KR101811529B1 - Development of Issatchenkia orientalis starter by using Ca-alginate bead after air-blast drying - Google Patents

Abstract

The present invention relates to a method for producing a Isocyanichia orientalis strain as a yeast strain, and more particularly, to a method for producing a yeast strain by mixing a bacterial strain with sodium alginate at a concentration of 1 to 3% (w / v) ); (S2) spraying a solution of calcium chloride on the seed solution to prepare calcium-alginate beads; And a step (S3) of immersing the calcium-alginate beads in a dry protective composition, followed by drying in a blow dryer. The yeast strain produced by the production method of the present invention is characterized in that the survival rate is improved.

Description

[0001] The present invention relates to a method of producing a seed culture using Isocyanic acid orientalis starter by Ca-alginate bead after air-blast drying,

The present invention relates to a method for producing Ischia kia orientalis seeds.

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 labrusca B-derived Campbell Early is grown in about 70% of the whole 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 maintains 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, By encapsulating an indigenous wine yeast isolate KMBL5774 ( Issatchenkia orientalis KMBL5774) isolated from domestic grapes in a human-free Ca-alginate bead, and by developing a method of drying by adding a dry protective agent to blow dry the present invention, Completed.

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 is characterized in that it contains Ischarchenkia orientalis KMBL5774 (Accession number: KACC93124P).

In another embodiment of the present invention, the dry protective composition is a mixture of skim milk and saccharides, and the saccharide is glucose, fructose, and raffinose. 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 live bacteria before and after 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 broth includes a Ischchenkia orientalis strain, and the strain used in the present invention is deposited at the National Institute of Agricultural Science and Technology, and is Ischchenkia orientalis KMBL5774 (Accession No .: KACC93124P) .

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 glucose, fructose, and raffinose. Or more, and more preferably exhibits excellent survival rate when glucose is used.

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).

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 into a 100 mL yeast peptone dextrose (YPD) medium, followed by shaking culture at 30 ° 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. Ca-alginate beads containing yeast were immersed in a solution containing 10% skim milk, glucose, fructose or raffinose as a drying agent for 1 hour, ° C for 5 hours and then dried 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, (Glucose), fructose, or raffinose is used as a saccharide in the dry protective composition, and when the concentration of glucose is 10% (w / v) And showed the best survival rate.

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

In the case of domestic indigenous yeasts, separated from Gyeongbuk Yeongcheon Campbell Early grape to Issatchenkia orientalis KMBL5774, deposited with the National Institute of Agricultural Science and Technology on November 23, 2011, and granted accession number KACC93124P.

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 in a mixture of 10% skim milk and saccharide as a drying protectant for 1 hour and then dried for 5 hours at 37 ° C 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

When the control (1.2 × 10 ⁸ CFU / mL, 10 mL) without Ca-alginate bead was dried for 5 hours at 37 ° C., the survival rate was as low as 0.27% (FIG. 3, □: 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 which did not undergo skim milk immersion treatment was significantly improved by the skim milk immersion treatment, unlike the case of the 2.67% survival rate. The survival rate was improved to 17.90% in 5% skim milk treatment and 34.07% 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 29.97%.

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 cells in a barren environment without water. 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.

Survival rates were found to increase when concentrations were increased in all test sites. Issatchenkia orientalis KMBL5774 showed the best survival rate of 83.44% when 10% glucose was used, and excellent survival rate of fructose and raffinose (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 experiment, moisture content was 9.7% when dried for 5 hours, and moisture content was suitable for long - term storage and survival rate was 90.63%. 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

The survival rate and the viable cell count of the 2% Ca-alginate bead yeast strain prepared under conditions showing optimal survival rate were observed at 4 ° C for a long period of time, and the results are shown in FIG. In the case of storage at 4 ℃, the survival rate was decreased to the extent that it was possible to use as yeast until 4 months in all yeast germs, and the survival rate was 49.64%. 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: KACC93124

Payment date: 20111123

Claims (6)

Mixing the cells with a sodium alginate having a concentration of 1.5 to less than 2.5% (w / v) to prepare a seed solution (Sl);
(S2) spraying a solution of calcium chloride on the seed solution to prepare calcium-alginate beads; And
(S3) of immersing the calcium-alginate beads in a dry protective composition, followed by drying in a blow dryer,
Characterized in that the dry protective composition comprises 8 to 12% (w / v) of skim milk and 5 to 10% (w / v) of glucose, Issatchenkia orientalis KMBL5774 : KACC93124P) PROCESS FOR PRODUCING YEAST KYOCERA.
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. The method for producing a yeast strain according to claim 1, wherein the yeast strain is Kankyou Orientalis KMBL5774 (Issatchenkia orientalis KMBL5774, Accession No .: KACC93124P).
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