Sensory Evaluation

For 20 adult men and women, the sensory properties of the main strain and the distilled soju stock solution prepared using the parent strain and ethyl caproate high productivity mutant JY218 in Examples 3 and 4 were evaluated, and the results are shown in Table 6 below. It was.

Sensory Preferences of Medicinal Alcohol and Distilled Soju Stocks Prepared Using JY174 and JY218 division incense zest preference Yakju JY174 3.1 3.2 3.1 JY218 3.9 4.2 4.0 Distilled Shochu JY174 3.2 3.3 3.3 JY218 4.5 4.4 4.4 5: very good, 4: good, 3: normal, 2: bad, 1: very bad

As shown in Table 6, the sensory preferences of medicinal liquor and distilled soju prepared using JY218 of the present invention were found to be greatly improved as compared with those of medicinal liquor and distilled soju prepared using the parent strain JY174. . Therefore, it can be seen that using the JY218 strain of the present invention can greatly contribute to the improvement of flavor and flavor of the main alcohol to produce high-quality liquor preferred by consumers.

Example 6 Comparison of Ethyl Caprate and Ethyl Mistrate

In order to compare the ability to produce ethyl capric acid and ethyl myristic acid of the JY218 strain of the present invention with the parent strain JY174, ethyl capric acid and ethyl myristic acid in the chemical stocks were prepared by the same method as described in Example 3. The content was measured. The measurement results are shown in Table 7 below.

Ethyl caprate and ethyl myristic acid content in medicinal liquors prepared using JY174 and JY218 JY174 JY218 Ethyl Caprate (mg / L) 0.06 4.9 Ethyl myristin (mg / l) 0.04 9.4

As shown in Table 7, the parent strain JY174 failed to produce ethyl capricate and ethyl myristic acid in the chemical strain, but the JY218 strain of the present invention produced a considerable amount of ethyl capricate and ethyl myristic acid. I could confirm that.

Example 7 Comparison of Ethyl Caprolate Production Capacity of JY218 and JY210 (KFCC-11287)

In order to compare ethyl caprolate production ability of the JY218 strain of the present invention and the existing high productivity of the caprolate ethyl bacterium strain Saccharomyces cerevisiae JY210 (KFCC11287, Jinro Co., Ltd.), in the same manner as in Examples 3 and 4 After preparing the soju stock solution, the ethyl caprolate content was compared. The measurement results are shown in Table 8 below.

Comparison of Ethyl Caprolate Production Capacity of JY218 and JY210
Ethyl caproate (mg / l) Yakju Distilled shochu stock solution JY218 10.0 27.3 JY210 5.1 12.2

As shown in Table 8, the JY218 strain of the present invention was confirmed to produce ethyl caprolate at a high concentration of about 2 times as compared to the conventional high yielding patented capillary protein JY210.

Agricultural Biotechnology Research Institute KACC91696 20111223

Claims (4)

Cerulenin-resistant strain Saccharomyces cerevisiae JY218 (KACC91696P), which produces ethyl caprolate, ethyl caprate and ethyl myristic acid. delete A method of preparing liquor by ethanol fermentation of starch and sugar raw materials using Saccharomyces cerevisiae JY218 (KACC 91696P). The method of claim 3, wherein
The liquor is characterized in that the wine, sake, Takju, fruit wine or shochu.