JP2003219862A - Method for improving storage stability of freeze-dried bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for improving storage stability of a freeze-dried bacterium, to obtain a storage stability improver of the freeze-dried bacterium and to provide a method for preserving a bacterium. <P>SOLUTION: This method for improving storage stability of the freeze-dried bacterium comprises freeze-drying the bacterium in the presence of arginine, ornithine, serine or their salt. This storage stability improver of the freeze-dried bacterium comprises arginine, ornithine, serine or their salt. This method for preserving the bacterium comprises freeze-drying and preserving the bacterium in the presence of arginine, ornithine, serine or their salt. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for improving the storage stability of freeze-dried microorganisms useful for foods, a storage stability improver for freeze-dried microorganisms and a method for storing microorganisms.

[0002]

2. Description of the Related Art Lactic acid bacteria and bifidobacteria are known as useful bacteria in the intestine, and in recent years, many yogurt and powder-type health foods containing lactic acid bacteria and bifidobacteria are commercially available. Live cells of lactic acid bacteria and bifidobacteria are used as raw materials for these products.However, when live cells are used, the bacteria are damaged or killed during storage of the product, and the number of live cells decreases. There is. When a freeze-dried microorganism is used, the storage period can be extended as compared with the case where a live cell is used, but the cell may be damaged or killed during freeze-drying. Therefore, a method of adding various protective substances has been studied for the purpose of protecting cells during freeze-drying.

As a method for reducing damage or death of cells during freeze-drying, for example, a method of adding sodium glutamate, L-ascorbic acid, whey and glucose (Japanese Patent Laid-Open No. 2-86769), aspartic acid, arginine. Of a mixture of at least three amino acids selected from the group consisting of amino acids consisting of amino acid, glutamic acid, proline, lysine, leucine, and methionine (JP-A-8-
205857) is known. However, the damage or killing of the bacterial cells often occurs not only during freeze-drying but also during the subsequent storage period. In particular, when stored at a temperature of 37 ° C. or higher, there is a problem that the viable cell count is significantly reduced during the storage period.

To solve this problem, a method of adding silica gel or skim milk powder is generally used as a method for improving the storage stability of freeze-dried microorganisms. In addition, a method of adding vitamin E during freeze-drying of microorganisms (Japanese Patent Laid-Open No. 50-9)
4185), a method of adding raw starch (JP-A-58-149675),
Phenylalanine, histidine, citric acid, succinic acid,
Method of adding a compound selected from the group consisting of tartaric acid and salts thereof, and alkali carbonate (JP-A-61-26508)
5) etc. are known. However, a method for further improving the storage stability of freeze-dried microorganisms is desired.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for improving the storage stability of freeze-dried microorganisms, an agent for improving the storage stability of freeze-dried microorganisms, and a method for storing microorganisms.

[0006]

The present invention includes the following (1).
Regarding (5). (1) A method for improving the storage stability of a freeze-dried microorganism, which comprises freeze-drying the microorganism in the presence of arginine, ornithine or serine, or a salt thereof. (2) The storage stability of the freeze-dried microorganism according to (1) above, wherein the amount of arginine, ornithine or serine, or a salt thereof is 1 to 90 parts by weight with respect to 100 parts by weight of wet bacterial cells of the microorganism. How to make. (3) The microorganism is Bifidobacteria
ium) genus Lactobacillus (Lactobacillus) genus Streptococcus (Streptococcus) sp., Lactococcus (L
Actococcus ) and Enterococcus ( Enterococcu
s ) A method for improving the storage stability of the freeze-dried microorganism according to the above (1) or (2), which is a microorganism selected from the group consisting of microorganisms belonging to the genus. (4) A storage stability improver for freeze-dried microorganisms, which comprises arginine or a salt thereof. (5) A storage stability improver for freeze-dried microorganisms, containing ornithine or serine, or a salt thereof. (6) A method for preserving a microorganism, which comprises freeze-drying the microorganism in the presence of arginine, ornithine or serine, or a salt thereof and storing the microorganism.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism used in the present invention may be any microorganism such as bacteria, filamentous fungi and yeast as long as it can be preserved by freeze-drying. For example, the genus Bifidobacterium , Lactobacillus (Lactobacillus) genus Streptococcus (Strep
tococcus) genus Lactococcus (Lactococcus) genus is a microorganism belonging to the Enterococcus (Enterococcus) genus, and the like. As a microorganism belonging to the genus Bifidobacterium, for example, Bifidobacterium longum (Bi
fidobacterium longum ), Bifidobacterium infantis ), Bifidobacterium adressentitis ( Bifidobacterium ad
olescentis ), Bifidobacterium breve ( Bifi
dobacterium breve), Bifidobacterium lactis (Bifidobacteriumlactis), and the like. Examples of microorganisms belonging to the genus Lactobacillus include Lactobacillus acidophilu.
s), Lactobacillus casei (Lactobacillus case
i ), Lactobacillus delbrueckii s
p. bulgaricus ), Lactobaci
llus gasseri ), Lactobacillus plantarum ( Lact
obacillus plantarum ) and the like. Examples of the microorganism belonging to the genus Streptococcus include Streptococcus thermophilus.
us ) etc. Examples of microorganisms belonging to the genus Lactococcus include Lactococcus lactis ( Lact
ococcus lactis ) and the like. Examples of microorganisms belonging to the genus Enterococcus include Enterococcus
Faecium (Enterococcus fecium), Enterococcus faecalis (Enterococcus faecalis), and the like.

These microorganisms can be cultivated by an ordinary solid culture method or liquid culture method. As the microorganisms used in the present invention, when these microorganisms are cultured by a solid culture method, the cells grown in a solid medium may be used as they are together with the solid medium, or the cells may be collected by a method such as scraping from the solid medium. You may use the microbial cell obtained by doing.
When cultivated by the liquid culture method, the culture solution may be used as it is, or as a wet cell body obtained by separating the cell body by a method capable of separating the cell body from the culture solution such as filtration or centrifugation from the culture solution You may use. Further, the microorganism may be suspended in an aqueous medium such as a buffer and used. As the aqueous medium, water, phosphate buffer, HEPES (N-2 hydroxyethylpiperazine-N-ethanesulfonic acid) buffer, or Tris
[Tris (hydroxymethyl) aminomethane] Buffers such as hydrochloric acid buffer and the like can be mentioned. The pH of the buffer solution may be any value as long as it is suitable for survival of microorganisms, but usually pH 4
-10, preferably pH 7-9.

Arginine, ornithine or serine, or salts thereof are added to the above microorganisms and mixed. Examples of the salt of arginine, ornithine or serine include hydrochloride, sulfate, phosphate and the like. Arginine, ornithine or serine, or salts thereof may be used in the form of powder or dissolved in an aqueous medium used for suspending the above-mentioned microorganism. The concentration of arginine, ornithine or serine, or a salt thereof in the solution may be any concentration, but 1% to
It is preferable to adjust the concentration to 50% (w / v).
In addition, arginine, ornithine or serine, or a salt thereof can be used as a storage stability improver for the freeze-dried microorganism of the present invention by itself being added to or mixed with the microorganism.

Arginine, ornithine or serine,
Alternatively, the amount of the salt added is preferably 1 to 90 parts by weight with respect to 100 parts by weight of wet microorganism cells.
More preferably, it is about 50 parts by weight. Further, in order to reduce the hygroscopicity of the freeze-dried microorganism, silica gel, starch, skim milk powder, gluconate, cellulose or the like may be added as a substance other than arginine, ornithine or serine, or salts thereof. Examples of the gluconate salt include, for example, sodium salt, potassium salt,
Examples include calcium salts and magnesium salts. There is no particular limitation on the amount of arginine, ornithine or serine, or a substance other than salts thereof added, for example, arginine, ornithine or serine to be added,
Alternatively, it is preferable to add 0.1 to 10 times the weight of the salt, and more preferably 0.5 to 2 times the weight.

Arginine, ornithine or serine,
Alternatively, the substances other than their salts may be added either before or after the freeze-drying treatment as long as they do not affect the storage stability of the freeze-dried microorganisms. Arginine, ornithine or serine, or a salt thereof, and a substance other than these, if necessary, are added to the microorganisms and mixed, and then the pH is adjusted if necessary and freeze-dried. As a method for mixing arginine, ornithine or serine, or a salt thereof and a microorganism, any method may be used as long as they can be mixed, and examples thereof include a method using a stirrer. You can The pH may be any value as long as it is suitable for survival of microorganisms, but usually pH 4
-10, preferably pH 7-9.

Freeze-drying can be carried out by using a freeze-dryer,
It can be performed by a conventional method. After freeze-drying, it is preferable that the freeze-dried microorganisms obtained are immediately sealed and stored in a suitable sealable container. At this time, substances other than the above-mentioned arginine, ornithine or serine, or salts thereof that reduce the hygroscopicity of the freeze-dried microorganisms may be enclosed. The preservation may be performed by any method as long as the freeze-dried microorganism can be preserved, but it is preferable that the preservation is allowed to stand in a shaded container. The storage temperature may be any number within the normal range, but is preferably 50 ° C. or lower. Examples will be shown below, but the present invention is not limited to the following examples.

[0013]

Example 1 Bifidobacterium seed-cultured in an MRS medium (manufactured by Difco, adjusted to pH 7.7 with sodium hydroxide solution) supplemented with 0.025% (w / v) cysteine, in the same medium・ Longum ( Bifidobacterium longum ) ATCC15707 strain 10% (v / v)
The cells were inoculated and cultured at 37 ° C for 8 hours with stirring under anaerobic conditions by blowing carbon dioxide. The pH was adjusted with a sodium bicarbonate solution so that the lower limit value was 5.5. After culturing,
The culture broth was centrifuged to collect bacterial cells to obtain wet bacterial cells. To 15 g of the wet cells, 75 ml of an aqueous solution of each of the 20 kinds of amino acids shown in Table 1 [2% (w / v)] was added, mixed and suspended, and the pH was adjusted to 8.0.

The obtained cell suspension was placed in a stainless steel vat and frozen at -80 ° C. overnight, and then freeze-dried machine TFD-550.
Freeze-drying was performed using -8 (manufactured by Takara Seisakusho) to obtain a mixture of freeze-dried microorganisms. In addition, a mixture of freeze-dried microorganisms was obtained by the same method as above except that no amino acid was added. The viable cell count in the mixture was measured according to a conventional method, and was taken as the viable cell count immediately after freeze-drying. 5 g of the mixture of freeze-dried microorganisms and 2.5 g of silica gel were packed in an aluminum pouch and stored at 50 ° C. About the sample opened 2 days after the start of storage,
The viable cell count was measured in accordance with a conventional method and used as the viable cell count after storage. The survival rate during storage was calculated from the following formula. Survival rate (%) during storage = Number of viable cells in 1 g of freeze-dried mixture after storage / Number of viable cells in 1 g of freeze-dried mixture immediately after freeze-drying × 100 The results are shown in Table 1.

[0015]

[Table 1]

The survival rate during storage was high when arginine hydrochloride, ornithine hydrochloride and serine were added, especially when arginine hydrochloride was added. Example 2 Wet cells of the Bifidobacterium longum ATCC15707 strain were prepared in the same manner as in Example 1. In 10 g of the wet cells, 6.7% (w / v) arginine hydrochloride aqueous solution 15 ml (containing arginine hydrochloride 1 g), 10% (w / v) arginine hydrochloride aqueous solution 30 ml (containing arginine hydrochloride 3 g), 15 % (w / v) arginine hydrochloride aqueous solution 30 ml (containing arginine hydrochloride 5 g), 21% (w / v) arginine hydrochloride aqueous solution 30
ml (containing 7 g of arginine hydrochloride) and 30% (w / v) aqueous solution of 30% arginine hydrochloride (containing 9 g of arginine hydrochloride) were added, mixed and suspended, and then the pH was adjusted to 8.0. . Put the resulting suspension in a stainless steel vat,
After freezing overnight at ℃, it was freeze-dried using a freeze dryer TFD-550-8 (manufactured by Takara Seisakusho) to obtain a mixture of freeze-dried microorganisms. The mixture of the freeze-dried microorganisms was enclosed with silica gel in the same manner as in Example 1 and stored at 50 ° C., and the survival rate 2 days after the start of storage was measured by the same method as in Example 1.
The results are shown in Table 2.

[0017]

[Table 2]

Example 3 In Example 2, a mixture of freeze-dried microorganisms obtained by adding 30 ml of a 10% (w / v) aqueous solution of arginine hydrochloride (containing 3 g of arginine hydrochloride) to 10 g of wet cells was prepared. The silica gel was stored at 50 ° C. without enclosing it in the same manner as in Example 1, and the survival rate 2 days after the start of storage was measured by the same method as in Example 1, and the survival rate was 41.0%. A high survival rate was obtained even when silica gel was not enclosed.

Example 4 Wet cells of Bifidobacterium longum ATCC 15707 strain were prepared in the same manner as in Example 1. To 10 g of the obtained wet cells, 100 ml of 3% (w / v) arginine hydrochloride aqueous solution was added, mixed and suspended, and then the pH was adjusted to 8.0.
Adjusted to. The resulting suspension was placed in a stainless steel vat and frozen overnight at -80 ° C, then freeze-dried by TFD-550-8.
Lyophilization was performed using (manufactured by Takara Seisakusho) to obtain a mixture of freeze-dried microorganisms. After the mixture of the freeze-dried microorganism and silica gel was enclosed and stored at 20 ° C. for 8 months, the survival rate of the freeze-dried microorganism was measured by the same method as in Example 1. The survival rate was 50.9%. Met.

[0020]

Industrial Applicability According to the present invention, it is possible to provide a method for improving the storage stability of freeze-dried microorganisms, a storage stability improver for freeze-dried microorganisms, and a method for storing microorganisms.

Claims (6)

[Claims] 1. A method for improving the storage stability of a freeze-dried microorganism, which comprises freeze-drying the microorganism in the presence of arginine, ornithine or serine, or a salt thereof. 2. The storage stability of the freeze-dried microorganism according to claim 1, wherein the amount of arginine, ornithine or serine, or a salt thereof is 1 to 90 parts by weight with respect to 100 parts by weight of wet bacterial cells of the microorganism. How to improve. 3. The microorganism is Bifidobacterium (Bifido
bacterium) genus Lactobacillus (Lactobacillus) genus,
Streptococcus (Streptococcus) sp., Lactococcus (Lactococcus) genus and Enterococcus (Enter
The method for improving the storage stability of a freeze-dried microorganism according to claim 1 or 2, which is a microorganism selected from the group consisting of microorganisms belonging to the genus ococcus ). 4. An agent for improving the storage stability of freeze-dried microorganisms, which comprises arginine or a salt thereof. 5. A storage stability improver for freeze-dried microorganisms, containing ornithine or serine, or a salt thereof. 6. Lyophilizing a microorganism in the presence of arginine, ornithine or serine, or salts thereof,
A method for preserving microorganisms, characterized by preserving.