KR101588282B1 - Method for mass production of saccharomyces cerevisiae 157-1 - Google Patents

Abstract

The present invention relates to a mass production method of Saccharomyces cerevisiae 157-1 and more particularly to a method for mass production of yeast including a step of inoculating yeast into a medium and culturing for 12-18 hours .
According to the present invention, the yeast selected by selecting an optimal yeast can be mass-produced under conditions of optimal culture and optimal culture.

Description

[0001] METHOD FOR MASS PRODUCTION OF SACCHAROMYCES CEREVISIAE 157-1 [0002]

The present invention relates to a mass production method of Saccharomyces cerevisiae 157-1 and the production of makgeolli using the same.

Alcohol is naturally occurring and has evolved into several types of unique sake depending on region, ethnicity, climate, climate and cultural differences. Since there was no modern fermentation technology before the existence of bacteria, yeast, etc., The fermented bean was prepared by the native yeast present in the yeast. However, there is a problem in that it is difficult to maintain good and constant efficacy by using native yeasts. In order to solve this problem, yeast having excellent characteristics among the native yeasts in modern times was selected and separated to carry out fermentation, and excellent and constant efficacy was maintained to produce excellent fermented beverage.

Currently, typical fermented beverages, such as beer, sake or wine, are fermented by selected fermenting yeast, and new fermenting yeast is continuously being explored.

In order to perform such a search, it is necessary to ferment the strains selected in the laboratory and to measure the characteristics of each efficacy, so that it takes a lot of time and cost. Accordingly, the present inventors have reached the present invention in order to select an optimal yeast and mass-produce it, thereby easily and easily producing a fermented juice.

It is an object of the present invention to provide an optimal culture medium and an optimal culture time for selecting an optimal yeast for producing a fermented juice and mass-producing the same.

In order to accomplish the above object, there is provided a method for culturing yeast comprising inoculating yeast into a medium and culturing for 12 to 18 hours in one embodiment.

In the present invention, the yeast is Saccharomyces cerevisiae 157-1 (accession number: KCTC12290BP), wherein the medium contains fructose in an amount of 7 To 11 parts by weight of the culture medium, wherein the culture medium contains 3 to 7 parts by weight of yeast extract per 100 parts by weight of the culture medium.

In one embodiment, there is provided a method for mass production of yeast comprising the steps of inoculating yeast into a medium, followed by culturing for 12 to 18 hours.

In the present invention, the yeast is Saccharomyces cerevisiae 157-1 (accession number: KCTC12290BP), wherein the medium contains fructose in an amount of 7 To 11 parts by weight of the culture medium, wherein the culture medium contains 3 to 7 parts by weight of yeast extract per 100 parts by weight of the culture medium.

In one embodiment, a medium is provided that includes yeast, fructose, and yeast extract.

In the present invention, the yeast is Saccharomyces cerevisiae 157-1 cerevisiae 157-1. Wherein the culture medium contains 7 to 11 parts by weight of fructose relative to 100 parts by weight of the culture medium, wherein the culture medium comprises yeast extract (yeast extract (KCTC12290BP), yeast extract extract is contained in an amount of 3 to 7 parts by weight.

In the present invention, the strain 157-1 refers to Saccharomyces cerevisiae 157-1 strain.

The fermentation product of the present invention is not limited thereto, but refers to any one selected from the group consisting of Yakju, Takju, Makgeolli, sake, wine and fruit wine.

The fermentation liquor according to the present invention comprises the steps of: 1) adding water to a starch raw material (for example, glutinous rice), yeast, and the strain of the present invention to prepare a slurry; 2) a fermentation step in which fermented starch material, koji and water added in the above step 1) are further added and fermented; And 3) filtering the fermentation broth obtained in the step 2). After the step 1) or 2), the phytopharmaceuticals such as the cordyceps, red seaweed, fermented red ginseng, jujube, licorice, Plums, grapes, etc. may be added. For example, the step of preparing the vitreous can be performed by fermenting at 5 to 30 ° C for 2 to 20 days, and the immersion step may be performed at 5 to 30 ° C for 2 to 30 days. However, the method for producing the fermented beverage according to the present invention is not limited to these specific methods.

The present invention can mass-produce the yeast selected by selecting an optimal yeast under the conditions of optimal culture and optimal culture.

Fig. 1 is a graph showing the growth curve of the cells according to the fermentation agent (coenzyme).
FIG. 2 is a graph showing the type of culture medium of the strain and the degree of growth according to the culture time.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  1. Selection and Identification of Strain

1-1. Strain  Primary selection

A total of 1,000 strains of yeast isolated from 300 strains collected nationwide were subjected to fermentation at 25 ℃ for 3 days. The immigrated glycation solution was prepared by mixing 1: 3 (w / v) of water with 1: 3 (w / v) and stirring for 5 hours at 62 ° C. After centrifugation at 6,000 rpm for 20 minutes, the yeast was diluted to 14 ° brix. In a second subculture at 29 ° C for 24 hours, and then suspended in sterilized 0.85% NaCl at a concentration of 2 × 10 ⁷ / ml. The CO ₂ gas production rate of the inoculated cells was compared and measured, and it was determined that the gas production was recognized as alcohol fermentation. The fragrance production ability was sensed after fermentation for 7 days.

1-2. Strain  2nd selection

A total of 206 isolates were selected and mixed with 1: 3 (w / v) of water at a volume of 300 mL and fermented at 25 ℃ for 7 days to compare the alcohol content, pH, acidity, solids content, Respectively.

1-3. Strain  Tertiary selection

Twenty - five strains were selected and makkolli was prepared with a capacity of 1,500 mL. As the fermenting agent, koji of sp300 was used. Yeast (0.02%) and water were added and fermented at 25 ℃ for 2 days. After the addition of rice and water, the paste was prepared and fermented for 5 days. The yield was 200% . Alcohol content, sensory characteristics and aroma components of makgeolli were analyzed.

1-4. Selection of strain by fermentation agent

Mungbean was prepared from 10 strains selected from the 3rd stage. Yeast (0.02%) and water were added and fermented at 25 ℃ for 2 days. After the addition of rice and water, the paste was prepared and fermented for 5 days. The yield was 200% . Based on the alcohol content, physicochemical analysis, organic acid content, sensory characteristics, aroma component analysis and microbial identification results of Makgeolli prepared by this method, 3 strains suitable for makgeolli were selected for each fermentation agent.

When yeast was used as an inoculum, 157-1 strains were found to be suitable. The sensory evaluation of makgeolli made with selected strain and suitable fermentation agent. The rice wine immersed in koji and 157-1 strain showed carbonic acid, fresh, sweet and bitter taste.

1-5. Identification of the strain

The identification of the strains was carried out using 18s rDNA sequencing method, which is mainly used for identification of microorganisms. The isolates were isolated from whole genomic DNAs and cultured on a medium, and then amplified by PCR. The primers used for PCR amplification were amplified using ITS1 (TCCGTAGGTGAAC CTGCGG) and ITS4 (TCCTC CGCTTATTGATATGC), which are mainly used for yeast amplification. The PCR was repeated 35 times for 5 minutes at 95 ° C, 45 seconds at 94 ° C, 1 minute at 55 ° C, 1 minute at 72 ° C, and 10 seconds at 72 ° C. The PCR prodect was analyzed using an ABI 3730XL sequencing machine. The analyzed nucleotide sequences were aligned using BioEdit seqencing program and the results were compared with the data base of NCBI.

As a result of the 18s RNA sequencing, the strain was found to be Saccharomyces cerevisiae .

Example  2. Mass culture of strains

2-1. Carbonic  selection

The selection of the carbon source for the establishment of the mass culture condition of the yeast and the composition of the medium was carried out as follows. One colony of the strain cultured in PDA for 24 hours at 30 DEG C was inoculated into 100 mL of PDB and cultured at 25 DEG C for 24 hours with shaking at 120 rpm. 1.0 mL of the cultured strain was inoculated into a medium (Table 1) having a different carbon source composition (Table 1), and cultured at 25 DEG C and 120 rpm for 24 hours.

The reagent used as a carbon source in the culture medium produced is _{glucose (C 6 H 12 O 6} , Sigma-ALDRICH, USA), fructose (C 6 H 12 O 6, Sigma-ALDRICH, USA), sucrose (C 12 H 22 O 11, Sigma-ALDRICH, USA), high fructose syrup (DAESANG, Korea) and molasses (EVERMIRACLE, Korea).

Medium Composition Blank 1% yeast extract, 2% bacto-peptone, 0.1% MgSO ₄ .7H ₂ O, 0.5% KH ₂ PO ₄ - Control 3% glucose, Glucose 3,6,9% glucose Fructose 3,6,9% fructose Sucrose 3,6,9% sucrose H.F.S. 3,6,9% high fructose syrup Molasses 3,6,9% molasses

Growth of the microorganism and dry cell mass were observed in the medium of Table 1 with different types and contents of carbon sources (Table 2).

Strains 157-1 Increase compared to control% Medium Growth (660 nm) Dry cell (g / L) Growth (660 nm) Dry cell (g / L) Blank 0.200 1.44 - - Control 1.736 10.84 - - Glucose 3% 1.736 10.84 100% 100% 6% 2.208 13.72 127% 127% 9% 2.392 14.85 138% 137% Fructose 3% 1.677 10.48 97% 97% 6% 2.244 13.95 129% 129% 9% 2.468 　 15.31 142% 141% 12% 2.15 13.38 124% 123% Sucrose 3% 1.557 9.74 90% 90% 6% 2.267 14.09 131% 130% 9% 2.401 14.91 138% 138% H.F.S. 3% 1.692 10.57 97% 98% 6% 2.062 12.83 119% 118% 9% 2.373 14.73 137% 136% Molasses 3% 1.192 7.51 69% 69% 6% 1.514 9.48 87% 87% 9% 1.957 12.19 113% 112%

The strain 157-1 was found to have the greatest effect on the growth of the strain. control culture medium, the dry cell mass of 157-1 strain was as high as 10.84 g / L. Glucose, fructose and high fructose syrup showed similar tendency. When sucrose was added more than 6%, the effect of sucrose on the growth was insignificant. The optimum carbon source for growth of 157-1 strain was fructose>sucrose>glucose> high fructose syrup> molasses. The optimum medium for growth of fructose was 9%.

2-2. Selection of nitrogen source

The selection of nitrogen source for the culture medium for the establishment of mass culture conditions of yeast was performed after the optimum carbon source was selected for each strain first. One colony of the strain cultured in PDA for 24 hours at 30 DEG C was inoculated into 100 mL of PDB and cultured at 25 DEG C for 24 hours with shaking at 120 rpm. 1.0 mL of the cultured strain was inoculated into a medium (Table 3) having a different composition of nitrogen source, and cultured at 25 DEG C and 120 rpm for 24 hours.

The reagents used as nitrogen sources for the preparation of the medium were yeast extract (Difco, USA), bacto-peptone (Difco, USA), urea (NH ₂ CONH ₂ , Junsei Chemical Co Ltd, Japan), casein (Wako Pure Chemical Industries Ltd, Japan ), tryptone (Difco, USA), ammonium nitrate (NH ₄ NO ₃ , Fisher Scientific, PA, USA), ammonium sulfate ((NH ₄ ) ₂ SO _{4 and} Junsei Chemical Co Ltd, Japan).

Medium Composition Blank 0.1% MgSO ₄ .7H ₂ O, 0.5% KH ₂ PO ₄ - 3% glucose Control


The optimal carbon source selected first for each yeast 1% yeast extract, 2% bacto-peptone, Y.E 1,3,5% yeast extract B.P. 1,3,5% bacto-peptone Urea 1,3,5% urea Casein 1,3,5% casein Tryptone 1,3,5% tryptone A.N. 0.1, 0.3, 0.5% NH ₄ NO ₃ A.S. _{0.1,0.3,0.5% (NH 4) 2 SO} 4

The optimal carbon source for each yeast was determined. The growth and dry cell mass of the selected carbon source (fructose 9%) and seven nitrogen sources were investigated (Table 4).

Strains 157-1 Increase compared to control% Medium Growth (660 nm) Dry cell (g / L) Growth (660 nm) Dry cell (g / L) Blank 0.248 1.73 - - Control 2.434 15.10 - - Y.E. One% 2.093 13.02 86% 86% 2% 2.311 14.35 95% 95% 5% 2.380 14.78 98% 98% 8% 2.076 12.92 85% 86% B.P One% 1.635 10.22 67% 68% 3% 2.141 13.31 88% 88% 5% 2.352 14.60 97% 97% Urea One% 0.128 1.00 5% 7% 3% 0.070 0.65 3% 4% 5% 0.028 0.39 One% 3% Casein One% 2.389 14.73 98% 98% 3% 2.388 14.73 98% 98% 5% 2.378 14.76 98% 98% Tryptone One% 1.842 11.49 76% 76% 3% 2.430 14.08 100% 93% 5% 2.149 13.36 88% 88% A.N. 0.1% 0.272 1.88 11% 12% 0.3% 0.289 1.98 12% 13% 0.5% 0.289 1.99 12% 13% A.S. 0.1% 0.168 1.24 7% 8% 0.3% 0.261 1.81 11% 12% 0.5% 0.330 2.23 14% 15%

In the yeast extract, bactopeptone and casein supplemented with 157-1 yeast, the amount of dry cell mass increased with increasing the amount of yeast extract, 14.78, 14.60 and 14.76 g / L, respectively. Urea showed no effect on the growth of the cells, and tryptone was higher at 14.08 g / L when added at 3%, but less than the amount of dry cells in medium supplemented with 5% yeast extract.

2-3. Selection of incubation time

The incubation time setting experiment for establishing the optimal culture condition of the yeast was performed as follows. One mL of the selected culture medium (one loop in PDA / 100 mL in PDB) was inoculated into 50 mL of each optimal medium, incubated at 25 ° C with shaking, and the degree of growth and the amount of dried cells were measured at 3-hour intervals.

The measurement of the growth of the strain and the measurement of the dry cell mass are as follows. The cells recovered from the liquid medium were centrifuged (3000 rpm, 2 hr) and the pellet was washed twice with distilled water and dried at 80 ° C until it became constant. The dried cells were suspended in distilled water, and the absorbance at 660 nm was measured using a spectrophotometer to calculate the dry cell mass of the culture from the standard curve (FIG. 1).

Fig. 2 shows the culture type of the 157-1 strain, which is the most suitable yeast when using yeast as an inoculum for the production of makgeolli, and the degree of growth expressed by the amount of dried cells according to the incubation time.

The amount of dry cell mass of 157-1 strain increased rapidly after 6 hours of culture in the control medium and reached a maximum value of 10.74 g / L at 12 hours of culture, and the change was insignificant thereafter. The culture in the optimal medium showed a less amount of dry cell mass than the control until 9 hours of culture, but then increased rapidly until 15 hours after culture and reached a maximum value of 13.80 g / L and a slight decrease until 24 hours But it was more suitable than culture in control medium. As a result, the strain 157-1 was found to be most suitable for growth for 15 hours in the optimum medium.

Korea Biotechnology Research Institute KCTC12290BP 20121015

Claims (12)

A method for mass production of Saccharomyces cerevisiae 157-1 (accession number: KCTC12290BP) for preparing a fermented juice comprising the following steps:
a) seeding Saccharomyces cerevisiae 157-1 in a medium comprising 7-11 parts by weight of fructose and 3-7 parts by weight yeast extract per 100 parts by weight of the medium; And
b) at 25 < 0 > C and 120 rpm For 12 to 18 hours. delete delete delete A method for culturing Saccharomyces cerevisiae 157-1 (accession number: KCTC12290BP) for producing fermented juices comprising the steps of:
a) seeding Saccharomyces cerevisiae 157-1 in a medium comprising 7-11 parts by weight of fructose and 3-7 parts by weight yeast extract per 100 parts by weight of the medium; And
b) incubating at 25 < 0 > C and 120 rpm for 12-18 hours. delete delete delete 7 to 11 parts by weight of fructose and 3 to 7 parts by weight of yeast extract relative to 100 parts by weight of the culture medium were mixed with Saccharomyces cerevisiae 157-1 No. KCTC12290BP) Medium for culture. delete delete delete

Images