KR102168992B1 - Novel Yeast for Long Term Preservable Makgolli - Google Patents

Abstract

The present invention relates to a low-temperature susceptible mutant yeast for the production of raw makgeolli capable of low-temperature long-term storage, and to a method for producing raw makgeolli using the same, and even if the raw makgeolli prepared according to the present invention is stored for a long period, the original flavor of makgeolli is lost. It is easy to transport and store for a long time because there is no change in alcohol quality and no increase in alcohol content.

Description

Novel Yeast for Long Term Preservable Makgolli {Novel Yeast for Long Term Preservable Makgolli}

The present invention relates to a low-temperature susceptible new yeast for preparing raw makgeolli capable of long-term storage at low temperature, and more particularly, to a low-temperature-sensitive mutant yeast for preparing raw makgeolli capable of low-temperature long-term storage, and a method for producing raw makgeolli using the same .

Makgeolli has been called takju (濁酒), nongju (農酒), craft or hoeju since ancient times, and the best yeast for makgeolli is also called Ihwaju (梨花酒) because it is made when pear flowers are in bloom.

As is well known, even when makgeolli is distributed in a container such as a bottle, the taste changes due to the growth of yeast and the action of starch-degrading enzymes derived from koji, and eventually the quality changes with the increase of acidity and alcohol. Until now, it could not but be sold only within the produced region without crossing the boundaries of the region where it was produced, and despite its explosive popularity from foreigners, long-distance transportation to Japan and the United States was impossible.

Usually, makgeolli is final packaged for long-term storage and long-distance transportation

At the stage, high-temperature instantaneous sterilization is performed to commercialize it, and it is often classified as sterilized makgeolli according to the Liquor Tax Act. On the other hand, makgeolli (so-called raw makgeolli) in a state in which microorganisms such as yeasts are alive because it does not go through the sterilization process is made into a product. It is called raw makgeolli).

In the case of sterilized makgeolli, the shelf life of makgeolli can be extended compared to raw makgeolli, but due to high-temperature heat treatment, the tangy taste of the original makgeolli produced by fermentation disappears, and the freshness and refreshing sensation of the makgeolli are deteriorated. Therefore, in order to prevent deterioration during distribution, a sterilization process is carried out, but through the packaging stage, a separate carbon dioxide gas is injected through the investment of enormous facility cost to inject carbonic acid to restore the original taste of makgeolli. It was impossible to completely reproduce the taste of makgeolli.

The raw makgeolli is a cause of shortening the shelf life of the product by using the sugar contained in the mash due to the nature of the product, so that the alcohol content increases and the alcohol quality changes by using sugar even at low temperatures (less than 10℃). Has become.

Therefore, for the development of the makgeolli industry, it is essential to extend the shelf life of raw makgeolli, and research is continuing in various aspects to solve this problem. For example, in Korean Patent Nos. 166 and 395, natural additives such as licorice, garlic and ginger are added to makgeolli to increase the preservation period by adding natural additives such as licorice, garlic, and ginger to preserve the properties of makgeolli while maintaining the characteristics of makgeolli. In Korean Patent Laid-Open No. 1994-0011531, when the manufactured makgeolli is packaged, the air in the space between the can lid is put into carbon gas or nitrogen gas at a constant pressure, and It has also been proposed to prevent corruption or rancidity during long-term storage.

For the purpose of deteriorating the quality of these products, a technology called complete fermentation that completely consumes sugar during the fermentation process, as well as synthetic sweeteners (aspartame, etc.), which are not used by yeasts, are added to preserve the sweet taste when bottling the product. Is being used.

In addition, a method of prolonging the preservation period of raw makgeolli by inducing the activity of the yeast to a temporary stop state by keeping various beneficial bacteria and microorganisms such as yeast inside the raw makgeolli alive but maintaining a sleeping state has also been proposed. (Korea Patent Registration No. 1,218,903)

However, the methods known so far have lost the original flavor of makgeolli, resulting in lower marketability, and the effect of extending the actual shelf life of makgeolli was insignificant, and thus long-term transportation and storage problems could not be solved.

Accordingly, the present inventors developed a yeast mutant strain with reduced fermentability of yeast at low temperature, and when manufacturing makgeolli using the yeast mutant strain, the change in the quality of the alcohol and the increase in alcohol content even without the addition of synthetic sweeteners. By suppressing it, it was confirmed that the shelf life of the product could be extended, and the present invention was completed.

An object of the present invention is to provide a low temperature sensitive mutant yeast for the production of raw makgeolli.

Another object of the present invention is to provide a method of preparing raw makgeolli capable of long-term storage at a low temperature using the low temperature sensitive mutant yeast.

Another object of the present invention is to provide a raw makgeolli prepared by the above method and capable of long-term storage at a low temperature.

In order to achieve the above object, the present invention provides S-105 KCTC18678P to the low temperature sensitive mutant yeast Saccharomyces cerevisiae.

The present invention also provides a method for producing raw makgeolli capable of long-term storage at a low temperature, characterized in that the low-temperature-sensitive mutant yeast is used.

The present invention also provides a raw makgeolli prepared by the above method and capable of long-term storage at a low temperature containing the low-temperature-sensitive mutant yeast.

Even if the raw makgeolli manufactured according to the present invention is stored for a long time, the original flavor of makgeolli is not lost, and there is no change in alcohol quality and no increase in alcohol content, so long-term transportation and storage are easy.

1 shows the degree of glucose remaining in the culture medium after culturing a mutant strain selected as a low temperature sensitive strain according to the present invention at 15° C. for 3 days.

In the present invention, yeast having low temperature sensitivity was developed from yeast used for fermentation of raw makgeolli, and fermentation is normally performed at a normal fermentation temperature and the fermentability of yeast is weakened at low temperature. In order to develop a yeast mutant strain whose main quality is not altered, the yeast strain was mutated by UV treatment, and a strain whose growth stopped at low temperature was selected from among the mutated strains. Among the selected strains, a strain having reduced glucose consumption at 10° C., which is a storage temperature of raw makgeolli, was finally selected and selected as a low-temperature sensitive strain. In the case of makgeolli prepared by using the selected low-temperature-sensitive strain, and makgeolli prepared by the low-temperature-sensitive strain, there is little increase in alcohol content and little change in sugar content even during long-term storage, and makgeolli using wild-type yeast and In comparison, it was confirmed that the amount of glucose reduction during bottling storage significantly decreased.

Accordingly, in one aspect, the present invention relates to a low-temperature-sensitive mutant yeast Saccharomyces cerevisiae S-105 KCTC18678P.

In the present invention, the parent strain, Saccharomyces cerevisiae (SILesaffre, France) (product name: Lafayette) strain was treated with ultraviolet rays to mutate and grow at low temperature among the mutated strains. The strain to stop was selected. Among the selected strains, a strain having reduced glucose consumption at 10° C., which is a storage temperature of raw makgeolli, was finally selected and selected as a low-temperature sensitive strain.

In the present specification, "showing low temperature sensitivity" means "normally fermented at 20 to 30°C, and exhibited a fermentation capacity of less than 1/3 of commercial yeast at 10 to 15°C." The yeast used in the present invention can be obtained, for example, by the following method. For example, commercially available yeast (e.g., baker's yeast, sake yeast, wine yeast, brewer's yeast, miso soy sauce, etc.) is irradiated with known mutation induction methods, for example, ultraviolet rays, etc. Mycin, nystatin, etc.) were brought into contact and cultured at a low temperature (10°C), and cells that were unable to proliferate at this low temperature or very weak in proliferation were selected.

Colonies isolated by primary selection were transferred onto a YPG plate medium (1% yeast extract, 2% polypeptone, 3% glycerol, 2% agar), cultured at 30° C. for 24 hours to grow colonies, and dye agar medium thereon. (Yeast extract 0.5%, peptone 1%, sucrose 10%, bromcresorbabul 0.02%, agar 1%) was layered and incubated at 10°C for 6 to 12 hours, in the meantime, the color tone around the colony was When observed, the color tone of the strong fermentation ability of 10°C changes from purple to yellow, but in the strains lacking or weak fermentation ability, there is no change in color tone or can be discriminated in very little, and the strain with weak fermentation ability is selected I did.

In another aspect, the present invention relates to a method for producing raw makgeolli capable of long-term storage for 3 to 25 days at a low temperature of 5 to 15° C., characterized in that the low temperature-sensitive mutant yeast is used.

In the present invention, the low temperature is preferably 0 to 18°C, more preferably 2 to 15°C, and more preferably 5 to 15°C.

In the present invention, the long-term storage is preferably 3 to 30 days, more preferably 5 to 25 days, more preferably 6 to 15 days.

In the present invention, the method of manufacturing makgeolli comprises the steps of: (a) adding low-temperature-sensitive mutant yeast to a composition containing rice, rice, coenzyme, and water, and fermenting it to prepare a mash; And (b) forming the fermented mash.

In the present invention, the low-temperature-sensitive mutant yeast may be characterized in that S-105 (KCTC18678P) is a saccharomyces cerevisiae.

In another aspect, the present invention relates to a raw makgeolli prepared by the above method and capable of long-term storage at a low temperature comprising S-105 (KCTC18678P) in a low-temperature-sensitive mutant yeast saccharomyces cerevisiae.

When the raw makgeolli according to the present invention is stored for a long time at a low temperature, there is little increase in alcohol content, and there is little change in sugar content or glucose concentration, so that long-term storage is possible without altering the quality of the liquor.

In another aspect, the present invention relates to a method for producing raw wine capable of long-term storage at a low temperature, characterized by using the low-temperature sensitive mutant yeast.

In the present invention, the saengju may be characterized in that yakju or cheongju.

Hereinafter, the present invention will be described in more detail by examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

Example 1: Preparation of yeast mutant strain having temperature sensitivity

In order to produce a temperature-sensitive yeast mutant whose growth is significantly inhibited at low temperatures, a yeast strain variant was produced by the method of Kyogoku et al. (Kyogoku K. & Ouchi K. Applied and Environ. Microbio. 639-642, 1995; Japanese Patent Laid-Open Patent Publication No. 2001-78655).

Saccharomyces cerevisiae strain (SILesaffre, France, product name: Rapparijang) was inoculated with YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) and cultured at 30°C for 12 hours. Then, the cells were obtained by centrifugation. After washing the obtained cells with a KH ₂ PO ₄ solution, the cells were suspended in a KH ₂ PO ₄ solution with an absorbance of 1.0, that is, the number of cells per ml is 1x10 ⁷ , and the cell suspension was irradiated at a wavelength of 254 nm for 4 minutes to achieve viability 1 Ultraviolet ray was irradiated so that it might become -30%.

The ultraviolet-treated cell suspension was transferred to 5 mL of YPD medium and incubated at 30° C. for 12 hours. After the cultivation was completed, the cells were obtained by centrifugation, and then the cells were washed, and 1 mL of a nitrogen-free medium (Yeast nitrogen base w/o amino acid and ammonium sulfate (Deifuko) 0.17%, glucose 1%) Incubated at 30° C. for 12 hours (G1 step).

After completion of the culture, the cells were collected by centrifugation again, and the collected cells were inoculated into 0.9 ml of YPD medium containing 1 μM of Antimycin A, and cultured at 10° C. for 36 hours. Nistatin 10mg/ml was added to the culture solution and incubated at 10°C for 2 hours. The culture solution was centrifuged to obtain cells, and plated on a YP5D agar medium (1% yeast extract, 2% polypeptone, 5% glucose, 2% agar).

After incubation at 30°C for 48 hours, colonies grown on YP5D plates and colonies of wild-type strain were taken with a toothpick and inoculated on YPG Plates ((yeast extract 1%, polypeptone 2%, glycerol 3%, agar 2%), 30 After incubation for 24 hours at °C, the colonies were overlaid and covered with soft agar (0.02% bromoercresol purple, 10% sucrose, 0.5% yeast extract, 1% polypeptone, 1% agarose) After 6-12 hours, wild-type colonies were 10 °C. Also, the color around the colonies changed to yellow, and the colonies remaining in a dark purple state were selected as the low temperature sensitive mutant strain.

It was confirmed that the selected low-temperature-sensitive mutant strain turned yellow within 2 hours when allowed to stand at room temperature, and that the strain was grown normally at room temperature.

Example 2: Confirmation of glucose consumption at low temperature of mutant strains

For the mutant strains selected in Example 1, the residual amount of glucose in the medium during low temperature culture was measured.

17 of the low-temperature-sensitive strains were dotting on a YPD plate and incubated for 48 hours at 30°C, 17 colonies were inoculated in YPD liquid medium and cultured at 30°C for 24 hours, and 100 µl of the culture medium was added to Con liquid medium (yeast extract 1%, 2% polypeptone, 2.5% glucose) After suspending in 5 mL, the change of glucose over time was measured while incubating at 10°C.

As a result, as shown in Table 1, the low-temperature-sensitive mutant strains had a higher glucose residual amount in the culture medium than the wild-type strain, and the S-105 strain showed the highest glucose residual amount.

Glucose residual amount in the culture medium of the mutant strain at low temperature Strain No Glucose (mg/dL) Day 1 Day 3 Day 4 Day 5 Day 6 8 2,750 2,470 1,910 1,370 340 11 2,700 2,440 1,800 1,080 250 21 2,830 2,670 1,760 1,150 290 38 2,650 2,680 2,220 1,520 570 47 2,750 2,400 1,690 1,030 178 60 2,720 2,550 1,960 1,290 410 68 2,680 2,420 1,990 1,150 280 83 2,690 2,690 2,090 1,500 670 85 2,710 2,590 2,080 1,400 500 86 2,620 2,620 2,200 1,770 830 100 2,410 2,690 1,920 1,660 680 101 2,520 2,530 2,020 1,390 660
S-105
2,580
2,340
2,180
1,860
1,280
110
2,610
1,980
1,680
770
165 122 2,630 2,180 1,880 1,270 350 153 2,620 2,350 2,100 1,840 1,180 154 2,730 2,280 2,110 1,730 1,130 blank 2,760 2,820 2,440 2,470 2,540 Wild type
(control) 2,690 2,000 1,350 440 116

In addition, strains S-86, S-105, S-153, and S-154, which had a high glucose residual amount in the results of Table 1, were suspended in 5 mL of Con liquid medium, and then incubated at 15°C with time-dependent changes in glucose. The measurement results are shown in Table 2 and FIG. 1.

As a result, S-105 strain showed the highest residual amount of glucose, so S-105 strain was finally selected and used for production of raw makgeolli, and the strain was transferred to KCTC18678P in the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center as of March 15, 2018. Deposited.

Glucose residual amount of mutant strains when cultured at 15℃ Strain No Glucose (mg/dL) Day 3 S-86 46.95% 1,000 S-105 82.16% 1,750 S-153 69.48% 1,480 S-154 61.97% 1,320 Wild 6.34% 135 Blank 100.00% 2,130

Example 3: Preparation of Makgeolli using S-105 mutant strain

Using the strain selected in Example 2 (S-105), makgeolli was prepared, and the effect of extending the shelf life was confirmed.

Makgeolli was performed by immersing in two stages according to the method of Table 3, and wild-type yeast and S-105 yeast were used as yeast, respectively.

Rice (kg) Entry (kg) Yeast (g) Coenzyme (g) Water (ℓ) 1st stage 0.40 0.10 5.00 18.00 0.75 2nd stage 1.00 - - 18.00 1.50 Sum 1.40 0.10 5.00 2.25

As a result, as shown in Table 4, there was no significant difference between the wild-type control strain and the mutant in the alcohol content of the 6th day of soaking.

analysis Use of wild-type control bacteria yeast Use of mutant strains Alc. (%) Alc.(%) Immersion Day 6 16.0 16.9

When the fermentation of the mash was completed, the preparation was performed, and the conditions for the preparation were 5.4% alcohol and sugar and water were added so that the sugar content of the mash was 10 Brix. After making, it was bottled in a 750ml pet and stored in an incubator at 10°C to measure changes in alcohol and components over time. In the bottling analysis, day 1 refers to the day of bottling as day 1.

As a result, as shown in Table 5, it was confirmed that the alcohol content of makgeolli using the mutant strain was maintained at 7.0 even on the 13th day after bottling, the decrease in glucose concentration was significantly less than that of the wild type, and the sugar content was also maintained.

Bottling
analysis Day 1 Day 2 Day 6 Day 8 Day 10 Day 13 transition contrast transition contrast transition contrast transition contrast transition contrast transition contrast Alc. 5.3 5.4 5.4 6.0 6.1 8.8 6.4 10.1 6.5 10.5 7.0 10.8 Sugar content
(Brix.) 10.0 9.2 10.0 9.0 9.4 6.0 9.0 4.9 9.0 4.9 8.6 4.9

Under the Liquor Tax Act, the allowable value of alcohol change in raw makgeolli is from -0.5 to +1.0% of the standard alcohol concentration, and if the alcohol standard of the product is 6%, the range of alcohol allowed by law is 5.5% to 7.0%.

[Accession number]

Name of donated institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC18678P

Consignment Date: 20180315

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (7)

Low temperature sensitive mutant yeast Saccharomyces cerevisiae S-105 KCTC18678P.
A method for producing raw makgeolli capable of long-term storage at a low temperature, characterized in that the low-temperature-sensitive mutant yeast of claim 1 is used.
The method of claim 2 comprising the following steps:
(a) adding a low-temperature-sensitive mutant yeast to a composition containing rice, rice, coenzyme, and water, and fermenting it to prepare a mash; And
(b) preparing the fermented mash.
Raw makgeolli prepared by the method of claim 2 or 3 and capable of long-term storage at a low temperature containing S-105 KCTC18678P, a low-temperature-sensitive mutant yeast Saccharomyces cerevisiae.
The raw makgeolli according to claim 4, wherein long-term storage is possible for 3 to 30 days at a low temperature of 2 to 15°C.
A method for producing raw wine capable of long-term storage at low temperatures, characterized in that the low-temperature-sensitive mutant yeast of claim 1 is used.
The method of claim 6, wherein the saengju is yakju or cheongju.

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