JP3504365B2 - Microbial protective agent and method for producing frozen or lyophilized microorganism using the protective agent - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a process for producing a frozen or freeze-dried microbial cell which has less damage and death of a microorganism during freeze-thawing and freeze-drying and has high survival property. More specifically, the present invention relates to a bacterial cell dispersion liquid of a microorganism, aspartic acid, arginine, glutamic acid,
A microbial protective agent containing a mixture of at least three amino acids selected from a specific amino acid group consisting of proline, lysine, leucine and methionine or a derivative thereof, and a mixture of the amino acids or their derivatives A method for producing a frozen or lyophilized microbial cell, which comprises adding at a concentration of at least 0.5% (by weight) as a total amount of amino acids, mixing, and freezing or lyophilizing;
Is.

In the present specification, the percentage is a value by weight, excluding the survival rate of microorganisms, unless otherwise specified. Bifidobacterium is a microorganism belonging to the genus Bifidobacterium and lactic acid bacteria other than Bifidobacterium are lactic acid bacteria. Lactic acid bacteria, etc., bifidobacteria, lactic acid bacteria, etc. may be collectively referred to as lactic acid bacteria, and cells or cells of microorganisms may be collectively referred to as bacterial cells.

[0003]

BACKGROUND OF THE INVENTION Lactic acid bacteria are widely known as one of the useful enterobacteria, and there are many reports on the physiological significance of Bifidobacteria. In the intestine, organic acids such as lactic acid and acetic acid were detected. The effects of producing and suppressing the growth of harmful bacteria, production of vitamins, activation of immunity, etc. have been clarified (Mitsuoka Tomohushi, "Research on Bifidobacteria", Japan Bifidobacteria Center, 1994). ).

Therefore, various foods containing bifidobacteria, such as yogurt, confectionery, and beverages, have been developed for the purpose of maintaining health by ingesting bifidobacteria (edited by Chizu Mitsuoka, "Research on Bifidobacteria", Japan Bifidobacteria Center, 1994
Year). When adding Bifidobacteria to these foods, pre-cultured Bifidobacteria are processed and added, but the processing requires a large amount of labor and time, and a high survival rate of Bifidobacteria is maintained in the processing process. In order to do so, there were technically extremely difficult points.

On the other hand, lactic acid bacteria have been used as a starter for a very large number of dairy products such as yogurt and cheese since ancient times, and in recent years, like the bifidobacteria, they are expected to have an intestinal regulating action. By doing so, lactic acid bacteria are added to many foods (microorganisms, vol. 16, pages 44-55,
1990). Conventionally, these lactic acid bacteria, etc. are obtained by culturing them in milk etc., but this requires a lot of labor and time, and sufficient attention must be paid to the quality control of the strains. There was a problem that it had to be.

[0006] Therefore, foods containing bifidobacteria or lactic acid bacteria, such as dairy products such as yogurt and cheese, can be easily produced by using bifidobacterium or lactic acid bacteria viable cells that have been previously frozen or lyophilized. It becomes possible to do. However, the production of frozen or freeze-dried lactic acid bacteria having the required viable cell concentration for these products must prevent damage and / or death of the cells during freeze-thawing or freeze-drying. There was a big problem.

In the past, many cryoprotectants and freeze-dried protectants have been developed to solve these problems.
These substances include skim milk, sodium glutamate,
Gelatin and sucrose (Japanese Patent Publication No. 53-8793),
Phenylalanine, histidine, citric acid, succinic acid,
Tartaric acid and alkali carbonate (JP-A-61-265085) are known, and in addition, for example, amino acids alone, combinations of amino acids and substances other than amino acids,
Peptone, glucose, trehaloose, skimmed milk powder, ascorbic acid soda, etc. are also being investigated [Cryobiology, Vol. 26, 149-153.
Page, 1989].

However, even with these conventional techniques, the damage or death of the cells during freezing or freeze-drying is great, and it is not possible to produce stable frozen cells or freeze-dried cells with a high viable cell concentration. It was difficult. In addition, there is a problem that freeze-thawing and freeze-drying have different mechanisms of damage or death of cells, and therefore an effective protective agent for freeze-drying cannot necessarily be a protective agent for freeze-thawing. On the contrary, an effective protectant against freeze-thawing may not necessarily be an effective protectant against freeze-drying, and for the preparation of frozen cells or freeze-dried cells, individual protective agents should be searched. There was the inconvenience of having to.

Furthermore, in the process of producing frozen or freeze-dried cells, the rate of freezing the cell dispersion has a great influence on the damage or death of the cells, so that the freeze- or freeze-dried cells are industrially used. When manufacturing the body, there were many problems that had to be overcome in controlling the freezing rate.
As described above, various types of microbial protective agents have been developed so far, but amino acid mixtures are not known at all, and microbial cells are damaged or killed in the process of freezing or freeze-drying the microorganisms. There is no general-purpose protective agent that can prevent the above-mentioned problems, and its development has been long awaited.

[0010]

In view of the above-mentioned prior art, the present inventors have conducted earnest research on a general-purpose protective agent capable of preventing damage or death of microbial cells in the steps of freezing and lyophilizing microorganisms. As a result, rapid freezing, which lowers the liquid temperature as quickly as possible, and slow freezing, which gradually lowers the liquid temperature, cause different damage to microbial cells due to the difference in the crystal structure of the ice produced. , It is necessary to select a cryoprotective agent suitable for the freezing rate, and there are amino acids that damage microbial cells by freezing or freeze-drying, and amino acids that damage microbial cells. It was found that a specific amino acid mixture other than the above can prevent the damage or death of microbial cells in the process of freezing or freeze-drying the microorganism, and completed the present invention.

An object of the present invention is to provide a microbial protectant capable of obtaining a frozen or lyophilized microbial cell having a high survival rate with less damage or death of the microbial cell during freezing or lyophilization. That is.

[0012] Another object of the present invention is to provide a method for producing a frozen or freeze-dried microbial cell which is less likely to be damaged or killed in the freezing or freeze-drying step and has a high survival rate.

[0013]

The first invention of the present invention for solving the above problems is a composition for protecting microorganisms from damage during freezing or lyophilization, which comprises aspartic acid, arginine, glutamic acid, proline, lysine, a microorganism protection agent, characterized by comprising a mixture or mixtures of their derivatives of at least three amino acids selected from a specific group of amino acids consisting of leucine and methionine,
The mixture of three amino acids is a mixture of aspartic acid, glutamic acid and lysine, the mixture of three amino acid derivatives is an equal mixture of sodium aspartate, sodium glutamate and lysine hydrochloride, and the microorganism is , Bifidobact
erium) microorganisms or Lactobacillus
illus), a microorganism belonging to the genus Strepto
Lactococcu, a microorganism belonging to the genus coccus
s) Enterococcus, a microorganism belonging to the genus
s) a microorganism belonging to the genus or a mixed microorganism thereof is also a preferred embodiment.

A second aspect of the present invention for solving the above-mentioned problems is to provide a bacterial cell dispersion of a microorganism in which washed wet bacterial cells are dispersed in water, wherein aspartic acid, arginine, glutamic acid, proline, lysine, leucine and methionine are added. A mixture of at least three amino acids selected from the following specific amino acid group or a mixture of derivatives thereof is added at a concentration of at least 0.5% (by weight) as the total amount of amino acids, mixed, and then freeze-dried or freeze-dried. A method for producing a frozen or freeze-dried microorganism, characterized in that the total amount of amino acids is 2
˜20% (by weight), the mixture of three amino acids is a mixture of aspartic acid, glutamic acid and lysine, and the mixture of three amino acid derivatives is a mixture of sodium aspartate, sodium glutamate and lysine hydrochloride. Equivalent mixture, and the microorganisms are microorganisms belonging to the genus Bifidobacterium (Bifidobacterium) or Lactobacillus (Lactobacillus), microorganisms belonging to the genus Streptococcus, microorganisms belonging to the genus Lactococcus The microorganism is a microorganism belonging to the genus Enterococcus or a mixed microorganism thereof, which is also a preferable embodiment.

Next, the present invention will be described in detail. At first,
The first aspect of the present invention will be described. Microorganisms to which the microbial protectant of the present invention is applied are useful microorganisms that are frozen or lyophilized and used, and the type thereof is not particularly limited, but specifically, for example, Bifidobacteria, lactic acid bacteria, etc., bacteria, mold, yeast, etc. More specifically, Bifidobacteria are Bifidobacterium (Bi
A microorganism belonging to the genus fidobacterium, for example, Bifidobacterium longu
m), Bifidob
acterium infantis), Bifidobacterium adolescentis, Bifidobacterium breve
And the like, and all of them are strains that are commercially available or can be easily obtained from depository institutions.

Lactic acid bacteria and the like are microorganisms generally classified as lactic acid bacteria. For example, Lactobacillus (Lactobacillus)
microorganisms belonging to the genus obacillus [eg, Lactobacillus acidophilus, Lactobacillus bulgaricu
s), Lactobacillus casei)
Etc.], a microorganism belonging to the genus Streptococcus [eg,
Streptococcus
thermophilus), etc.], a microorganism belonging to the genus Lactococcus [eg, Lactococcus lactis (Lactococcus
lactis), etc.], a microorganism belonging to the genus Enterococcus [eg, Enterococcus faecium (Enterococcus f
aecium), Enterococcus faecalis (Enteroco)
ccus faecalis) etc.] and the like, all of which are commercially available strains or can be easily obtained from depository institutions.

Further, as bacteria other than lactic acid bacteria, microorganisms belonging to the genus Bacillus [eg, Bacillus
Bacillus subtilis etc.] and the like, and the mold is a microorganism belonging to the genus Penicillium [eg,
Penicillium roquefort
i) etc.] etc., and yeast is Saccharomyces (Saccharomyc
es) microorganisms belonging to the genus [eg, Saccharomyces cerevisiae, etc.] and the like, all of which are commercially available strains or can be easily obtained from depository institutions.

The amino acid constituting the microbial protectant of the present invention is a mixture of at least three kinds of amino acids selected from a specific amino acid group consisting of aspartic acid, arginine, glutamic acid, proline, lysine, leucine and methionine, or A mixture of these derivatives, which may be a mixture of four or more, is preferably a mixture of aspartic acid, glutamic acid and lysine, particularly preferably a mixture of sodium aspartate, sodium glutamate and lysine hydrochloride. Yes, the amino acid is L
It may be any type, D type or DL type optical isomer. The ratio of the three or more kinds of amino acids to be mixed is arbitrary, but an equivalent mixture is particularly preferable, and as the amino acid derivative, sodium salt, hydrochloride, potassium salt, other salts, hydrate, etc. Can be illustrated as a suitable example.

As will be apparent from the test examples described below, the larger the number of mixed amino acids or their derivatives, the higher the survival rate of microorganisms.
For example, it is possible to exemplify a suitable combination in the range of 3 to 7 types. Commercially available amino acids and amino acid derivatives used in the microbial protection agent of the present invention can be used, and the microbial protection agent of the present invention is sealed in an appropriate amount (eg, 1 kg unit) by a known method. It can be packaged into a final product. The microbial protectant of the present invention having the above-mentioned constitution makes it possible to obtain frozen cells or freeze-dried cells of a microorganism having less damage or death in the freezing or freeze-drying step and having a high survival rate. Therefore, it has an effect that frozen or freeze-dried cells of a microorganism having a high survival rate can be obtained regardless of the freezing rate such as quick freezing and slow freezing.

Furthermore, since the microbial protectant of the present invention can be composed only of amino acids or derivatives thereof, it also has excellent effects such as dispersibility, solubility and stability after dissolution, which are not found in known protectants. Have If necessary, the microorganism protecting agent of the present invention may be used in combination with a known protecting agent, and other optional components may be appropriately blended.

Next, the second invention of the present invention will be described. In the method for producing a frozen or freeze-dried microorganism of the present invention, first, bacterial cells of the microorganism are produced by a conventional method. The medium necessary for growing the microorganism varies depending on the microorganism, but for example, a liquid medium containing glucose, yeast extract, peptone or the like is inoculated with one or more kinds of lactic acid bacteria,
Usually, the cells can be cultured at 25 to 45 ° C. for 4 to 24 hours, and the bacterial cells are collected from the culture solution and washed to obtain wet bacterial cells.

In the method of the present invention, the wet bacterial cells obtained as described above are usually dispersed at a concentration of 0.5 to 12% (for the purpose of preserving the bacterial cells, mass-producing the bacterial cells, etc.). Disperse in water at different concentrations) to prepare a microbial cell dispersion. Next, the microorganism-protecting agent is added to the bacterial cell dispersion liquid at a concentration of at least 0.5% as the total amount of amino acids and uniformly mixed. The total amount of amino acid mixture added is 0.5-30%, preferably 2-20%. In the microbial cell dispersion concentration range, the added concentration of the amino acid mixture is irrelevant to the microbial cell dispersion concentration, and the protective effect is exerted by the concentration in the microbial cell dispersion liquid. At this time, a known cryoprotective agent other than the microorganism protection agent (for example, glycerin, sucrose, ascorbic acid, etc.) can be used in combination. When the pH of this mixed solution is acidic, a pH adjuster such as sodium hydroxide, sodium carbonate, potassium hydroxide, etc.
The pH is preferably adjusted to 6-8.

Next, in the method of the present invention, the mixed solution to which the microorganism protecting agent is added and mixed is frozen or lyophilized by a conventional method.
When it is carried out at 0 ° C to -160 ° C or lower (for example, use of liquid nitrogen) and freeze-dried, for example, it is performed by a known method under a vacuum at a shelf temperature of 35 ° C or lower and a vacuum degree of about 50 to 400 hPa. be able to.

In the method of the present invention, by adding and mixing the microorganism protecting agent as described above, damage or death of microorganisms during freezing or freeze-drying is reduced, and a high survival rate and a high concentration are obtained. Frozen cells or freeze-dried cells of microorganisms can be obtained. Further, in the method of the present invention, the microbial protective agent of the present invention may be used in combination with a known protective agent, if desired, and other optional components may be appropriately blended.

Next, the present invention will be described in detail by showing test examples. Test Example 1 This test was conducted in order to examine the protective effect of each amino acid or a derivative thereof at the time of freeze-thawing at two freezing rates of quick-freezing and slow-freezing against lactic acid fungal cells. 1) Preparation of wet cells Peptone (manufactured by Difco) 1%, yeast extract (manufactured by Difco) 2%, glucose (manufactured by Difco) 3%, monosodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) ) 0.5%, monosodium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.5%
Liquid culture medium (pH 6.5) containing Lactobacillus bulgaricus ATCC11
842 (obtained from ATCC) was inoculated and incubated at 37 ° C for 12 hours. After the completion of the culture, the bacterial cells were collected from the culture solution, washed with water, and collected again to obtain wet lactic acid bacterial cells.

2) Preparation of sample This wet microbial cell was added to an aqueous solution of 20 kinds of amino acids shown in Table 1, sodium glutamate and lysine hydrochloride (all manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to a concentration of 1%. A sample in which the microbial cell solid content concentration was 10% and a sample in which the same wet microbial cell was similarly dispersed in water (control) were prepared. The pH of all samples was adjusted to 6.5 with a 10% sodium hydroxide solution (Nacalai Tesque).

3) Test method Freezing method Each sample was rapidly frozen using liquid nitrogen, thawed in hot water at 37 ° C., and the freeze-thaw operation was repeated 5 times, and slowly in a freezer at −30 ° C. Frozen samples were thawed at room temperature and tested for viability by the method described below. Survival rate The number of viable bacteria before and after freezing of each sample was measured by a conventional method, and the survival rate was calculated from the following formula. Survival rate (%) = (the number of viable cells per 1 g of the sample after freeze-thawing / the number of viable cells per 1 g of the sample before freezing) × 100

4) Test Results The test results are shown in Table 1. As is clear from Table 1, the survival rate was significantly different depending on the type of amino acid. In particular, 7 amino acids, aspartic acid, arginine, glutamic acid, lysine, leucine, methionine, and proline, showed no decrease in survival rate compared to control water under both conditions of quick freezing and slow freezing. It was On the other hand, for other amino acids,
Both rapid and slow freezing were found to reduce viability more than control water. This indicates that amino acids other than the above-mentioned seven kinds of amino acids are more damaged by the freezing depending on freezing conditions.

The effects of the amino acid derivatives sodium glutamate and lysine hydrochloride are not different from those of the above-mentioned seven kinds of amino acids, and the amino acid derivatives such as salts and hydrates of each amino acid prepared in the same manner as above. The results of the test showed that salts of each amino acid, amino acid derivatives such as hydrates, etc. also have similar effects.

From the above results, aspartic acid, arginine, glutamic acid, lysine, leucine, methionine,
Unlike the amino acids other than these, 7 kinds of amino acids of proline or their derivatives have an action of reducing damage to cells due to freezing, and further, the damage is subjected to both quick freezing and slow freezing conditions. It was also confirmed that there was no action to increase even in. Similar results were obtained when the type of lactic acid bacteria was changed and when freeze-dried.

[0031]

[Table 1]

Test Example 2 This test was conducted to examine the cryoprotective effect of the amino acid mixture against lactic acid bacteria. 1) Preparation of wet bacterial cells Wet bacterial cells were obtained by the same method as in Test Example 1.

2) Preparation of sample As shown in Table 2, 2 to 8 kinds of amino acids were mixed from 20 kinds of amino acids, and the total amount of amino acids was 4%.
H was adjusted to 6.5), and the same wet bacterium was added to a sample in which the wet microbial cell was dispersed at a microbial cell solid concentration of 10%, and skim milk powder was dissolved in water at a concentration of 10%. A body dispersed sample (control) and a sample dispersed in 4% peptone water (control) were prepared.

3) Test method By the same method as in Test Example 1, a sample in which the cells were freeze-thawed was prepared and the survival rate was tested.

4) Results The results of this test are shown in Table 2. In Table 2, X represents any of 7 kinds of amino acids of aspartic acid, arginine, glutamic acid, lysine, leucine, methionine and proline, and Z is any of 13 kinds of amino acids excluding the above 7 kinds of amino acids. Indicates X or Z
The sample containing the amino acid indicated by indicates the minimum value and the maximum value of the test results of the 7 or 13 kinds of amino acid mixture samples, respectively.

As is clear from Table 2, in a mixture of 3 or more kinds of amino acids selected from 7 kinds of amino acids of aspartic acid, arginine, glutamic acid, lysine, leucine, methionine and proline, both slow-freezing and quick-freezing were performed. It was found that the cell survival rate was high and damage due to freeze-thawing of the cells was reduced. These survival rates were clearly higher than those of the sample containing each amino acid alone or the sample containing a mixture of two kinds of amino acids. In addition, as a mixture of two kinds of amino acids, the test was conducted for combinations other than those shown in Table 2, but the results were almost the same.

On the other hand, when amino acids other than the above-mentioned 7 kinds were added, the survival rate was remarkably lowered in all cases. From these facts, a mixture of three or more kinds of the above amino acids has an action of significantly reducing damage to lactic acid bacteria against freezing, and a mixture of amino acids other than the above seven kinds of amino acids has a reduced protective effect. Was shown. Although other lactic acid bacteria and other microorganisms were also tested, almost the same results were obtained. Also, almost the same results were obtained when freeze-dried.

[0038]

[Table 2]

Test Example 3 This test was carried out in order to investigate the influence of the concentration of the amino acid mixture on the survival rate of the cells during freeze-thawing. 1) Preparation of sample Wet cells were prepared in the same manner as in Test Example 1.

2) Preparation of sample Aspartic acid, arginine, glutamic acid, lysine,
From the 7 kinds of amino acids of leucine, methionine and proline, two sets of 3 kinds shown in Table 3 are selected and mixed, and the total amount of amino acids is 0.1, 0.5, 2.0, 10.0, 20.
0, 30.0, 50.0% amino acid mixed aqueous solution (p
H was adjusted to 6.5) to prepare a sample in which wet cells were dispersed at a cell solid concentration of 12%, and a sample in which the same wet cells were similarly dispersed in 4% peptone (control). Prepared.

3) Test Method By the same method as in Test Example 1, a sample in which the cells were freeze-thawed was prepared and the survival rate was tested.

4) Results The results of this test are shown in Table 3. As is clear from Table 3, in the sample in which the concentration of the amino acid mixture in the bacterial cell dispersion liquid is less than 0.5%, no increase in the survival rate due to freeze-thawing of lactic acid bacteria was observed, and the concentration was 30%. In the samples exceeding the above range, the survival rate did not increase with the increase of the addition ratio. On the other hand, in the sample in which the concentration of the amino acid mixture in the bacterial cell dispersion liquid is in the range of 0.5 to 30%, the survival rate is increased, and particularly in the range of 2 to 20%, the high survival rate is observed. Admitted. Therefore, the ratio of the amino acid mixture to be added to the bacterial cell dispersion liquid is 0.5 to 30%, preferably 2 to 20% as the total amount of amino acids. In addition, the lactic acid bacteria and the types of amino acids were changed and tested, but almost the same results were obtained. Also, almost the same results were obtained when freeze-dried.

[0043]

[Table 3]

Test Example 4 This test was conducted to examine the effect of the amino acid mixture on freeze-thawing of various lactic acid bacteria. 1) Preparation of wet bacterial cells G to which glucose (manufactured by Difco) was added at a rate of 2%
Streptococcus thermophilus AT in AM liquid medium (manufactured by Nissui)
CC19258 strain (obtained from ATCC), Lactobacillus acidophilus AT
CC4356 strain (obtained from ATCC), Enterococcus faecalis ATCC1
9433 strain (obtained from ATCC), Bifidobacterium longum ATCC15
Strain 707 (obtained from ATCC) was separately inoculated and cultured at 37 ° C. for 12 hours. After the completion of the culture, the bacterial cells were collected from the culture solution, washed with water, and collected again to obtain wet bacterial cells of each lactic acid bacterium.

2) Preparation of samples Wet cells were L (+) sodium aspartate monohydrate (Wako Pure Chemical Industries, Ltd.), L-lysine monohydrochloride (Wako Pure Chemical Industries, Ltd.), L (+) arginine. Disperse the aqueous solution containing 4% of each of hydrochloride (Wako Pure Chemical Industries, Ltd.) and L proline (Wako Pure Chemical Industries, Ltd.) at a concentration of 10% of the solid content of the cells to adjust the pH to 6. Sample (sample 1) adjusted to 5; skim milk powder and sodium glutamate dissolved in water at a concentration of 8% each, the same wet bacterial cells were dispersed, and the pH was adjusted to 6.5 (control) Was prepared and then slowly frozen and rapidly frozen in the same manner as in Test Example 1.

3) Test method The same method as in Test Example 1 was used.

4) Test Results The test results are shown in Table 4. As is clear from Table 4, in all of the strains, the sample 1 using the microbial protective agent of the present invention is higher than the control using only the known protective agent under both slow freezing and quick freezing conditions. Survival was observed. From these results, it was proved that the microbial protective agent of the present invention is extremely effective for freeze-thawing various lactic acid bacteria. It should be noted that, although the types of lactic acid bacteria and the amino acid mixture were changed and tested, almost the same results were obtained.

[0048]

[Table 4]

Test Example 5 This test was conducted to examine the effect of the amino acid mixture on the freeze-drying of various lactic acid bacteria. 1) Preparation of wet cells The same method as in Test Example 4 was used.

2) Preparation of sample L monosodium glutamate (manufactured by Wako Pure Chemical Industries, Ltd.), L lysine monohydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.), L proline (manufactured by Wako Pure Chemical Industries, Ltd.), DL methionine (Wako Pure Chemical Industries, Ltd.) (Made by Yaku Kogyo)
To an aqueous solution containing 3% of each, and a solid concentration of 1
A sample (Sample 2) in which the wet bacterial cells were dispersed at a ratio of 0% and the pH was adjusted to 6.5, and the same wet bacterial cells were dissolved in skim milk powder and sodium glutamate at a concentration of 8% each in water. The same wet bacterial cells were dispersed in the solution, and a sample (control) in which the pH was adjusted to 6.5 was prepared.

3) Test Method 1 ml of each sample was placed in a 3 ml vial bottle, frozen in a freezer at -30 ° C, and then freeze-dried at a temperature of 30 ° C in a vacuum degree of 100 to 200 hPa. The number of bacteria in each freeze-dried sample was measured by a conventional method, and the protective effect of the amino acid mixture on freeze-drying was tested.

4) Test Results The results of this test are shown in Table 5. As is clear from Table 5, it was confirmed that the amino acid mixture had a remarkably high survival rate against any lactic acid bacteria and had a freeze-drying protective effect on the lactic acid bacteria. The other amino acid mixtures and other lactic acid bacteria were also tested, but almost the same results were obtained.

[0053]

[Table 5]

[0054]

EXAMPLES Next, the present invention will be described in more detail by showing examples, but the present invention is not limited to the following examples. Example 1 100 g of L (+) sodium aspartate monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.), 100 g of L sodium glutamate (manufactured by Wako Pure Chemical Industries, Ltd.) and L lysine monohydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) 100 g of the mixture was uniformly mixed, filled in a synthetic resin bag (manufactured by Dainippon Printing Co., Ltd.) and sealed to obtain about 300 g of a microbial protection agent.

Example 2 L (+) arginine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) 100
g, L leucine (Wako Pure Chemical Industries, Ltd.) 100 g, L lysine monohydrochloride (Wako Pure Chemical Industries, Ltd.) 100 g, L proline (Wako Pure Chemical Industries, Ltd.) 100 g, and DL methionine (Wako Pure Chemical Industries, Ltd.) Are evenly mixed, filled in a bag made of synthetic resin (manufactured by Dai Nippon Printing Co., Ltd.) and sealed, and a microbial protection agent of about 40
0 g was obtained.

Example 3 Monosodium L-glutamate (manufactured by Wako Pure Chemical Industries, Ltd.) 50
g, L-lysine monohydrochloride (Wako Pure Chemical Industries, Ltd.) 50 g, L
(+) Arginine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) 50 g and L-leucine (manufactured by Wako Pure Chemical Industries, Ltd.) 50 g are uniformly mixed and filled in a synthetic resin bag (manufactured by Dai Nippon Printing Co., Ltd.) and sealed. Then, about 200 g of a microbial protection agent was obtained.

Example 4 Monosodium L-glutamate (Wako Pure Chemical Industries, Ltd.) 40
40 g of L lysine monohydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.), L
(+) Arginine hydrochloride (Wako Pure Chemical Industries, Ltd.) 40 g,
L-proline (Wako Pure Chemical Industries, Ltd.) 40 g and DL methionine (Wako Pure Chemical Industries, Ltd.) 40 g were uniformly mixed, filled in a synthetic resin bag (Dainippon Printing Co., Ltd.), sealed, and protected with microorganisms. About 200 g of the agent was obtained.

Example 5 80 g of L (+) sodium aspartate monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.), 80 g of L-lysine monohydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.), L (+) arginine hydrochloride (sum Kogaku Yakuhin Kogyo Co., Ltd.) 80 g, L proline (Wako Junyaku Kogyo KK) 80 g
Further, 80 g of DL methionine (manufactured by Wako Pure Chemical Industries, Ltd.) was uniformly mixed, filled in a bag made of synthetic resin (manufactured by Dai Nippon Printing Co., Ltd.) and sealed to obtain about 400 g of a microbial protectant.

Example 6 20 g of sodium L (+) aspartate monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.), 20 g of monosodium L glutamate (manufactured by Wako Pure Chemical Industries, Ltd.), L (+) arginine hydrochloride (sum Kogaku Yakuhin Co., Ltd.) 20 g, L-proline (Wako Pure Chemical Industries, Ltd.) 20 g and DL methionine (Wako Pure Chemical Industries, Ltd.) 2
0 g was uniformly mixed, filled in a synthetic resin bag (manufactured by Dainippon Printing Co., Ltd.), and sealed to obtain about 100 g of a microbial protection agent.

Example 7 30 g of L (+) sodium aspartate monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.), 30 g of monosodium L glutamate (manufactured by Wako Pure Chemical Industries, Ltd.), L lysine monohydrochloride (Wako Pure Chemical Industries, Ltd.) 30 g, L (+) arginine hydrochloride (Wako Pure Chemical Industries, Ltd.) 30 g, L proline (Wako Pure Chemical Industries, Ltd.) 30
g and DL methionine (manufactured by Wako Pure Chemical Industries, Ltd.) were uniformly mixed, filled in a bag made of synthetic resin (manufactured by Dai Nippon Printing Co., Ltd.) and sealed to obtain about 180 g of a microbial protectant.

Example 8 20 g of L (+) sodium aspartate monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.), 20 g of monosodium L glutamate (manufactured by Wako Pure Chemical Industries, Ltd.), L lysine monohydrochloride (Wako Pure Chemical Industries, Ltd.) 20 g, L (+) arginine hydrochloride (Wako Pure Chemical Industries, Ltd.) 20 g, L proline (Wako Pure Chemical Industries, Ltd.) 20
g, L leucine (manufactured by Wako Pure Chemical Industries, Ltd.) 20 g and DL methionine (manufactured by Wako Pure Chemical Industries, Ltd.) 20 g are uniformly mixed,
Fill a synthetic resin bag (manufactured by Dai Nippon Printing Co., Ltd.) and seal it.
About 140 g of a microbial protection agent was obtained.

Example 9 Peptone (manufactured by Difco) 1%, yeast extract (manufactured by Difco) 2%, glucose (manufactured by Difco) 3%, disodium monohydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.5%, sodium dihydrogen phosphate 0.5% (manufactured by Wako Pure Chemical Industries, Ltd.)
Lactobacillus bulgaricus AT in 300 ml of liquid medium (pH 6.5) containing
CC11842 (obtained from ATCC), 37 ° C
After culturing for 12 hours, 300 ml of 2.5 × 10 8 CFU / ml lactic acid bacterium solution was obtained. Cool this bacterial solution in the refrigerator,
Put in a 300 ml centrifuge tube, centrifuge (Tomi-Seiko Co., Ltd.)
By centrifugation at 6,000 xg for 20 minutes, the supernatant is removed by decantation, and 300 ml of sterilized water (4 ° C) is added to the precipitated wet cells to disperse the wet cells. Centrifugation was performed again under the same conditions, and the supernatant was similarly removed to obtain about 0.5 g of wet bacterial cells.

The wet bacterial cells were treated with the microbial protective agent of Example 1.
30 ml of a solution prepared by dissolving 6 g in water was added to disperse the cells, and the pH was adjusted to 6.5 with a 10% sodium hydroxide (Nacalai Tesque, Inc.) solution to prepare a cell dispersion. 3 ml of this dispersion was filled in a 10 ml vial bottle (manufactured by Nichiden Rikagaku Co., Ltd.), three of which were immersed in liquid nitrogen for 2 minutes and rapidly frozen to obtain lactic acid bacteria rapidly frozen cells. Also, put three other vials in a freezer (Sanyo-
The product was left for 1 hour in S.A. and slowly frozen to obtain lactic acid bacteria slowly frozen cells.

The frozen cells thus obtained were left to thaw at room temperature for 30 minutes to be thawed, and the survival rate was measured by the same method as in Test Example 1. As a result, in the rapidly frozen vial, it was 92.2%, slowly frozen. It was 94.1% in the vials, and all of them maintained a high survival rate.

Example 10 0.5 g of wet lactic acid bacteria obtained by the same method as in Example 1
To this, 20 ml of a solution prepared by dissolving 2.4 g of the microorganism protecting agent of Example 2 in water was added to disperse the wet cells, and the pH was adjusted to 6.5 with a 10% sodium hydroxide (Nacalai Tesque, Inc.) solution.
To prepare a bacterial cell dispersion liquid. Trace this dispersion
Pour into a lyophilizer and use a freeze dryer (Edwards Co., Ltd.)-
Cool to 40 ℃ and freeze, then 30 ℃, vacuum 10Pa
Freeze-drying was performed under the conditions described above to obtain about 1.5 g of freeze-dried cells.

The survival rate of the freeze-dried microbial cells thus obtained was measured according to the following formula, and as a result, it was 33.4%, and the high survival rate was maintained. Survival rate (%) = [Number of viable bacteria per 1 g after freeze-drying x Solid content of solution before freeze-drying (%) / Number of viable bacteria per 1 g before freeze-drying x)
100] x 100

Example 11 Peptone (manufactured by Difco) 1%, yeast extract (manufactured by Difco) 2%, glucose (manufactured by Difco) 2%, disodium monohydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.2%, monosodium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.4%
Bifidobacterium longum AT in 300 ml of liquid medium (pH 6.5) containing
CC15707 strain (obtained from ATCC) was inoculated and 37
After culturing for 16 hours at .degree. C., 300 ml of a 1.5.times.10@9 CFU / ml bifidobacteria solution was obtained. This bacterial solution was cooled in a refrigerator, placed in a 300 ml centrifuge tube, centrifuged at 6,000 xg for 20 minutes by a centrifuge (Tomi Seiko Co., Ltd.), and the supernatant was removed by decantation. Then, 300 ml of sterilized water (4 ° C.) was added to the precipitated wet cells to disperse the wet cells, centrifuge again under the same conditions, and the supernatant was removed in the same manner. 0.5 g was obtained.

To the wet cells, 30 ml of a solution prepared by dissolving 2.4 g of the microorganism-protecting agent of Example 3 in water was added to disperse the cells, and then 10% sodium hydroxide (Nacalai Tesque) solution was used. The pH was adjusted to 6.5 to prepare a bacterial cell dispersion liquid. 3 ml of this dispersion was filled in a 10 ml vial bottle (manufactured by Nichiden Rikagaku Co., Ltd.), three of which were immersed in liquid nitrogen for 2 minutes and rapidly frozen to obtain bifidobacterium rapidly frozen cells. Further, the other three were left in a freezer (manufactured by Sanyo Co., Ltd.) at -30 ° C. for 1 hour and slowly frozen to obtain bifidobacterium slowly frozen cells.

The frozen cells thus obtained were allowed to thaw at room temperature for 30 minutes to thaw them, and the survival rate was measured by the same method as in Test Example 1. As a result, in a rapidly frozen vial, it was 95.5%, slowly frozen. It was 93.1% in all the vials, and the high survival rate was maintained in each case.

Example 12 Peptone (manufactured by Difco) 1%, yeast extract (manufactured by Difco) 1%, glucose (manufactured by Difco) 1%, disodium monohydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.2%, dihydrogen phosphate monosodium (Wako Pure Chemical Industries, Ltd.) 0.2%
To 300 ml of liquid medium (pH 6.5) containing
Bacillus subtilis ATCC6633 strain (A
(Obtained from TCC), incubate at 30 ° C for 12 hours,
300 ml of a bacterial solution of 2.2 × 10 8 CFU / ml was obtained.
This bacterial solution was cooled in a refrigerator, placed in a 300 ml centrifuge tube, centrifuged at 5,000 xg for 15 minutes by a centrifuge (Tomi Seiko Co., Ltd.), and the supernatant was removed by decantation. Then, 300 ml of sterilized water (4 ° C.) was added to the precipitated wet cells to disperse the wet cells, centrifuge again under the same conditions, and the supernatant was removed in the same manner. 0.
4 g was obtained.

To the wet cells, 30 ml of a solution prepared by dissolving 1.8 g of the microorganism-protecting agent of Example 4 in water was added to disperse the cells, and then 10% sodium hydroxide (Nacalai Tesque) solution was used. The pH was adjusted to 6.5 to prepare a bacterial cell dispersion liquid. 3 ml of this dispersion was filled into a 10 ml vial bottle (manufactured by Nichiden Rikagaku Co., Ltd.), three of which were immersed in liquid nitrogen for 2 minutes, and quick frozen to obtain Bacillus subtilis rapidly frozen bacterial cells. Also, the other three were left in a freezer (manufactured by Sanyo Co., Ltd.) at -30 ° C for 1 hour to slowly freeze them,
Slowly frozen cells of Bacillus subtilis were obtained.

The frozen cells thus obtained were allowed to thaw for 30 minutes at room temperature and thawed, and the survival rate was measured by the same method as in Test Example 1. As a result, 97.7% was slowly frozen in the rapidly frozen vial bottle. It was 95.7% in the vials, and all of them maintained a high survival rate.

Example 13 Peptone (manufactured by Difco) 0.3%, yeast extract (manufactured by Difco) 0.4%, glucose (manufactured by Difco) 3
%, Disodium hydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.)
300 ml of liquid medium (pH 6.5) containing 0.2% and 0.2% of sodium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.)
Saccharomyces calsvar Jensis (Sacckaro
myces carlsbergensis) ATCC9080 strain (ATCC
1.2) and incubate at 25 ° C for 20 hours.
300 ml of a bacterial solution of × 10 7 CFU / ml was obtained. This bacterial solution was cooled in a refrigerator, placed in a 300 ml centrifuge tube, and centrifuged at 7,000 xg for 10 minutes by a centrifuge (Tomi Seiko Co., Ltd.), and the supernatant was removed by decantation. Then, 300 ml of sterilized water (4 ° C) was added to the precipitated wet cells.
Was added to disperse the wet microbial cells, centrifugation was again performed under the same conditions, and the supernatant was similarly removed to obtain about 0.4 g of wet microbial cells.

To the wet cells, 30 ml of a solution prepared by dissolving 2.8 g of the microorganism-protecting agent of Example 5 in water was added to disperse the cells, and then 10% sodium hydroxide (Nacalai Tesque) solution was used. The pH was adjusted to 6.5 to prepare a bacterial cell dispersion liquid. 3 ml of this dispersion was filled into a 10 ml vial bottle (manufactured by Nichiden Rikagaku Co., Ltd.), three of which were immersed in liquid nitrogen for 2 minutes, and quick freeze was performed to obtain yeast freeze-frozen cells. In addition, another three are stored in a freezer at -30 ° C (manufactured by Sanyo Electric Co., Ltd.)
It was left inside for 1 hour and slowly frozen to obtain yeast slowly frozen cells.

The frozen cells thus obtained were left to thaw at room temperature for 30 minutes to be thawed, and the survival rate was measured by the same method as in Test Example 1. As a result, in the rapidly frozen vial bottle, 94.1%, slowly frozen. It was 93.2% in all the vials, and the high survival rate was maintained in each case.

[0076]

INDUSTRIAL APPLICABILITY As described in detail above, the present invention uses a microbial protecting agent containing a mixture of specific amino acids or a derivative thereof during freezing or lyophilization, and the microbial protecting agent. The present invention relates to a method for producing a frozen or freeze-dried microbial cell having a high survival rate, and the effects of the present invention are as follows. 1) Frozen cells or freeze-dried cells of microorganisms which are less damaged or killed in the freezing or freeze-drying process and have a high survival rate can be obtained. 2) Frozen cells or freeze-dried cells of microorganisms having a high survival rate can be obtained regardless of the freezing rate such as quick freezing and slow freezing. 3) Since the microbial protective agent is composed of a mixture of specific amino acids or a mixture of derivatives thereof,
The workability at the time of dissolving the protective agent is good, the stability is high, and the dissolved water is colorless and transparent, so that the state of the bacteria after dispersion of the bacterial cells can be easily observed. 4) Since the solution of the microorganism protecting agent has a buffering action, it is possible to suppress the fluctuation of pH due to the dispersed microorganisms.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI (C12N 1/20 C12R 1:01) (72) Inventor Sayuri Miyaura 5-83 Higashihara, Zama City, Kanagawa Prefecture Mori Naga Milk Industry Co., Ltd. (56) References JP 60-172280 (JP, A) JP 54-76886 (JP, A) JP 5-292943 (JP, A) (58) Fields investigated ( Int.Cl. ⁷ , DB name) C12N 1/20 JST plus file (JOIS)

Claims (9)

(57) [Claims] 1. A composition for protecting microorganisms against damage during freezing or lyophilization, which is selected from at least 3 amino acids selected from the group consisting of aspartic acid, arginine, glutamic acid, proline, lysine, leucine and methionine. microbial protection agent characterized by comprising a mixture or mixtures of their derivatives species acids. 2. The mixture of three amino acids is a mixture of aspartic acid, glutamic acid and lysine.
The microbial protectant according to. 3. The agent for protecting microorganisms according to claim 1, wherein the mixture of three kinds of amino acid derivatives is a mixture of equal amounts of sodium aspartate, sodium glutamate and lysine hydrochloride. 4. The microorganism is Bifidobacterium (Bifi)
Lactobacillus, a microorganism belonging to the genus dobacterium
bacterium belonging to the genus obacillus, Streptococcus (Str
Lactococcus (Lacto), a microorganism belonging to the genus eptococcus
Enterococcus (Enterococcus), a microorganism belonging to the genus coccus
The microorganism protecting agent according to claim 1, which is a microorganism belonging to the genus occus) or a mixed microorganism thereof. 5. A microbe prepared by dispersing washed wet cells in water .
In the bacterial cell dispersion, a mixture of at least three kinds of amino acids selected from a specific amino acid group consisting of aspartic acid, arginine, glutamic acid, proline, lysine, leucine and methionine, or a mixture of their derivatives is used as the total amount of amino acids. A method for producing a frozen or freeze-dried microorganism, which comprises adding at a concentration of at least 0.5% (by weight), mixing, and freezing or freeze-drying. 6. The total amount of amino acids is 2 to 20% (by weight).
The method for producing a frozen or freeze-dried microorganism according to claim 5, wherein 7. The mixture of three amino acids is a mixture of aspartic acid, glutamic acid and lysine.
Alternatively, the method for producing a frozen or freeze-dried microorganism according to claim 6. 8. The method for producing a frozen or freeze-dried microorganism according to claim 5 or 6, wherein the mixture of the three kinds of amino acid derivatives is an equal mixture of sodium aspartate, sodium glutamate and lysine hydrochloride. 9. The microorganism is Bifidobacterium (Bifi).
microorganisms belonging to the genus dobacterium), microorganisms belonging to the genus Lactobacillus, microorganisms belonging to the genus Streptococcus, microorganisms belonging to the genus Lactococcus, enterococcus
The method for producing a frozen or lyophilized microorganism according to claim 5, which is a microorganism belonging to the genus (Enterococcus) or a mixed microorganism thereof.