KR920008363B1 - Method for freeze-drying - Google Patents

Abstract

A freeze dried cell is prepd. by (a) culturing Saccharomyces cerevisiae ATCC 60583, (b) concentrating the cultured broth and freeze drying, and (c) mixing 30-100 g freeze-dried cells with 100-250 g lactose, 150-250 g galactose, skim milk or peptone and 30-70 g citric acid. The survival rate of saccharomyces cerevisiae cells is very high.

Description

Method for preparing lyophilized cells

The present invention relates to a method for preparing lyophilized cells, and more particularly, by freeze-drying a Saccharomyces cerevisiae ATCC 60583 strain belonging to Saccheromycetaceae, using a freeze-drying aid. It relates to a method for producing lyophilized cells to reduce the instability of the strain during dry preservation.

In general, the storage of industrially used strains mainly use two methods of freeze-preservation and freeze-drying. The freeze-preservation of the strain in liquid nitrogen is effective and inexpensive, but it is stored because it must be kept frozen. And mobile phase problems. On the other hand, freeze-drying has no problem in storage and mobile phase, but there is a problem in that the number of viable cells is reduced during freeze-drying and strain preservation.

That is, conventionally, the lyophilization method of the strain is made using a scheme milk, glucose or a mixture thereof as a lyophilization aid (Maintenance of Microorganisms. Academic Press). In this case, due to the low freeze effect, the number of viable bacteria is inevitably reduced.

Therefore, the present invention, in order to improve the above-mentioned conventional problems, in particular caused by instability when lyophilized Saccharomyces cerevisiae cells of the diffuser yeast, which is known to have a recent dressing effect for a long time storage The purpose is to prepare lyophilized cells by using a new lyophilized preservative that can solve the problem of reducing the number of viable cells.

Hereinafter, the present invention will be described in detail.

The present invention is cultured in a YM liquid medium, the Saccharomyces cerevisiae (ATCC 60583) strain of the diffuser yeast, concentrated in lyophilization, based on 100 parts by weight of the skim milk or peptone or a mixture thereof 30 ~ It is a method for producing a lyophilized microorganism, characterized in that the freeze-drying aid consisting of 100 parts by weight, 250 to 500 parts by weight of sugar and 30 to 70 parts by weight of citric acid is added to the concentrated culture solution.

As described above, the present invention will be described in more detail.

The present invention uses a freeze-drying aid of a new composition at the time of freeze-drying Saccharomyces cerevisiae (ATCC 60583) strain, generally the regeneration ability of the freeze-dried strain by the degree to withstand the freezing and drying procedure It's decided. Fatal damage to the strains that occur during this process causes most of the strains to die during lyophilization or storage.

This phenomenon can be reduced by reducing the live strain by using a freeze-drying aid, by using the freeze-drying aid provided in the present invention, the respective freeze-drying protection functions in harmony with the strain regeneration during freeze-drying or strain preservation It is to increase.

Therefore, in the present invention, the known Saccharomyces cerevisiae (ATCC 60583) bacteria belonging to the diffuser yeast are activated culture in YM liquid medium consisting of, for example, dextrose 1%, peptone 0.5%, yeast extract 0.3% malt extract 0.3% The resulting cells were concentrated and lyophilized in a commonly used lyophilizer by adjusting the pH. At this time, the lyophilization aid of the new composition was added to the scheme milk or peptone 30-100 before concentrating the cells and adjusting the pH. It is added in an amount of 250 parts by weight to 250 parts by weight and 30 to 70 parts by weight of citric acid, and the addition method thereof is in the order of skim milk or peptone and citric acid.

At this time, the skim milk or peptone added as a main component of the freeze-drying aid to prevent damage to the strain by preventing the East Sea, each 30 to 100 parts by weight, preferably 50 to 70 to 100 parts by weight of the dry cells It can be used by weight and can also mix and use these.

However, if less than 30 parts by weight of skim milk or peptone or a mixture thereof can not be expected to add the effect, even if it is used in excess of 100 parts by weight, the effect of the prevention of freeze is similar.

In addition, as a synergistic effect as a lyophilization adjuvant along with skim milk and peptone, sugars were used in an amount of 200 to 500 parts by weight, preferably 250 to 350 parts by weight, wherein the sugars increased the viscosity of the cell concentrate to prevent freeze damage. It is considered to have a synergistic effect, and it is preferable to use lactose and galactose, and its composition is 100-250 parts by weight of lactose, 150-250 parts by weight of galactose, preferably 150-230 parts by weight of lactose and 180-250 parts by weight of galactose. It is good to use.

If too little sugar is used, the anti-freeze effect is lowered, and if too much sugar is used, the viscosity of the culture solution becomes high, which is not good for lyophilization. In particular, the use of lactose and galactose in the above composition was found to have a very good synergistic effect.

In addition, citric acid is added to the composition. It is not necessarily a component to be used, but it is preferable to use it in order to give higher cell stability during lyophilization, and the amount of use may be used in an amount of 30 to 70 parts by weight based on 100 parts by weight of dry cells. If the amount is out of the above range, there is no big problem, but if excessively used, there may be a risk of cell damage due to acidity.

As described above, the present invention uses a lyophilization aid having a new composition as described above, and even though using only skim milk or peptone or a mixture thereof, the present invention has some effect, but it is used in combination with other auxiliary ingredients. Preferably, the use of such lyophilization aids was higher in viability of the strain than in the absence of the freeze-drying aid. After 30 days, the survival rate was at least 40% and even 60-70.

As described above, the present invention was able to significantly increase the survival rate of the cells by using a lyophilization aid of a new composition that was not used conventionally during freeze-drying of the cells, the freeze-drying aid used in the present invention according to various conditions Even if the amount used is changed to a range other than the scope of the present invention, similar effects can be obtained.

In addition, the present invention was performed only against Saccharomyces cerevisiae (ATCC 60583) strain, but it is thought that it can be widely applied to lyophilized cells having similar biological and bacteriological properties.

Hereinafter, the present invention will be described in detail with reference to Examples. The present invention is not limited by the examples.

In the following example, the measurement of viable cell count of lyophilized bacteria was diluted several times with sterile physiological saline, plated to 30-300 cells in YM agar medium, and cultured for 48 hours at 27 ° C incubator to measure the number of bacteria.

EXAMPLES 1-4

Saccharomyces cerevisiae (ATCC 60583) was preserved in YM liquid medium (1% dextrose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract), and then inoculated in a 3.7 liter fermentation bath to air Incubation amount was 100 In / hour, rotation speed 1,000rpm, and incubation for 24 hours at 27 ℃ culture temperature.

After completion of the culture, the culture solution is concentrated to 1/10, and 50 ml of the concentrated culture solution is stored at 4 ° C.

(Example 1) 10 ml of the following solution A was added to 50 ml of the concentrated culture broth, adjusted to pH 6.8 with 50% NaOH, and left at 4 ° C. for 1 hour.

(Example 2) 10 ml of the following solution B was added to 50 ml of the concentrated culture broth, adjusted to pH 6.8 with 50% NaOH, and left at 4 ° C. for 1 hour.

(Example 3) 10 ml of the following solution B was added to 50 ml of concentrated culture solution, mixed and left for 1 hour at 4 ° C., then 40 ml of the following solution C was added, adjusted to pH 6.8 with 50% NaOH, and left at 4 ° C. for 1 hour.

(Example 4) 10 ml of the following solution B was added to 50 ml of the concentrated culture solution, mixed and left for 1 hour at 4 ° C., then 40 ml of the following solution D was added thereto, adjusted to pH 6.8 with 50% NaOH, and left at 4 ° C. for 1 hour.

(Solution A): 10 g of skim milk is dissolved in 100 ml of distilled water to prepare a 10% skim milk solution.

(Solution B): 10 g of skim milk and 30 g of lactose are dissolved in 100 ml of distilled water to prepare a solution of 10% of skim milk and 30% of lactose.

(Solution C): 8 g of galactose was dissolved in 100 ml of distilled water to prepare an 8% galactose solution.

(Solution D): 8 g of galactose and 2 g of citric acid are dissolved in 100 ml of distilled water to prepare a 8% galactose and 2% citric acid solution.

However, the skim milk, lactose galactose Difco product and citric acid used Shinyo product were used.

As described above, each culture solution mixed with the lyophilization aid was lyophilized and stored in a polyethylene bottle, and then strain stability was observed while storing in a constant temperature and humidity chamber at a temperature of 20 ° C. and a humidity of 50%. The stability of the lyophilized cells over time is shown in the following Tables 1,2,3,4.

TABLE 1

TABLE 2

TABLE 3

TABLE 4

From the above results, when lactose or lactose and galactose were added to the skim milk, the survival rates after storage for 30 days were maintained at 22% and 20%, respectively, and were maintained at 43% when lactose, galactose, and citric acid were added simultaneously. .

[Examples 5 to 8]

Saccharomyces cerevisiae (ATCC 60583) was preserved in the same YM liquid medium as in Example 1, and then inoculated in a 3.7 L fermentation tank to inflate air at 100 In / hour, rotation speed 1,000 rpm, culture temperature 27 Incubated for 24 hours at ℃.

After completion of the culture, the culture solution is concentrated to 1/10, and 50 ml of the concentrated culture solution is stored at 4 ° C.

(Example 5) 10 ml of the following solution A 'was added to 50 ml of the concentrated culture medium, adjusted to pH 6.8 with 50% NaOH, and left at 4 ° C for 1 hour.

(Example 6) 10 ml of the following solution B 'was added to 50 ml of the concentrated culture broth, adjusted to pH 6.8 with 50% NaOH, and left at 4 ° C for 1 hour.

Example 7 10 ml of the following solution B 'was added to 50 ml of the concentrated culture solution, mixed, and then stored at 4 ° C. for 1 hour, 40 ml of the solution C was added thereto, adjusted to pH 6.8 with 50% NaOH, and left at 4 ° C. for 1 hour. do.

Example 8 10 ml of the following solution B 'was added to 50 ml of the concentrated culture solution, mixed, and then stored at 4 ° C. for 1 hour, and 40 ml of the solution D was added thereto, adjusted to pH 6.8 with 50% NaOH, and left at 4 ° C. for 1 hour. do.

(Solution A '): A 10% peptone solution was prepared by dissolving 10 g of peptone in 100 ml of distilled water.

(Solution B '): 10 g of peptone and 30 g of lactose are dissolved in 100 ml of distilled water to prepare a solution of 10% peptone and 30% of lactose.

However, the peptone used was a Difco product.

After lyophilization, each culture solution was lyophilized, and the cells were crushed and stored in a polyethylene bottle, and strain stability was observed while being kept in a constant temperature and humidity chamber at a temperature of 20 ° C. and a humidity of 50%. The stability of the lyophilized cells over time is shown in Tables 5, 6, 7, and 8.

TABLE 5

TABLE 6

TABLE 7

TABLE 8

From the above results, when lactose or lactose and galactose were added to peptone, the survival rates after 30 days of storage were maintained at 27% and 28%, respectively, and when lactose, galactose, and citric acid were simultaneously added, 60% were maintained.

[Comparative Example]

Saccharomyces cerevisiae (ATCC 60583) in the preservation liquor was activated in the same YM liquid medium as in Example 1 and inoculated in a 3.7 L fermentation tank (Bioengineering KLF 2000) air feed rate 100In / hour, rotation speed 1,000rpm , 24 hours incubation at 27 ℃.

After completion of the culture, the culture solution is concentrated to 1/10 and 50 ml of the concentrated culture solution is adjusted to pH 6.8 with 50% NaOH, and left at 4 ° C. for 1 hour, followed by freeze drying (freeze dryer: Edwards 501). The lyophilized cells were crushed and stored in a polyethylene bottle, and strain stability was observed while stored in a constant temperature and humidity chamber (product of Duckwoo Science) at a temperature of 20 ° C. and a humidity of 50%. The stability of the lyophilized cells over time is shown in Table 9 below.

TABLE 9

From the results of these examples and comparative examples, it was confirmed that the use of the lyophilization aid according to the present invention exhibited a survival rate of 100 times or more as compared with the prior art.

Claims (5)

Saccharomyces cerevisiae (ATCC 60583) strain, which is a diffuser yeast, is cultured in YM liquid medium and concentrated in lyophilization, and 30 to 100 parts by weight of skim milk or peptone or a mixture thereof based on 100 parts by weight of dry cells. And lyophilization of the lyophilization aid consisting of 250 to 500 parts by weight of sugar and 30 to 70 parts by weight of citric acid by adding the concentrated culture solution. The method of claim 1, wherein the sugar is used as 100 to 250 parts by weight of lactose and 150 to 250 parts by weight of galactose, based on 100 parts by weight of the dry cells. The method of claim 1, wherein the lyophilized adjuvant is added to the concentrated culture solution in order to add skim milk or peptone, sugar and citric acid in order. A freeze-drying aid for the cells comprising 30-100 parts by weight of skim milk or peptone or a mixture thereof, 250-500 parts by weight, and 30-70 parts by weight of citric acid, which is used to lyophilize the cells of the diffuser yeast. The lyophilization aid of cells according to claim 4, characterized in that the sugar lactose comprises 100 to 250 parts by weight and galactose 150 to 250 parts by weight.