KR20170138801A - Development of Saccharomyces cerevisiae starter by using Ca-alginate bead after air-blast drying - Google Patents

Abstract

The present invention relates to a method for preparing strains of Saccharomyces cerevisiae as yeast strains. More specifically, the method for preparing strains of Saccharomyces cerevisiae as yeast strains includes the steps of: mixing sodium alginate in a concentration rate of 1-3% (w/v) with mycelium to prepare a strain solution (S1); spraying a calcium chloride solution to the strain solution to prepare calcium-alginate beads (S2); and immersing the calcium-alginate beads in a drying-protection composition before drying the calcium-alginate beads with a blow dryer. The yeast strains prepared by using the method have an improved survival rate.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a saccharomyces cerevisiae starter using saccharomyces cerevisiae,

The present invention relates to a method for producing Saccharomyces cerevisiae.

The share of domestic wines is on the rise, but its reliability is lower than that of imported wines, which has a considerable impact on Korean farming industry. Major grape varieties in Korea include the American species Vitis Campbell Early from labrusca B is grown in about 70% of the total vineyard area. The main breed of Campbell, which is 14-15% sugar content, is low in pH, strong in acidity, weak in wine color and weak in fragrance, so it is urgent to develop wine yeast suitable for the production of Campbell wine.

As a yeast used in domestic wine production, Saccharomyces cerevisiae Fermivin (DSM Food Specialties, Heerlen, Netherlands) and S. cerevisiae EC1118 (Lallemand, Montreal, Quebec, Canada) are commonly used. The yeast sold on the market is a yeast that has been developed by European nations that have been actively studied for good yeast, and is sold in various forms such as dry yeast and liquid yeast by freeze drying. However, these exotic yeasts include domestic wine It is expected that it will not help to improve quality.

Most of the wine yeast is sold as powdery yeast by the freeze drying method. This freeze drying method is a drying method in which the material is frozen and the moisture is removed by sublimation of ice after decompression. This method keeps the original shape of the material as much as possible, There is a high advantage. However, the cost of energy and equipment is high and the drying time is long, making commercial mass production impossible. On the other hand, the low temperature blowing drying method shows a commercial superiority compared to the freeze drying method because it is less damaged by heat and is easy to control moisture content and reduce cost. In addition, freeze-drying showed a fixed cost and a manufacturing cost of about 10 times in blow drying.

On the other hand, Korean Patent Publication No. 2008-0109537 discloses a method for producing such a bacterium by inoculating a strain of Bacillus subtilis into soybean powder to produce a bacterium. However, this method also has a problem of hot air drying or lyophilization.

The present inventors have intensively studied to solve the problem of difficulty in preserving and culturing microorganisms in a small-scale primary processing of a farm in the conventional wine making. As a result, The present invention has been accomplished by developing a method for encapsulating seeds in a Ca-alginate bead which is harmless to humans and then drying by adding a dry protective agent to the native wine grains S. cerevisiae D8, M12 and S13 isolated from domestic grapes.

That is, the present invention aims at improving the stability and recovery rate of seedlings during distribution and storage by establishing a seeding method.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object of the present invention, the present invention provides a process for producing a yeast strain comprising the steps of:

Mixing the microbial cells with 1 to 3% (w / v) calcium alginate to prepare a seed solution (Sl);

(S2) spraying a solution of calcium chloride on the seed solution to prepare calcium-alginate beads; And

Dipping the calcium-alginate beads in a dry protective composition, and drying the dried calcium-alginate beads in an air dryer (S3).

In one embodiment of the present invention,

a) inoculating the yeast culture broth into a culture medium, followed by shaking culture;

b) separating the cells by centrifuging the shake cultured yeast culture; And

and c) washing the centrifuged cells.

In another embodiment of the present invention, the yeast culture solution contains Saccharomyces cerevisiae D8 (Accession No .: KACC93245P), Saccharomyces cerevisiae M12 ( Saccharomyces cerevisiae D8, S. cerevisiae M12, accession number: KACC93246P), and Saccharomyces cerevisiae S13 ( Saccharomyces cerevisiae S13, accession number: KACC 93247P).

In another embodiment of the present invention, the dry protective composition is a mixture of skim milk and saccharides, and the saccharide is sucrose, raffinose, and trehalose. And at least one kind selected from the group consisting of

In another embodiment of the present invention, the concentration of the skim milk is 5 to 20% (w / v).

In another embodiment of the present invention, the concentration of the saccharide is 3 to 15% (w / v).

The present invention aims at enhancing the survival rate and improving the shelf life of yeast starter used in the production of fruit wine, and differentiating it from the conventional method of producing freeze- The cells are beaded with Ca-alginate, water is removed by air drying, and calcium-alginate, skim milk and saccharides are used at optimal concentrations to improve storage stability.

1 is a view showing a process for producing a Ca-alginate bead.
FIG. 2 shows the size and appearance of the Ca-alginate bead prepared by the method of the present invention before and after blow drying.
Fig. 3 is a graph showing the survival rate of the live bacteria before and after the blow drying as a control group of Ca-alginate bead.
FIG. 4 is a graph showing the survival rates before and after blow drying of the seeds prepared with different concentrations of Ca-alginate beads and without addition of a dry protecting agent.
FIG. 5 is a graph showing the survival rate of 2% Ca-alginate bead prepared by the method of the present invention when the concentration of skim milk, which is a dry preservative, was added differently.
FIG. 6 is a graph showing survival rates before and after blow drying using 16 dry protection additives with different concentrations after adding 2% Ca-alginate bead seedlings and 10% skim milk prepared by the method of the present invention.
FIG. 7 is a graph showing the correlation between the moisture content and the drying time of the 2% Ca-alginate bead seed preparation prepared by the method of the present invention.
8 is a photograph showing the inside of a bead of a 2% Ca-alginate bead strain produced by the method of the present invention.
9 is a graph comparing survival rates during storage of 2% Ca-alginate bead seedlings produced by the method of the present invention.

The inventors of the present invention conducted studies to enhance the survival rate and improve the shelf life of the yeast starter used in the manufacture of fruit wine, and found that, unlike the conventional method of producing free seeds by freeze-drying, the use of calcium-alginate And the dry protection composition in which skim milk and saccharides are mixed is used, followed by blow-drying to remove moisture. Thus, the present inventors have completed the present invention.

That is, the present invention provides a method for producing a yeast strain comprising the steps of:

Mixing the microbial cells with 1 to 3% (w / v) calcium alginate to prepare a seed solution (Sl);

(S2) spraying a solution of calcium chloride on the seed solution to prepare calcium-alginate beads; And

Dipping the calcium-alginate beads in a dry protective composition, and drying the dried calcium-alginate beads in an air dryer (S3).

This manufacturing method is schematically shown in Fig. The calcium-alginate is a colorless and odorless polymer and is used in various fields such as pharmaceuticals and foods due to its low cost. When the calcium-alginate is less than 1% (w / v), the bead does not form a circular shape When the concentration exceeded 3% (w / v) concentration, the bead network was dense and showed a limit to be difficult to absorb various protectants used in the experiment in the beads. Preferably at a concentration of 1.5 to 2.5% (w / v).

In the present invention, the microbial cells may be produced by a method comprising the steps of: a) inoculating a yeast culture broth into a culture medium, followed by shaking culture;

b) separating the microbial cells by centrifuging the shake cultured medium; And

and c) washing the centrifuged cells.

In the present invention, the yeast culture solution contains a strain in Saccharomyces cerevisiae. The strain used in the present invention is deposited at the National Academy of Sciences, and Saccharomyces cerevisiae is added with D8 ( Saccharomyces cerevisiae D8, accession number: KACC93245P), Saccharomyces cerevisiae M12 ( Saccharomyces cerevisiae M12, accession number: KACC93246P), or Saccharomyces cerevisiae S13 ( Saccharomyces cerevisiae S13, accession number: KACC 93247P).

In the present invention, the dry protective composition is a mixture of skim milk and saccharides, and the saccharide is one kind selected from the group consisting of sucrose, raffinose, and trehalose Or more.

The saccharides are preferably sucrose in the case of S. cerevisiae D8 in Saccharomyces cerevisiae and raffinose in the case of M12 ( S. cerevisiae M12) in Saccharomyces cerevisiae , And in the case of S13 ( S. cerevisiae S13) in Saccharomyces cerevisiae , it is preferable to use trehalose.

The concentration of the skim milk may be 5 to 20% (w / v). When the concentration of the skim milk is less than 5% (w / v), the improvement of the survival rate is rather poor, and when the concentration exceeds 20% (w / v), the survival rate is rather lowered. 18% (w / v), more preferably 8 to 12% (w / v).

In addition, the concentration of the saccharide may be 3 to 15% (w / v), more preferably 5 to 10% (w / v).

In Example 1, 5% (w / v) yeast seed culture medium was inoculated in a 100 mL yeast peptone dextrose (YPD) medium, followed by shaking culture at 30 DEG C for 24 hours. After culturing the yeast maintains a viable count of about 10 ⁸ CFU / mL. After centrifugation, the yeast pellet recovered was washed twice with 0.85% (w / v) aqueous solution of NaCl. After washing, the cells were mixed with 100 mL of sterilized water and sodium alginate to prepare a final concentration of 2% (w / v) of the seed solution and sprayed on a 0.1 M CaCl ₂ solution using a syringe (21 gauge) (Calcium-alginate bead) is formed.

At this time, as Na ⁺ and Ca ^{2 +} are substituted, two strands of alginate form a network around Ca ^{2 +} , and encapsulation proceeds. The Ca-alginate bead containing the yeast is 10% skim milk and dried protective agent sucrose (S.cerevisiae D8), raffinose ( S. cerevisiae M12), trehalose (S. cerevisiae S13) for each mixed solution was immersed in 1 hours And dried at 37 ° C for 5 hours using a blow dryer to a moisture content of 10% or less.

In addition, the present invention was carried out to examine the correlation between the concentration of Ca-alginate bead and the survival rate of yeast in Example 2, The survival rate of Ca-alginate beads was 10% (w / v). The survival rate of skim milk in Ca-alginate bead, , And that the saccharides used in the dry protective composition were sucrose, raffinose and trehalose and showed the best survival rate when the concentration was 10% (w / v).

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1. Domestic Indigenous  Isolation and preservation of yeast

1.1. Domestic Indigenous  Isolation of yeast

Domestic indigenous yeasts were identified as S. cerevisiae D8, M12, and S13, respectively, from Campbell Early grapes.

1.2. Domestic Indigenous  Cultivation and preservation of yeast

Purified a native yeast was used by the microbial viable count of 24 hours incubation at 30 ℃ inoculated in suitable YPD broth (Table 1) in the culture was to be about 10 ⁸ log CFU / mL, while sub-culturing as 4 weeks storage Respectively. For the long-term storage of pure microorganisms, yeast seed culture was prepared with glycerol final concentration of 20% and stored at -70 ℃.

ingredient Concentration (%) Dextrose 2 Peptone 2 Yeast extract One

1.3. Domestic Indigenous  Production method of yeast seeds

The production of the seeds in the present invention was carried out by the following method.

100 mL YPD medium was inoculated with the 5% (w / v) yeast seed culture medium prepared in 1.2 above, followed by shaking culture at 30 ° C for 24 hours. After culturing the yeast the bacterial count was maintained to approximately 10 ⁸ CFU / mL. After centrifugation, the yeast pellet recovered was washed twice with 0.85% aqueous NaCl solution to separate the culture and the cells.

Back after the cell is a mixture of sterile water 100mL and each sodium alginate (Sodium alginate) to prepare a final concentration of 2% (w / v) inoculum solution washing, a syringe in 0.1 M CaCl ₂ solution using a (syringe, 21 gauges) To prepare a calcium-alginate bead. At this time, Na ⁺ and Ca ^{2 +} are substituted and Ca ^{2 +} is centered, and alginate of two strands forms a network and encapsulation proceeds.

Ca-alginate beads containing yeast were immersed for 1 hour in a mixture of 10% skim milk and sucrose ( S. cerevisiae D8), raffinose ( S. cerevisiae M12), and trehalose ( S. cerevisiae S13) And dried at 37 ° C for 5 hours using a blower dryer (moisture content 10% or less).

The vitreous device and the reagent used for the preparation of the Ca-alginate bead were sterilized by using a high-pressure sterilizer at 121 ° C for 15 minutes. The prepared Ca-alginate bead was blow dried at 37 ° C for 5 hours using a blow dryer (HB-509C, Hanbaek Scientific Technology, Seoul, Korea), and the size of the Ca-alginate bead was 0.4-0.5 mm.

Example  2. Preparation and survival rate of Ca-alginate bead

2.1. Survey of free cell survival rate

The survival rate of each yeast strain was 0.24% for S. cerevisiae D8, 0.24% for S. cerevisiae M12, and 0.24% for S. cerevisiae D8 when the control (about 1.2 × 10 ⁸ CFU / mL, 10 mL) 0.28%, and S. cerevisiae S13 was 0.27%. (Fig. 3: before drying, after drying).

2.2. Effect of concentration of Ca-alginate bead

Ca-alginate beads were prepared by mixing sodium alginate with a yeast culture solution and dried for 5 hours at 37 ° C. (FIG. 4).

There was no significant difference in the survival rates between treatment groups. However, when 1% (w / v) sodium alginate was used, the bead did not form a circular shape and tended to easily collapse. When 3% (w / v) sodium alginate was used, the bead had a dense network structure, which made it difficult to absorb the various protectants used in the experiment. The concentration of Ca-alginate bead was set to 2% (w / v).

2.3. Influence of skim milk

In general, skim milk is widely used as a freezing agent for lyophilization of cells. It is known that the protein structure of skim milk is protected by coating the cells under extreme environmental conditions. In this example, skim milk was used as a protecting agent to improve the survival rate of yeast cells in the absence of water during air drying. To investigate the effect of skim milk on cell protection, 5% (w / v), 10% (w / v), and 20% (w / v) beads prepared with 2% (w / v) sodium alginate, Of skim milk solution for 1 hour, so that skim milk was absorbed into the bead. The results of the survival rate and viable cell count according to each treatment condition are shown in FIG.

The survival rate of the 2% Ca-alginate bead seedlings without skim milk immersion treatment was about 2.1-2.6%, but the survival rate was significantly improved under skim milk immersion treatment. The survival rate was improved to 17.7-18.9% in 5% skim milk treatment and 31.9-34.3% in 10% skim milk treatment. The survival rate of 20% skim milk treatment was lower than that of 10% skim milk treatment, and the survival rate was 28.2-28.6%.

Therefore, 10% skim milk was the best preservative for Ca-alginate bead yeast strain.

2.4. Effect of drying inhibitor

It is known that protectant, which is added during the production of seeds such as freeze-drying, blow-drying, etc., plays an important role in protecting the cells in a barren environment without moisture. In order to examine the effect of the type and concentration of the protectant on Ca-alginate bead yeast strain, 5% (w / v) and 10% (w / v) ) Protectant, Ca-alginate bead was immersed in the solution for 1 hour to treat skim milk and protectant to be absorbed into the bead. The change in viable count and survival rate after 5 hours of air drying was shown in FIG.

The kinds of protectants used in this example are glucose, fructose, galactose, sucrose, lactose, trehalose, maltose, raffinose, dextrin and ten kinds of sugars such as starch, four kinds of sugar alcohols such as mannitol, sorbitol, inositol and adonitol, MSG, and peptone were used as protectants.

The survival rate was found to be increased when the concentration was increased in all test groups, although there was a difference between strains. The survival rate of S. cerevisiae D8 was 90.67% when 10% sucrose was used. The survival rate of S. cerevisiae M12 was 87. 63% when 10% raffinose was used. S. cerevisiae S13 showed 92.05% survival in 10% trehalose (Fig. 6).

2.5. Effects of Drying Time and Moisture Content on Ca-alginate bead Yeast-seeded Prototype

One of the most important factors in the seed product prototype is the moisture content. Proper moisture content minimizes the change of physical properties during long - term storage and reduces the rate of cell death during storage of the prototype. Therefore, the experiment was conducted by setting the drying time to 5 hours based on the preliminary experiment. However, since there is a possibility of changing the moisture content in the bead according to addition of 10% skim milk and 10% protectant, The necessity of resetting the water content suitable for long-term storage has been raised.

Each experiment was carried out using Ca-alginate bead yeast strain, which was set up to exhibit the optimal survival rate for each strain. The result of observing the change in the survival rate and moisture content per hour while drying at 37 ° C for 7 hours is shown in FIG. As a result of the experiment, moisture content was 9.8-10.1% when dried for 5 hours, and water content was suitable for long - term storage and survival rate was also excellent as 83.4 - 92.4%. This is because 10% skim milk and 10% protectant do not significantly affect the total moisture content of the Ca-alginate bead yeast strain prototype.

2.6. Transmission electron microscope ( SEM ) Of Ca-alginate bead yeast strain

8 shows the result of observing the interior of the bead at a magnification of 1,000 times using a scanning electron microscope (SU8220, Hitachi, Japan) in order to examine the form of each Ca-alginate bead yeast strain stored in the bead. .

As a preparation step for microscopic observation, the moisture of each bead was completely removed from the oven, and then the bead was finely cut with a cutter knife and the inside was observed. As a result of the observation, it can be seen that each yeast is very tightly packed in a bead coated with skim milk or saccharides.

In addition, it was found that some yeasts were observed to have proliferative products at the stage where the culture was vigorous, and it was confirmed that when the seed product prepared by the method of the present invention was used, the cell activity would be obtained in a short time.

2.7. Survival rate of Ca-alginate bead yeast strain as a result of long-term storage experiment

Fig. 9 shows the results of observing the survival rate and the number of viable cells while keeping the 2% Ca-alginate bead yeast strain at 4 ° C for the optimum survival rate for each yeast. The survival rate of S. cerevisiae D8 was 29%, that of S. cerevisiae M12 was 34.7% and that of S. cerevisiae S13 was 39.3% Respectively. At 5 months, the number of bacteria decreased sharply and the log CFU / mL value decreased to a maximum of 2.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Depositor Name: Agricultural Biotechnology Research Institute

Accession number: KACC93245

Checked on: 20160308

Depositor Name: Agricultural Biotechnology Research Institute

Access number: KACC93246

Checked on: 20160308

Depositor Name: Agricultural Biotechnology Research Institute

Accession number: KACC93247

Checked on: 20160308

Claims (6)

A method for producing Saccharomyces cerevisiae yeast strain comprising the steps of:
Mixing the microbial cells with 1 to 3% (w / v) calcium alginate to prepare a seed solution (Sl);
(S2) spraying a solution of calcium chloride on the seed solution to prepare calcium-alginate beads; And
Dipping the calcium-alginate beads in a dry protective composition, and drying the dried calcium-alginate beads in an air dryer (S3).
The method according to claim 1,
The above-
a) inoculating the yeast culture broth into a culture medium, followed by shaking culture;
b) separating the cells by centrifuging the shake cultured yeast culture; And
and c) washing the centrifuged cells. < RTI ID = 0.0 > 8. < / RTI >
3. The method of claim 2,
The yeast culture broth was supplemented with saccharomyces cerevisiae D8 ( Saccharomyces cerevisiae D8, accession number: KACC93245P), Saccharomyces cerevisiae M12 ( Saccharomyces S. cerevisiae M12, accession number: KACC93246P), and Saccharomyces cerevisiae S13 ( Saccharomyces cerevisiae S13, accession number: KACC 93247P). The yeast strain according to claim 1, wherein the yeast strain is selected from the group consisting of:
The method according to claim 1,
The dry protection composition is a mixture of skim milk and saccharides. The saccharide is at least one selected from the group consisting of sucrose, raffinose, and trehalose. By weight.
5. The method of claim 4,
Wherein the concentration of the skim milk is 5 to 20% (w / v).
5. The method of claim 4,
Wherein the concentration of the saccharide is 3 to 15% (w / v).

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