KR20140079199A - Saccharomyces cerevisiae jy230 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 JY230 yeast (KACC93165P) producing 2-phenyl ethanol and 2-pen ethyl acetate at a high concentration, and a method for manufacturing alcohol using same, wherein the yeast produces 2-pen ethyl acetate known as a main flavor component of alcohol at a high concentration, and thus, alcohol with an excellent flavor can be manufactured by using the same.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a yeast, and to a method for producing the same. [0002] The present invention relates to a yeast,

The present invention relates to Sacchromyces cerevisiae JY230 yeast (KACC93165P) which produces aroma components at a high concentration, a process for producing a liquor using the yeast, and a liquor produced by the process.

In recent years, the mainstream market has been changing drastically as consumers' demands for mainstream products have become more diverse and more luxurious, and demand for makgeolli has increased explosively.

One of the important factors that determine the quality of alcoholic beverages is the aroma property determined by the type and content of the aroma ingredient. The aroma components of alcoholic beverages can be roughly classified into those derived from raw materials and those produced by microorganisms used for fermentation.

Examples of the aroma component produced by microorganisms during alcohol fermentation include higher alcohols such as butyl alcohol, isoamyl alcohol and 2-phenethyl alcohol, and esters thereof, such as ethyl acetate ethyl acetate, isoamyl acetate and 2-phenethyl acetate. In addition, ethyl caproate, ethyl caprate, And ethyl caprylate are known. Among them, flavor components such as isoamyl acetate, ethyl caproate, and 2-phenethyl acetate are abundant in the finest Japanese sake, It is called "Yin - Yang" and it is known as a fragrance ingredient that is very important for the sensual characteristics of alcohol.

Particularly, 2-phenylacetate, which is an ester of 2-phenylethanol and 2-phenylethanol and acetic acid, is a very important perfume ingredient in the mainstream, which has a similar aroma to rose flower and honey.

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

However, according to the results of many researches so far, unlike the fruit fermented fruit, in which the aroma sensation is greatly influenced by the raw material, in the case of cereal fermented wine, the aroma characteristic of the raw material is not only weak but also the raw material is pretreated, fermented, It is known that there is a limit to improvement of aroma sensation through process improvement. Therefore, it is considered to be 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 these grains.

Korean Patent Application Laid-Open No. 2003-0079480 discloses a saccharomyces cerevisiae mutant yeast having high productivity of ethyl caproate and ethyl caprylate in relation to a yeast with improved aroma production characteristics, Open Publication No. 2003-0079481 discloses a saccharomyces cerevisiae mutant yeast having high productivity of isoamyl alcohol and isoamyl acetate.

On the other hand, 2-phenylethanol is biosynthesized by using phenylalanine, which is one of aromatic amino acids, as a precursor, and 2-phenylethanol synthesized in this way is converted into 2-phenethyl acetate by receiving acetic acid from acetyl-CoA through the action of alcohol acetyltransferase .

Kazuro Fukuda et al. Reported that the content of 2-phenylethanol and 2-phenethyl acetate in yeast fermentation broth was resistant to p-fluorophenylalanine (an analog of phenylalanine, hereinafter referred to as "PFP"), (Kazuro Fukuda et al. , Agric. Biol. Chem. , 54 (1), 269-271, 1990).

Therefore, in order to breed brewer's yeast which is biosynthesizing a large amount of 2-phenylethanol and 2-phenylacetate, the present inventors selected saccharomyces cerevisiae mutants having resistance to PFP, The present inventors have completed the present invention by developing a saccharomyces cerevisiae yeast which is capable of biosynthesizing 2-phenylethanol and 2-phenethyl acetate at a high concentration through an alcohol fermentation test.

Korean Patent Application Publication No. 2003-0079480 Korean Patent Application Publication No. 2003-0079481

Kazuro Fukuda et al., Agric. Biol. Chem., 54 (1), 269-271, 1990

Therefore, an object of the present invention is to provide a novel saccharomyces cerevisiae yeast with high productivity of a perfume ingredient.

Another object of the present invention is to provide a method for producing a mainstream having excellent perfume characteristics by using the saccharomyces cerevisiae yeast.

Another object of the present invention is to provide a main stream having excellent perfume characteristics produced by the above method.

According to the above object, the present invention provides Saccharomyces cerevisiae JY230 yeast (KACC93165P) which biosyntheses 2-phenylethanol and 2-phenylacetate at a high concentration.

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 JY230 yeast (KACC93165P).

According to still another aspect of the present invention, there is provided a main stream having excellent perfume characteristics produced by the method.

The saccharomyces cerevisiae JY230 yeast (KACC93165P) according to the present invention produces 2-phenylethanol and 2-phenethyl acetate, which are known as major fragrance components of the mainstream, at a high concentration, have.

The present invention provides Saccharomyces cerevisiae JY230 yeast (KACC93165P) resistant to p-fluorophenylalanine (PFP), which produces 2-phenylethanol and 2-phenethyl acetate at a high concentration.

The yeast can be obtained by repeatedly treating a mutant such as ethylmethane sulphonate (EMS) or the like with Saccharomyces cerevisiae JY174 yeast, which is a yeast strain, and then selecting a PFP resistant mutant strains. Then, 2-phenylethanol and 2- In a high concentration.

The saccharomyces cerevisiae JY230 yeast, a 2-phenylethanol and 2-phenylacetate high productivity PFP tolerance mutant of the present invention, has a mycological property and ethanol Efficacy is similar, but differs in the following respects:

1) PFP Resistance: The JY230 yeast of the present invention is resistant to PFP 10 mM whereas the yeast strain JY174 is resistant to 10 mM PFP;

2) Contents of 2-phenylethanol and 2-phenethyl acetate: The rice saccharification solution culture and the ethanol fermentation were carried out using Saccharomyces cerevisiae JY230 yeast of the present invention. As a result, the content of 2-phenylethanol And the content of 2-phenethyl acetate was increased about 4 to 6 times (see Tables 4 and 5).

As mentioned above, the Saccharomyces cerevisiae JY230 yeast of the present invention has the same ethanol efficacy as the parent yeast, but is resistant to PFP and has excellent 2-phenylethanol and 2-phenethyl Acetate production ability. 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.

The present invention also provides a method for producing a mainstream comprising fermenting starch and saccharide raw materials with ethanol using Saccharomyces cerevisiae JY230 yeast.

A specific method for producing the above-mentioned alcohol may be a conventional method well known to those skilled in the art. For example, the saccharomyces cerevisiae JY230 yeast of the present invention may be used instead of the saccharomyces cerevisiae, .

Also, according to the present invention, there is provided a main flavor excellent in flavor produced by the above-described method. The mainstream is preferably, but not exclusively, cauliflower, sake, takju, fruit wine or shochu.

Hereinafter, the present invention will be described in detail based on the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Reference Example 1: Confirmation of yeast growth inhibition concentration of PFP

To determine the minimum inhibitory concentration (MIC) of PFP against Saccharomyces cerevisiae JY174 yeast (Hite Jinro Co., Ltd.), yeast nitrogen base w / o amino acids (Difco ) Was added to 5 ml of a minimal medium containing 0.2% (w / v) glucose and 2.0% (w / v) glucose, and the yeast was inoculated and cultured at 30 ° C for 2 days After that, the amount of microbial cells was measured at the absorbance (600 nm) of each culture solution to determine the minimum inhibitory concentration (MIC) of PFP against the yeast.

As a result, as shown in Table 1, the MIC of PFP to the Saccharomyces cerevisiae JY174 yeast was confirmed to be 0.3 mM.

Reference Example 2: Mutation treatment

Yeast cells were recovered from 10 ml of the culture medium of Saccharomyces cerevisiae JY174 yeast cultured in a YM liquid medium for 24 hours by centrifugation (3,000 rpm, 5 minutes), and then repeatedly washed three times with the same amount of sterilized water Respectively. The washed cells were suspended in the same volume of sterilized water containing 5% (v / v) EMS (Sigma), and then treated with shaking (100 rpm) in a shaking incubator at 30 ° C for 70 minutes. The treated cells were recovered by centrifugation (3,000 rpm, 5 minutes), washed once with a solution of 5% (w / v) sodium thiosulfate in the same amount and twice with sterilized water, ≪ / RTI > in sterile water.

Reference Example 3: Selection of PFP-resistant mutants

200 μl of the mutant-treated yeast suspension as in Reference Example 2 was added to the PFP-resistant mutant strain-selecting agar plate medium (amino acid-free yeast nitrogen base (Difco) 0.2% (w / v), glucose 2.0% w / v), agar 1.8% (w / v)) and cultured at 30 ° C for 5 days.

Reference Example 4 Analysis of Fragrance Characteristics of PFP Resistant Mutant

(Step 1) Analysis of primary perfume characteristics

The PFP-resistant mutants selected in Reference Example 3 were inoculated in 5 ml of the saccharified solution (10 brix), respectively, and cultured at 25 캜 for 5 days. Each culture solution was subjected to sensory evaluation, and the content of 2-phenylethanol and 2-phenethyl acetate in the culture medium having excellent sensory properties was analyzed to select a second-perfume characteristic analysis candidate.

(Step 2) Secondary perfume characteristics analysis

The PFP-resistant mutants selected through the analysis of the primary aroma characteristics were subjected to ethanol fermentation using the rice as a raw material according to the conventional method for preparing the rice wine, as shown in Table 2 below.

The content of 2-phenylethanol and 2-phenethyl acetate in the eluent was measured and the content of the fermented fermented product was high and the distillate (45-50% alcohol, ) Were selected as 2-phenylethanol and 2-phenethyl acetate high-yielding flavor yeast final candidate yeast.

Example 1: Selection of PFP resistant mutants

In order to confirm the concentration of PFP most suitable for selecting 2-phenylethanol and 2-phenethyl acetate high productivity PFP resistant mutant strains, EMS was used as a mutagenic source in the same manner as in Reference Example 2 to prepare saccharomyces cerevisiae The JY174 yeast was mutagenized five times and then plated on a PFP resistant mutant selection medium containing PFP with different concentrations ranging from 0.5 mM to 10.0 mM and cultured.

The number of colonies grown in the selection medium containing PFP at each concentration was confirmed and three colonies having good growth among the colonies grown in each selection medium were selected to determine the amount of 2-phenethyl acetate in each colony And confirmed by the method of Reference Example 4. [ As a result, as can be seen from Table 3, the lower the PFP concentration of the selection medium, the higher the occurrence frequency of the PFP-resistant mutant but the productivity of 2-phenethyl acetate was not significantly improved. Therefore, the PFP concentration of the selection medium for selection of 2-phenylethanol and 2-phenethyl acetate high productivity PFP resistant mutants was found to be greatly improved although the frequency of the resistant mutants was very low, but the productivity of 2-phenethyl acetate was greatly improved 10 mM.

Example 2: Evaluation of primary perfume characteristics of PFP-resistant mutant strains

In the same manner as in Reference Example 2, EMS was used as a mutagen inducing agent, and the saccharomyces cerevisiae JY174 yeast was mutagenized 15 times in total. Then, the transformant was transformed into a transformant containing 10 mM PFP in the same manner as in Reference Example 3 And cultured to obtain a total of 19 colonies of PFP (10 mM) resistant mutant strains. Each of these mutants was subjected to sensory evaluation using the olfactory sense of each of the culture broths of each resistant mutant strain using the method of step 1 of Reference Example 4, and a culture solution having strong fragrance similar to rose flower or honey was selected, 2-phenylethanol and 2-phenethyl acetate content were analyzed.

As a result, as shown in Table 4, it was confirmed that the content of 2-phenylethanol and 2-phenethyl acetate of JY230 among the PFP-resistant mutants of 19 colonies was the highest.

The PFP resistant mutant Saccharomyces cerevisiae JY230 was deposited with the National Institute of Agricultural Biotechnology on Dec. 17, 2012 under accession number KACC93165P.

Example 3: Evaluation of secondary aroma characteristics of PFP resistant mutant strains

Ethanol fermentation was carried out using the three step method as in step 2 of Reference Example 4 using Saccharomyces cerevisiae JY230 yeast resistant to PFP 10 mM selected in Example 2.

As a result, as shown in Table 5, the content of 2-phenylethanol and 2-phenylacetate in the yeast fermentation broth of Saccharomyces cerevisiae, a yeast strain used for PFP resistant mutant selection, was found to be 61 ppm and 1.7 ppm, respectively . On the other hand, it was confirmed that the JY230 mutant selected from the above contained 159 ppm of 2-phenylethanol and 7.4 ppm of 2-phenethyl acetate in the fermentation broth.

Therefore, it was confirmed that a mainstream having excellent aroma characteristics can be produced using Saccharomyces cerevisiae JY230 yeast.

Agricultural Biotechnology Research Institute KACC93165 20121217

Claims (5)

Sacchromyces cerevisiae JY230 yeast (KACC93165P) producing 2-phenylethanol and 2-phenethyl acetate at high concentration.
Which comprises fermenting starch and saccharide raw materials with ethanol using Saccharomyces cerevisiae JY230 yeast (KACC93165P).
3. The method of claim 2,
Wherein the mainstream is a sake, sake, wine, fruit wine or shochu.
A liquor produced by the method of claim 2.
5. The method of claim 4,
Characterized in that the mainstream is liquor, sake, takju, fruit wine or shochu.