KR20160115224A - Saccharomyces cerevisiae jy231 strain with high productivity of flavor components and process for preparing alcoholic liquors using same - Google Patents

Abstract

The present invention relates to a Saccharomyces cerevisiae JY231 yeast and a manufacturing method of alcoholic liquors using the same. The yeast exhibits the resistance to TFL, PFP and cerulenin, and produces high concentrations of isoamyl acetate, 2-penethyl acetate, and ethyl caproate, known as main flavor components of alcoholic liquors, thereby manufacturing the alcoholic liquors with excellent flavor by using the same.

Description

 FIELD OF THE INVENTION [0001] The present invention relates to a process for producing a high-yielding saccharose,

The present invention relates to a method for producing a perfume composition having high resistance to 5,5,5-trifluoro-DL-leucine (TFL), p-fluorophenylalanine (PFP) and cerulenin, Saccharomyces cerevisiae JY231 yeast, a method for producing a mainstream using the yeast, and a mainstream produced by the method.

Among the aroma components of alcoholic beverages, the fragrance components of esters such as isoamyl acetate, ethyl caproate and 2-phenethyl acetate, which are abundant in high-grade Japanese yin and yang, Is known to have a great influence on the quality of the liquor due to its excellent sensory properties. In particular, 2-phenethyl acetate, an ester of 2-phenylethanol and acetic acid, has a similar aroma to roses and honey, and is a very important aroma component in alcoholic beverages.

As a method for producing a mainstream product having excellent perfume sensory properties, it is possible to use excellent raw materials (fruits, grains, plants, etc.) containing a large amount of precursors such as the above-mentioned fragrant or perfume ingredients, pretreatment of raw materials, fermentation, , And utilization of brewing microorganisms (fungi, yeast, lactic acid bacteria, etc.) which produce fragrance components at high concentration.

However, in the case of cereal fermented liquor, not only is the aroma characteristic of the raw material weak, but also there is a limit to improving the perfume sensory property by improving the manufacturing process such as pretreatment, fermentation, distillation, aging and the like of the raw material. Therefore, it is considered that it is more effective to improve the aroma-producing characteristics of brewing microorganisms used for fermentation, in particular yeast, in order to improve the aroma sensation of the fermented mainstream of grain.

Generally, it is known that saccharomyces cerevisiae used for fermentation of liquor synthesizes isoamyl acetate. Because leucine is a major precursor of isoamyl acetate synthesis, the amount of isoamyl acetate increases with increasing leucine content in yeast cells. However, when the intracellular leucine content is increased, the α-isopropyl maleate synthase (hereinafter referred to as "α-IPM synthase"), which is a rate-regulating enzyme of the leucine synthesis pathway, is inhibited by leucine, Becomes difficult. Therefore, in order to select a yeast that produces isoamyl acetate at a high concentration by promoting the synthesis of leucine, the feedback inhibition mechanism of α-IPM synthesizing enzyme by leucine should be canceled. Therefore, a mutant having resistance to 5,5,5-trifluoro-DL-leucine (TFL), which is one of the derivatives of leucine, is released from the inhibition of the feedback inhibition of the? -IPM synthase to produce an excess of leucine, Isoamyl acetate can also be produced in excess (Ashida, S. et al ., Agric. Biol. Chem ., 51 (8), 2061-2065, 1987).

2-phenylethanol and 2-phenethyl acetate are inhibited by phenylalanine and tyrosine, respectively. Therefore, a mutant having resistance to p-fluorophenylalanine (PFP), which is one of the derivatives of phenylalanine, is a compound that inhibits the feedback of 3-deoxy-D-arabinoseheptulosonate-7- phosphate (DHAP) (Fukuda, K., et al ., Agric. Biol. Chem ., 54 (1), 269-271, 1990).

Since ethyl caproate is produced from medium chain fatty acids and ethanol, it is closely related to the content of medium chain fatty acids in yeast cells. For this reason, studies have been reported to release inhibition of the heavy chain fatty acid feedback of fatty acid synthase II to increase the amount of ethyl caproate produced. It has been reported that a mutant strain resistant to cerulenin, which is one of the double-chain fatty acid derivatives, releases the inhibition of the feedback of fatty acid synthase II by heavy chain fatty acids to produce ethyl caproate at a high concentration (Ichikawa E, et al . Agric. Biol. Chem ., 55 (8), 2153-2154, 1991).

The present inventors selected saccharomyces cerevisiae mutants having resistance to TFL, PFP and cerulenin, and conducted alcohol fermentation tests on these TFL, PFP and cerulenin resistant mutants to determine the presence of isoamyl acetate, 2-phenethyl The present invention has been accomplished by developing saccharomyces cerevisee yeast which is capable of biosynthesizing ingredients such as acetate and ethyl caproate at high concentration.

[Non-Patent Document 1] Ashida, S. et al ., Agric. Biol. Chem ., 51 (8), 2061-2065, 1987

[Non-patent Document 2] Fukuda, K., et al ., Agric. Biol. Chem ., 54 (1), 269-271, 1990

[Non-Patent Document 3] Ichikawa E, et al ., Agric. Biol. Chem ., 55 (8), 2153-2154, 1991

It is an object of the present invention to provide a brewing yeast which is highly resistant to TFL, PFP and cerulenin, and which produces isoamyl acetate, 2-phenethyl acetate and ethyl caproate at a high concentration.

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

It is still another object of the present invention to provide a liquor produced using the above method.

To achieve the above object, the present invention provides Saccharomyces cerevisiae JY231 yeast (Accession No .: KACC93218P).

According to another aspect of the present invention, there is provided a method for producing a mainstream, comprising fermenting starch and saccharide raw materials with ethanol using Saccharomyces cerevisiae JY231 yeast (accession number: KACC93218P).

According to another aspect of the present invention, there is provided a liquor produced by using the production method.

The saccharomyces cerevisiae JY231 yeast of the present invention exhibits resistance to TFL, PFP and cerulenin and produces isoamyl acetate, 2-phenylacetate and ethyl caproate, which are major fragrance components of the mainstream, at a high concentration, It is possible to produce a liquor with excellent flavor.

The present invention provides Saccharomyces cerevisiae JY231 yeast (accession number: KACC93218P).

The JY231 yeast of the present invention is TFL, PFP and cerulenin resistant mutants.

The JY231 yeast of the present invention is characterized by producing a high concentration of azaamyl acetate, 2-phenethyl acetate and ethyl caproate.

The content of the isoamyl acetate in the ethanol fermentation broth prepared using the JY231 yeast is 65 ppm or more, for example, 70 ppm, the content of 2-phenethyl acetate is 45 ppm or more, for example, 48 ppm, the content of ethyl caproate is 5 ppm or more, for example, 5.2 ppm.

The yeast is obtained by repeatedly treating yeast Saccharomyces cerevisiae JY174 yeast with a mutant such as ethylmethane sulfonate (EMS), and then isolating yeast producing high concentration of isoamyl acetate while showing tolerance to TFL (Reference Example 3). After the primary selection yeast was treated with a mutagen source, yeast that produced 2-phenethyl acetate at a high concentration while exhibiting resistance to TFL and PFP was secondarily selected (Reference Example 4). Finally, a yeast which is highly resistant to TFL, PFP and cerulenin, and which produces isoamyl acetate, 2-phenethyl acetate and ethyl caproate at a high concentration, is finally obtained by treating the mutant source with the secondarily selected yeast (Example 1).

The yeast of the present invention has similar bacteriological properties and ethanol efficacy as the yeast Saccharomyces cerevisiae JY174 yeast, but exhibits the following differences.

1) Resistance to TFL, PFP and cerulenin: Saccharomyces cerevisiae JY174 yeast was resistant to 0.3 mM TFL, 0.3 mM PFP and 5 μM cerulenin (see Table 1) Saccharomyces cerevisiae JY231 yeast shows versatility at concentrations of 10 mM, 8 mM and 10 μM against TFL, PFP and cerulenine, respectively (see Reference Examples 3 and 4).

2) Content of isoamyl acetate, ethyl caproate and 2-phenethyl acetate: The aroma component of the distillate prepared by using the Saccharomyces cerevisiae JY231 yeast of the present invention was measured. As a result, The content of isoamyl acetate was about 40 times, the content of ethyl caproate was about 4 times, and the content of 2-phenylacetate was about 44 times higher than that of using Serebee JY174 yeast (see Table 6).

In addition, compared with the fermentation components obtained from the fermentation broth obtained from the ethanol fermentation by the three-stage method, the content of isomyl acetate, ethyl caproate and 2-phenylacetate was about 28 Fold, about 10-fold, and increased about 28-fold, about 7-fold, and about 9-fold, respectively, compared with the fermentation broth of Saccharomyces cerevisiae JY230 yeast (KACC93165P) (Test Example 3).

As mentioned above, the yeast of the present invention has the same ethanol efficacy as the parent yeast but is resistant to TFL, PFP and cerulenin, and isoamyl acetate, 2-phenethyl acetate and ethyl Caproate production capacity. As a result, the sensory evaluation showed that the fruit and flower fragrance were stronger and the wake - up was relatively smaller than that of the parent yeast. Therefore, the yeast of the present invention can be usefully used in the production of a liquor having a high content of a perfume component.

On the other hand, the present invention provides a method for producing a mainstream comprising fermenting starch and saccharide raw materials with ethanol using Saccharomyces cerevisiae JY231 yeast (accession number: KACC93218P).

A specific method for producing the above-mentioned alcohol may be a conventional method well known to those skilled in the art. For example, JY231 yeast (accession number: KACC93218P) of the present invention may be used in place of saccharomyces cerevisiae, .

The present invention also provides a liquor produced by using the above production method.

The mainstream is preferably a sake, sake, wine, fruit wine or soju, but the present invention 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.

Reference example  One: TFL , PFP  And Cerulenine  About JY174  Confirm yeast growth inhibition concentration

In the present invention, in order to confirm the minimum inhibitory concentration (MIC) for TFL, PFP and cerulenin of Saccharomyces cerevisiae JY174 yeast (Hite Jinro Co., Ltd.), yeast nitrogen yeast containing no amino acid 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM and 0.5 (w / v) in 5 ml of a minimal medium containing 0.2% (w / v) nitrogen base w / o amino acids; Difco) and 2.0% mM TFL (Sigma) were added thereto, and the above-mentioned JY174 yeast was inoculated and cultured at 30 DEG C for 2 days. Then, the amount of cells was measured at the absorbance (600 nm) of each culture solution to determine the minimum inhibitory concentration of TFL against the yeast.

In the same manner as above, PFP (Sigma) and 0 μM, 5 μM, 10 μM, 15 μM and 20 μM of cerulenin (Sigma) were added at 0 mM, 0.1 mM, 0.2 mM, 0.3 mM, The MIC was measured. The results are shown in Table 1 below.

TFL PFP Cerulenine MIC 0.3 mM 0.3 mM 5 [mu] M

As shown in Table 1, it was confirmed that JY174 yeast did not proliferate in the presence of TFL of 0.3 mM or more, PFP of 0.3 mM or more, and cerulenin of 5 μM or more.

Therefore, in the selection agar plate culture medium for selection of mutants of resistance to TFL, PFP and cerulenin, the concentration of TFL, PFP and cerulenin in the JY174 yeast, considering their frequency of occurrence of MIC and resistant mutant, 10 mM, 8 mM and 10 μM, respectively.

Reference Example 2: Mutation treatment

Yeast cells were recovered from 1 ml of the culture medium of Saccharomyces cerevisiae JY174 yeast cultured in a YM liquid medium for 24 hours by centrifugation (3,000 rpm and 5 minutes), and then washed with sterilized water three times in the same volume Respectively. The washed cells were suspended in the same volume of sterilized water containing 5% (v / v) EMS (Sigma) and treated with shaking (100 rpm) in a shaking incubator at 30 ° C for 90 minutes. The treated cells were recovered by centrifugation (3,000 rpm and 5 minutes), washed once with 5% (w / v) solution of the same amount of sodium thiosulfate and twice with sterilized water, Suspended in sterile water.

Reference Example 3 Selection of TFL Resistant Isoamyl Acetate High Productivity Mutant

200 μl of the JY174 yeast suspension treated as described in Reference Example 2 was added to 0.2 ml of a 0.2% (w / v) amino acid-free yeast nitrogen base (Difco), 2.0% of glucose, a TFL- (w / v) and agar (1.8% (w / v)) and cultivated at 30 ° C for 5 days for growth. The 316 colonies were selected and selected as TFL resistant mutants.

The TFL resistant mutants selected in the above were inoculated in 50 ml of 20 brix each, and then cultured at 25 ° C for 5 days. The content of isoamyl acetate in the nutrient broth of each varieties was analyzed by gas chromatography (GC). Fourteen strains of isoamyl acetate increased by more than 5 times in comparison with JY174 yeast were selected as TFL resistant isoamyl acetate high productivity variant candidates.

Fourteen mutants selected from the above were subjected to ethanol fermentation using a three-stage injection method as shown in Table 2 according to a conventional method for producing rice bran using rice as a raw material.

Under Syrup Day 4 Day 5 Curse (ratio) 25g (25%) Increase (ratio) 37g (37%) 38g (38%) Water (water supply ratio) 40 (160%) 60 (160%) 60 (160%) Seed culture 0.5 * Fermentation for 25 to 14 days after the second filling

The fermentation broth of each fermented mutant strain was analyzed, and a mutant strain that produces isoamyl acetate 20 times or more as compared with JY174 yeast was selected as the isoamyl acetate high productivity TFL resistant mutant (hereinafter referred to as "JY174TR13026 mutant").

The content of isoamyl acetate and isoamyl alcohol in JY174 yeast and JY174TR13026 mutant strains was analyzed by GC and the results are shown in Table 3 below.

Strain name Isoamyl acetate (ppm) Isoamyl alcohol (ppm) JY174 2.6 234 JY174TR13026 68.3 431

Reference example  4: TFL  And PFP versatility  Resistant Isoamyl  Acetate and 2- Phenethyl  Selection of acetate high productivity mutants

JY174TR13026 selected in Reference Example 3 was subjected to mutation treatment in the same manner as in Reference Example 2 above. 200 μl of the thus obtained suspension was added to TFL containing 10 mM TFP and 8 mM PFP, 0.2% (w / v) amino acid-free yeast nitrogen base (Difco), glucose 2.0 The cultured colonies were cultured at 30 ℃ for 5 days and selected as TFL and PFP resistant mutants.

The selected TFL and PFP resistant variants were inoculated in 50 ml of 20 brix each, and then cultured at 25 ° C for 5 days. Analysis of the content of 2-phenylacetate in the nutrient broth of each mutant strain was analyzed by GC. Five species with increased 2-phenethyl acetate content by more than 10 times compared with JY174TR13026 were obtained from TFL and PFP resistant isoamyl acetate 2-phenylacetate high productivity variant candidates.

Then, the five kinds of mutant candidates were subjected to ethanol fermentation by a three-step method in the same manner as in Reference Example 3. Analysis of the fermentation broth of each fermented strain showed that the production of isoamyl acetate was similar to that of JY174TR13026, while the mutant strains producing about 13 times more of 2-phenylacetate were transformed with isoamyl acetate and 2-phenethyl acetate high productivity TFL and PFP resistant (Hereinafter referred to as "JY174TPR19073 mutant strain").

The content of isoamyl acetate, 2-phenethyl acetate and 2-phenylethanol in the mutants JY174TR13026 and JY174TPR19073 was analyzed by GC and the results are shown in Table 4 below.

Strain name Isoamyl acetate
(ppm) 2-phenethyl acetate
(ppm) 2-phenylethanol
(ppm) JY174TR13026 65 3.8 55 JY174TPR19073 67 48 93

Example  One: TFL , PFP  And Cerulenine versatility  Resistant Isoamyl  Acetate, 2- Phenethyl  Acetate and Ethylcaproate High Productivity Mutant Selection

Mutants were induced in the same manner as in Reference Example 2 using TFL and PFP multifunctional isoamyl acetate selected in Reference Example 4 and JY174TPR19073 as a 2-phenethyl acetate high productivity yeast strain as parent strains. Thereafter, TFL, PFP, and cerulenin versatile resistant strains containing 10 mM TFL, 8 mM PFP and 10 μM of cerulenin, 0.2% w / w amino acid-free yeast nitrogen base (Difco) 16 colonies cultured at 30 ° C for 5 days were plated on TFL, PFP and cerulenin-resistant mutant strain.

Sixteen multipurpose mutants obtained from the above were inoculated in 50 ml of 20 brix each, and then cultured at 25 ° C for 5 days. Analysis of the content of ethyl caproate in the nutrient culture medium having excellent sensory properties among the nutrient cultures of the respective mutant strains showed that the content of ethyl caproate increased more than twice as compared with JY174TR19073 as TFL, PFP, and isoamyl acetate resistant to cerulenin, Ethyl acetate and ethyl caproate.

Next, the three selected mutants were subjected to ethanol fermentation using a three-stage method using rice as a raw material as in Reference Example 3, followed by the addition of isoamyl acetate, 2-phenethyl acetate, and ethyl caproate Was analyzed by GC. The JY174TPCR1419 mutant, which is similar to JY174TPR19073 and has an increased amount of ethyl caproate about 9-fold, was treated with TFL, PFP, and isoamyl acetate resistant to cerulenin, 2-phenethyl acetate and ethyl caproate Were selected as high productivity variants.

The results of analysis of isoamyl acetate, 2-phenethyl acetate and ethyl caproate contents of the mutants JY174TPR19073 and JY174TPCR1419 are shown in Table 4 below.

Strain name Isoamyl acetate
(ppm) 2-phenethyl acetate
(ppm) Ethyl caproate
(ppm) JY174TPR19073 67 47 0.6 JY174TCPR1419 70 48 5.2

The present inventors named the final selected JY174TPCR1419 yeast as Saccharomyces cerevisiae JY231 and deposited it with the deposit number KACC93218P on Nov. 11, 2014 at the Korean Center for Agricultural Genetic Resources.

Test Example 1: Pilot test of JY231 yeast

A scale-up test was conducted on a second scale using a 500 L pilot to confirm the possibility of field application of the JY231 yeast obtained in Example 1. As a control, JY174 Yeast was used.

In the case of JY231 yeast, the alcohol fermentation rate was not significantly different from that of JY174 yeast, and the alcohol content in the final fermentation broth was not significantly different.

As shown in the following Table 6, the contents of isoamyl acetate, 2-phenylacetate and ethyl caproate, which are components of the flavor and aroma components contained in the distillation stock solution in which the final fermentation broth of JY231 yeast and JY174 yeast was distilled, In the distilled water of JY231. Specifically, isoamyl acetate increased about 40-fold, 2-phenethyl acetate increased about 44-fold, and ethyl caproate increased about 4-fold.

Strain name Alcohol content Isoamyl acetate 2-phenethyl acetate Ethyl caproate JY174 47.8% 9.3 ppm 4.1 ppm 2.9 ppm JY231 47.5% 359.0 ppm 174.5 ppm 11.2 ppm

From the above results, it was found that when the JY231 yeast according to the present invention was used, a distilled liquor having a significantly increased yin and yan flavor could be obtained without significantly changing the alcohol yield compared to the conventional JY174 yeast.

Test Example 2: Sensory evaluation of fermentation and distillation stock of JY231 yeast

In order to compare the sensory performances of the fermentation / distillation stock solution of the JY231 yeast obtained in Test Example 1 and the fermentation / distillation stock solution of the JY174 yeast, the fermentation / distillation stock solution was diluted to have an alcohol content of 20% (7 point scale) and preference test (7 point scale).

Table 7 shows the results of comparing the sensory characteristics of the pilot fermentation and distillation stock solutions of JY231 yeast and JY174 yeast.

Strain name Fruit flavor Fountain Wake preference JY174 3.87 3.91 3.90 3.08 JY231 5.66 5.08 3.03 3.73 1 point: very weak; 2 to 4 points: normal; 5 to 7: very strong

As shown in Table 7, the distillation stock of JY231 yeast was stronger than that of the distillation stock of JY174 yeast, and the fruit aroma and floral aroma were stronger and the wake was relatively smaller. In addition, preference for the distillation stock of JY231 yeast was higher than that of the fermentation and distillation of JY174 yeast. Therefore, it was confirmed that a main stream having excellent aroma characteristics could be prepared using Saccharomyces cerevisiae JY231 yeast.

Test Example  3: Analysis of fragrance content

(KACC91696P, Korean patent No. 1351431), 2-phenylacetate high-yield brewing yeast JY230 (KACC93165P, Korean patent No. 1497434), and biphenylacetate high yield brewing yeast JY174 Ethylene amyl acetate, 2-phenylacetate, and ethyl caproate high yield brewing yeast JY231 (accession number: KACC93218P) of the present invention were subjected to ethanol fermentation using a three-stage method using rice as a raw material as described in Reference Example 3 Respectively. The content of isoamyl acetate, 2-phenethyl acetate and ethyl caproate in the rice fermentation broth was analyzed and the results are shown in Table 8 below.

Strain name Isoamyl acetate
(ppm) 2-phenethyl acetate (ppm) Ethyl caproate
(ppm) JY174 2.6 1.7 0.5 JY218 2.6 1.7 10.0 JY230 2.5 7.4 0.6 JY231 70 48 5.2

The content of isoamyl acetate, 2-phenethyl acetate and ethyl caproate of the strain of the present invention was increased about 28 times, about 25 times and about 10 times, respectively, compared with the yeast fermented yeast strain JY174, and compared with that of JY218 yeast fermentation broth Amyl acetate was increased about 28 times and 2-phenylacetate was increased about 25 times. In addition, it was about 28 times, about 7 times, and about 9 times higher than that of JY230 yeast fermentation broth, respectively. Therefore, when the strain of the present invention is used, it contributes to the improvement of the quality of the food and the improvement of the flavor, and thus it is effective in manufacturing a high quality liquor rich in flavors preferred by consumers.

Korea Agricultural Genetic Resources Center KACC93218P 20141215

Claims (6)

Saccharomyces cerevisiae JY231 yeast (Accession No .: KACC93218P).
The method according to claim 1,
Wherein the JY231 yeast produces isoamyl acetate, 2-penetyl acetate, and ethyl caproate.
And fermenting the starch and saccharide raw materials with ethanol using Saccharomyces cerevisiae JY231 yeast (Accession No: KACC93218P).
The method of claim 3,
Wherein the mainstream is a sake, sake, wine, fruit wine or shochu.
A liquor produced by the method according to claim 3.
6. The method of claim 5,
Characterized in that the mainstream is liquor, sake, takju, fruit wine or shochu.