KR20190034190A - Manufacturing method of solid-type starter of Saccharomyces cerevisiae Y204 - Google Patents

Abstract

The present invention relates to a method for optimizing the production of a solid starter using Saccharomyces cerevisiae Y204 (KACC93237P). Particularly, as results of identifying the optimal conditions for producing a solid starter of Saccharomyces cerevisiae Y204 (KACC93237P), the following findings were confirmed: the growth rate and viable cell count were the highest when an S. cerevisiae Y204 starter was cultured in a YPD culture medium for 24 hours at 25°C at a stirring rate of 150 rpm; when preparing a solid starter, the viable cell count of the starter increased when 10% of skim milk, 1% of lactose, and 7 to 10% of trehalose were added as excipients; and when drying the solid starter of S. cerevisiae Y204 enzymes, freeze-drying showed approximately 1.2-1.4 times lower viable cell count, but a contamination rate reduced to approximately one half as compared to when drying with hot air. According to the present invention, the optimal conditions for producing the solid starter may be beneficially used in a production method of a solid starter of Saccharomyces cerevisiae Y204 (KACC93237P).

Description

[0001] The present invention relates to a method for producing solid seeds of Saccharomyces cerevisiae Y204 (Saccharomyces cerevisiae Y204)

The present invention relates to a method for optimizing the production of solid seeds using Saccharomyces cerevisiae Y204 (KACC93237P).

Each country in the world has been handed out unique liquor, which is local alcoholic beverages, such as traditional alcoholic beverages, traditional alcoholic beverages or regional beverages, depending on the climate, climate and customs of the area. In Korea, traditional wine called "takju" (makgeolli), yakju and distilled liquor has been passed down for a long time. Takju (makgeolli) is a type of fermented soybean fermented by using yeast made by cultivating fungi or yeast in cereals. It has higher nutritional value because it contains more dietary fiber and physiologically active substances than yakju and distilled liquor.

In general, the strains used for Takju fermentation include Aspergillus, Saccharomyces, Bacillus, and Lactobacillus, and Saccharomyces cerevisiae ( Saccharomyces cerevisiae) is a typical yeast belonging to the acanthaceous fungus. Most yeasts used for brewing, including baker's yeast and brewery yeast, are related to this species.

As mentioned above, despite the high nutritional value of Korean traditional liquor, Takju is not a typical mainstream. Recently, consumers' desire for liquor has diversified and the tendency to find high quality liquor has become stronger. Accordingly, there is a demand for development of superior seeds that have unique flavors and flavors.

Therefore, many researchers are conducting researches to improve the taste and quality of Takju. However, most of the existing research on Takju has been mainly concerned with the types of Takju raw materials and yeast, and research on yeast related to Takju fermentation has been rarely conducted (KOREAN J. FOOD SCI. TECHNOL. , pp. 56-62, 2010).

In recent years, attention has been focused on yeast which has excellent and constant efficacy, can improve flavor and taste, has a characteristic of excellent flavor or flavor, selects yeasts that maintain a constant efficacy, and performs fermentation The research on producing excellent fermented beans has attracted much attention.

Therefore, the inventors of the present invention have conducted studies to select a yeast having an excellent aroma-producing ability and an excellent aroma-producing ability, and to find a preparation method and an optimum condition for mass production. Saccharomyces cerevisiae Y204 ( KACC93237P), the present inventors have completed the present invention.

It is an object of the present invention to provide a method for optimizing the production of solid seeds using Saccharomyces cerevisiae Y204 (KACC93237P).

In order to achieve the above object, the present invention provides a method for culturing Saccharomyces cerevisiae Y204 (KACC93237P) at a stirring speed of 120 to 180 rpm at a temperature of 22 to 28 DEG C for 24 to 30 hours A method for producing Saccharomyces cerevisiae Y204 solid seeds is provided.

The present invention also provides a solid seed of Saccharomyces cerevisiae Y204 (KACC93237P) produced by the method of the present invention.

In addition, the present invention provides a method for preparing Takju, which comprises fermenting grains of Saccharomyces cerevisiae Y204 (KACC93237P) produced by the method of the present invention into cereals.

The present invention also provides Saccharomyces cerevisiae strain Y204 deposited with Accession No. KACC93237P.

The present invention relates to a method for optimizing the production of solid seeds using Saccharomyces cerevisiae Y204 (KACC93237P), and more particularly to a method for optimizing the production of solid seeds of Saccharomyces cerevisiae Y204 (KACC93237P) The optimum conditions for the production of S. cerevisiae Y204 were determined by cultivating S. cerevisiae Y204 strain at 25 ℃ for 24 hours at a stirring rate of 150 rpm in YPD medium. , lactose 1% and trehalose 7 to 10%, respectively, and the viability of the S. cerevisiae Y204 yeast solid was found to be 1.2 to 1.4 times lower than that of the hot-air drying method , And that the contamination degree of the germs is about twice as low as that of the present invention, the solid seeding optimization condition of the present invention is a solid of Saccharomyces cerevisiae Y204 (KACC93237P) solid It may be useful as a bacterium producing method.

1 is a diagram showing a yeast culture using a Jar-fermentor.
FIG. 2 is a diagram showing the growth rate and viable cell count of Saccharomyces cerevisiae Y204 (KACC93237P) yeast according to the medium.
FIG. 3 is a graph showing the growth rate and viable cell count of Saccharomyces cerevisiae Y204 (KACC93237P) yeast according to the stirring speed.
FIG. 4 is a graph showing the growth rate and viable cell count of Saccharomyces cerevisiae Y204 (KACC93237P) yeast according to the culture temperature.
Fig. 5 is a graph showing the growth of Saccharomyces cerevisiae Y204 (KACC93237P) yeast according to the incubation time.
Fig. 6 is a graph showing the number of live bacteria before and after storage of Saccharomyces cerevisiae Y204 (KACC93237P) yeast solid germ according to the type and concentration of the excipient.
FIG. 7 is a diagram showing Saccharomyces cerevisiae Y204 (KACC93237P) yeast solid seeds stored for each packaging container.
8 is a diagram showing a freeze-drying apparatus and a hot-air drying apparatus for a yeast solid-seeded bacterium.
Fig. 9 is a diagram showing the characteristics of a solid culture of Saccharomyces cerevisiae Y204 (KACC93237P) for the production of Takju according to the drying method and the excipient.
FIG. 10 is a graph showing the number of live cells of Saccharomyces cerevisiae Y204 (KACC93237P) solid organism according to the drying method and the excipient.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing Saccharomyces cerevisiae Y204 (KACC93237P), which comprises culturing Saccharomyces cerevisiae Y204 (KACC93237P) at a stirring rate of 120 to 180 rpm at a temperature of 22 to 28 ° C for 24 to 30 hours, Y204 solid seed culture.

Saccharomyces cerevisiae Y204 is a strain deposited by National Institute of Agricultural Science and Technology as the deposit No. KACC93237P on Jun. 15, 2015 as a strain isolated from traditional fermented foods.

It is preferable to use any medium selected from the group consisting of MEA, YPD, and YM, and it is most preferable to use a YPD medium.

The stirring speed is preferably 120 to 180 rpm, more preferably 150 to 160 rpm, and most preferably 150 rpm.

The incubation temperature is preferably 22 to 28 ° C, more preferably 25 to 26 ° C, and most preferably 25 ° C. If the incubation temperature is lower than 22 ° C or higher than 28 ° C, the growth rate of Saccharomyces cerevisiae Y204 seedlings may deteriorate.

The culture is preferably performed for 24 to 30 hours, more preferably for 24 to 26 hours, and most preferably for 24 hours. If the incubation time is less than 24 hours or more than 30 hours, the degree of growth of Saccharomyces cerevisiae Y204 seedlings may deteriorate.

The culture is preferably carried out by adding any one selected from skim milk, glutamate, lactose, trehalose and dextrin, and it is most preferable to add any one selected from skim milk, lactose and trehalose .

Preferably, the excipient is comprised of skim milk 8-12%, glutamate 0.5-2%, lactose 0.5-2%, trehalose 5-12% and dextrin 3-7%, wherein the excipient is skim milk 10%, glutamate 1 %, lactose 1%, trehalose 7 to 10% and dextrin 5%.

The saccharomyces cerevisiae Y204 (KACC93237P) solid seed can be stored in any one of the packaging materials selected from the group consisting of plastic, ordinary vinyl, aluminum bag and vacuum vinyl bag, and is stored at a temperature of -20 ° C to 30 ° C And is preferably stored at -20 ° C.

The method may further include drying the yeast cultured in the solid seed production method, wherein the drying is preferably freeze drying or hot air drying.

In a specific embodiment of the present invention, the present inventors selected Saccharomyces cerevisiae Y204 (KACC93237P), which is excellent in the ability to grow and grow in traditional fermented foods and has excellent alcohol and aroma-producing ability, as yeast for producing Takju, The optimal conditions for producing S. cerevisiae Y204 solid seeds were as follows. When the S. cerevisiae strain Y204 was cultured in YPD medium at a stirring speed of 150 rpm at 25 ° C. for 24 hours, (See FIG. 5), it was confirmed that when the skim milk 10%, the lactose 1% and the trehalose 7 ~ 10% were added as an excipient in the production of solid seeds, the survival rate was increased (see FIG. 6) Bacteria were detected in the order of ordinary vinyl, plastic, aluminum bag and vacuum vinyl bag. Bacterial contamination by storage temperature was 30 ℃, 4 ℃, -20 ℃ By this it was confirmed that the bacteria detection check (Table 2), S. cerevisiae Y204 yeast solids seed but did dry freeze drying the number of viable cells lower degree of 1.2 to 1.4 times that of a hot-air drying, contamination is twice as low ( See FIG. 10), the solid-seeded yeast optimization conditions of the present invention can be usefully employed in the production of Saccharomyces cerevisiae Y204 (KACC93237P) solid seeds.

The present invention also provides a solid seed of Saccharomyces cerevisiae Y204 (KACC93237P) produced by the method of the present invention.

In addition, the present invention provides a method for preparing Takju, which comprises fermenting grains of Saccharomyces cerevisiae Y204 (KACC93237P) produced by the method of the present invention into cereals.

The solid seed of Saccharomyces cerevisiae Y204 (KACC93237P) is characterized by excellent culture and growth ability and excellent alcohol and aroma-producing ability.

The cereal may be cereal such as rice, barley, wheat, rye, oats, barley, millet, sorghum and buckwheat rice; Papers such as corn, sweet potatoes; And mixtures thereof.

The present inventors have found that the optimum conditions for the production of solid seeds of Saccharomyces cerevisiae Y204 (KACC93237P), which is excellent in the ability to grow and grow in traditional fermented foods and has excellent alcohol and aroma-producing ability, were found to be S. cerevisiae Y204 strain Were cultured in YPD medium at a stirring speed of 150 rpm for 24 hours at 25 ° C. The highest degree of viability and viable cell count was observed (see FIGS. 2 to 5). Skim milk 10%, lactose 1% and trehalose 7 (See FIG. 6), bacterial contamination was detected in the order of ordinary vinyl, plastic, aluminum bag, and vacuum vinyl bag. storage temperature by bacterial contamination was confirmed that the 30 ℃, 4 ℃, -20 ℃ net bacteria is detected (see Table 2), the S. cerevisiae Y204 yeast solids seed drying during lyophilization heat (See FIG. 10), it was confirmed that the solid seedling optimization condition of the present invention was a solid of Saccharomyces cerevisiae Y204 (KACC93237P) solid And can be effectively used as solid seeds and takju prepared by using the method.

The present invention also provides Saccharomyces cerevisiae strain Y204 deposited with Accession No. KACC93237P.

Saccharomyces cerevisiae Y204 is a strain deposited by National Institute of Agricultural Science and Technology as the deposit No. KACC93237P on Jun. 15, 2015 as a strain isolated from traditional fermented foods.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples and Experimental Examples.

<Example 1> Yeast seed selection for producing takju

* Saccharomyces cerevisiae Y204, which has excellent culture and growth ability and excellent alcohol and aroma production ability among seven yeasts isolated from traditional fermented foods, was finally selected as a yeast for producing Takju. On June 15, 2015, No. KACC93237P to the National Academy of Sciences. The yeast was subcultured in a YPD medium and cultured in a large amount using a Jar-fermentor.

<Experimental Example 1> Preparation of solid seeds using yeast Saccharomyces cerevisiae Y204 (KACC93237P) for preparing takju

In order to identify the optimum conditions for the production of solid seeds of S. cerevisiae Y204 selected in Example 1, culturing conditions suitable for growth were confirmed.

<1-1>  S. cerevisiae  Y204 Selection of liquid medium for yeast culture

In order to find optimal conditions for S. cerevisiae Y204 yeast culture, viability and number of viable cells were analyzed by media.

Specifically, the cells were cultured in a Jar-fermentor according to the MEA, YPD and YM medium shown in Table 1, and cultured at 4 ° C for 24 hours at 150 rpm. For the measurement of growth, S. cerevisiae Y204 yeast was plated on each platinum strain, and pre-cultured. The cells were inoculated with 1% each of the MEA, YPD, and YM main culture media, cultured under the above culture conditions, Were measured. For the measurement of viable cell count, 10 g of the sample was taken in a sterilized bag, and 90 ml of sterile physiological saline was added thereto. The cell was dissolved in a homogenizer and diluted with sterilized physiological saline stepwise. Respectively.

ingredient Medium composition (%) MEA YM YPD Malt extract 3 0.3 - Yeast extract - 0.3 One Polypeptone - - 2 Peptone 0.5 0.5 - Dextrose - One 2 Agar 1.5 - 2

As a result, as shown in FIG. 2, S. cerevisiae Y204 growth of yeast and bacteria are also YPD medium, YM culture medium, MEA medium was a higher order (FIG. 2), and thus, the YPD culture medium with a carbon source and a nitrogen source evenly S were selected as the optimal medium for the cultivation of S. cerevisiae Y204 yeast.

<1-2>  S. cerevisiae  Y204 Selection of liquid culture stirring speed for yeast culture

In order to produce yeast seed culture, the amount of cells produced in the liquid culture should be high. For this, the agitation speed is also an important factor in addition to the abundant culture medium. Therefore, when the S. cerevisiae Y204 yeast was liquid cultured, the growth of the strain and the viable cell count were confirmed according to the stirring speed of the fermentation tank.

Specifically, S. cerevisiae Y204 yeast was cultivated using a Jar-fermentor. Culturing conditions were incubated at 4 ° C for 30 minutes at 30 ° C for 24 hours at 100, 150, and 200 rpm, The degree of growth and viable cell counts of these were measured by the method of Experimental Example < 1-1 &gt;.

As a result, as shown in FIG. 3, the growth rate of yeast and the number of viable cells tended to increase in the order of 150, 100, and 200 rpm, and the bacterial counts of S. cerevisiae Y204 yeast at 150 rpm were compared with those at 100 or 200 rpm It was confirmed that it had a cell number of 1.1 to 1.4 times or more (Fig. 3). Therefore, the optimum agitation speed for culturing S. cerevisiae Y204 yeast was selected at 150 rpm.

<1-3>  S. cerevisiae  Optimal temperature for liquid culture for Y204 yeast culture

During the liquid culture of S. cerevisiae Y204 yeast, the growth of the strain and the viable cell count were determined according to the incubation temperature of the fermenter.

Specifically, the culture conditions were as follows: the culturing temperature of the fermenter was divided into 4 kinds of YPD medium and the cultivation temperature was set at 25 ° C., 30 ° C. and 37 ° C., and the mixture was cultured at a stirring rate of 150 rpm for 24 hours. And their viability and viable cell count were measured.

As a result, as shown in Fig. 4, the S. cerevisiae Y204 yeast was able to grow and live at a temperature lower than 30 ° C and 37 ° C at 25 ° C (FIG. 4). Therefore, S. cerevisiae Y204 yeast is a yeast that grows well at a temperature in the range of 25 ° C to 30 ° C, which is lower than 37 ° C. Therefore, it is judged that alcohol fermentation is good at room temperature when preparing Takju.

<1-4>  S. cerevisiae  Y204 Optimal time for liquid culture for yeast culture

To determine optimal growth conditions of S. cerevisiae Y204 yeast for the production of Takju, cultivation was carried out at 4 ° C in YPD medium at 30 ° C and 150 rpm, and the culture was sampled at 6 hour intervals for 2 days Growth rate was investigated. The degree of growth was measured by the method of Experimental Example 1-1.

As a result, as shown in FIG. 5, it was confirmed that S. cerevisiae Y204 yeast grows rapidly during logarithmic growth between 6 and 12 hours, and maintains constant growth without significant change after 24 hours as a stopper (FIG. 5).

Therefore, when yeast Saccharomyces cerevisiae Y204 (KACC93237P) for the production of Takju is cultured in YPD medium at 30 ° C and 150 rpm for 24 hours, it is considered that not only the degree of growth of the strain but also the amount of cell mass is increased.

&Lt; Experimental Example 2 > Yeast for producing Takju Saccharomyces cerevisiae  Determination of the type and concentration of the optimal excipient for the production of solid seeds using Y204

In order to select the type of excipient suitable for the production of solid yeast seeds and the appropriate concentration, the change of the number of viable cells of the solid seedlings was measured according to the concentration of the four excipients and their concentrations.

Specifically, S. cerevisiae Y204 yeast solid germs prepared by mixing five types of excipients skim milk, glutamate, lactose, trehalose and dextrin at concentrations of 1, 3, 5, 7 and 10% were stored at 4 ° C for 30 days, And the change of the viable cell count of the solid organism according to the concentration. The viable cell count was measured by the method of Experimental Example 1-1.

As a result, as shown in FIG. 6, skim milk, lactose and trehalose were found to be useful as an excipient to maintain viable cell counts, although the viable cell count of S. cerevisiae Y204 yeast solid yeast strain varied depending on the type of excipient (FIG. 6 ). Survival rates of skim milk 10%, glutamate 1%, lactoses 1%, trehalose 7-10%, and dextrin 5% were higher than those of other excipients (FIG. 6).

&Lt; Experimental Example 3 > Yeast for producing Takju Saccharomyces cerevisiae  Y204 Selection of packaging paper for solid organism protection and identification of bacterial contamination according to storage temperature

The protective packaging container and the storage temperature which can efficiently store the prepared S. cerevisiae Y204 yeast solid germ were confirmed.

Specifically, the prepared S. cerevisiae Y204 yeast solid seeds were packed using plastic, ordinary vinyl, aluminum bag and vacuum plastic bags, and trehalose 10% and lactose 1% were added as excipients and preserved. The yeast solid seeds stored in the respective packaging containers were stored at -20 ° C, 4 ° C and 30 ° C After 3 months of storage, bacterial contamination was measured. Bacterial contamination was determined by dissolving 10 g of the sample in a sterilized bag, adding 90 ml of sterile physiological saline, dissolving in a homogenizer, diluting with sterilized physiological saline stepwise, plated on PCA medium and counting the number of bacterial cells in CFU / g Respectively.

As a result, as shown in Table 2, bacteria were detected in the order of ordinary vinyl> plastic> aluminum bag> vacuum plastic bag depending on the packaging container, and bacterial contamination by storage temperature was 30 ° C> 4 ° C> -20 ° C Bacteria were detected irrespective of packaging containers (Table 2).

Kinds
Storage temperature
Bacterial contamination Before storage After saving
plastic
-20 ° C 1.6 ± 0.5 × 10 1.4 占 0.7 占 10 4 ℃ 1.7 ± 0.4 × 10 2.5 ± 2.8 × 10 30 ℃ 1.5 ± 0.6 × 10 6.9 占 2.5 占 10
Regular vinyl
-20 ° C 1.7 ± 0.2 × 10 1.2 ± 0.3 × 10 4 ℃ 2.1 ± 0.8 × 10 3.2 占 2.5 占 10 30 ℃ 1.4 ± 0.6 × 10 7.7 ± 4.6 × 10
Aluminum bags
-20 ° C 1.5 ± 0.5 × 10 1.9 ± 0.6 × 10 4 ℃ 1.7 ± 0.5 × 10 1.9 ± 0.4 × 10 30 ℃ 1.9 ± 0.9 × 10 5.9 ± 2.9 × 10
Vacuum plastic bag
-20 ° C 1.9 ± 0.5 × 10 1.6 ± 0.7 × 10 4 ℃ 1.6 ± 0.3 × 10 1.8 ± 0.7 × 10 30 ℃ 1.7 ± 0.5 × 10 3.6 ± 4.5 × 10

&Lt; Experimental Example 4 > Yeast for producing Takju Saccharomyces cerevisiae  Determination of optimum drying conditions of Y204 solid seeds

In order to establish optimum conditions for industrial mass production of S. cerevisiae Y204 yeast solid seedlings, viable cell count and bacterial contamination degree were measured by drying method.

Specifically, the cells of the S. cerevisiae Y204 yeast solid phase strain were adjusted to about 3 × 10 ⁶ CFU / ml, 10% skim milk and 10% trehalose were each suspended in an excipient and equilibrated for 1 hour. Then, lyophilization and hot air drying were performed, respectively. Freeze-drying was performed by rapid freezing at -80 ° C, freeze drying for 48 hours using a freeze dryer, and hot air drying at 45 ° C for 48 hours. The moisture content, viable cell count and bacterial contamination degree of the solid seeds were measured according to the drying method in the obtained solid seeds. The water content was measured by weighing 10 g of a sample in a moisture analyzer (MX-50, A & D Co., Ltd., Tokyo, Japan) Respectively.

As a result, as shown in Figs. 9 and 10, in the case of S. cerevisiae Y204 yeast solid seed, the moisture content was 7 to 9% in the case of lyophilization and 5 to 6% in the case of hot air drying. When skim milk was treated with excipients, the viable cell counts of solid specimens were 3.1 × 10 ⁶ ± 7.2 CFU / ㎖ before drying, 1.7 × 10 ⁶ ± 4.4 CFU / ㎖ after lyophilization, 2.5 × 10 ⁶ ± 1.7 CFU / Ml of trehalose was treated with excipients, the viable cell counts of solid organisms were 2.2 × 10 ⁶ ± 1.7 CFU / ㎖ before drying, 1.6 × 10 ⁶ ± 2.6 CFU / ㎖ after lyophilization, 1.9 × 10 ⁶ 0.4 CFU / ml, indicating a survival rate as low as about 20% as compared to that before drying (Fig. 10). As a result, it was confirmed that the viable cell count of S. cerevisiae Y204 yeast showed a survival rate 1.2 to 1.4 times lower than that of the hot air drying.

On the other hand, when the skim milk was treated with excipient, it was found to be 2.0 × 10 ² ± 0.6 CFU / ㎖ before drying, 5.0 × 10 ² ± 2.5 CFU / ㎖ after lyophilization, 1.0 × 10 ² ± 0.5 CFU / ml (FIG. 10). When trehalose was treated with excipient, it was lyophilized to 3.0 × 10 ² ± 0.7 CFU / ㎖ before drying, 5.0 × 10 ² ± 1.0 CFU / ㎖ after lyophilization and 8.0 × 10 ² ± 1.5 CFU / ㎖ after hot air drying It is judged that freeze drying is more suitable than hot air drying (Fig. 10).

National Academy of Agricultural Sciences KACC93237P 20150615

Claims (2)

Saccharomyces cerevisiae Y204 deposited with accession number KACC93237P was added in YPD medium,
At a stirring speed of 150 to 155 rpm and at a temperature of 25 to 26 캜 for 24 to 26 hours, skim milk 8 to 12%; glutamate 1 to 3%; 1 to 3% lactose; trehalose 7 to 10%; And dextrin 1 or 5%; And a liquid culture; And
And a step of lyophilizing the liquid culture obtained in the above step. [Claim 10] A method for producing yeast solid seeds for the production of Takju, Saccharomyces cerevisiae Y204 Takju.
A yeast solid seed for the production of Takju of Saccharomyces cerevisiae Y204 produced by the method of claim 1.

Images