JPS615776A - Microorganism strain preservative and method for preservation using same - Google Patents

Abstract

PURPOSE:To preserve microbial cells with a high survival ratio for a long period, by using a microorganism strain preservative consisting of a protein modified with a higher alcohol, fatty acid or amino acid ester. CONSTITUTION:A microorganism strain preservative obtained by modifying a protein, e.g. casein, gelatin or soybean protein, with a higher alcohol, fatty acid or amino acid ester by an enzymic reaction or conventional chemical method. Microbial cells are dipped in a liquid containing the above-mentioned microorganism strain preservative and preserved at 2--10 deg.C, preferably 0--5 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a microbial strain preservative and a method for preserving microbial strains using the same.

[Prior Art] Conventionally, methods of freezing, drying, or freeze-drying microbial strains have been widely used to preserve microbial strains because they have a wide range of applicability and can be stored for long periods of time, but all of these methods are extremely complicated to operate. However, expensive equipment is also required.

Methods other than the above include the distilled water preservation method and the sucrose aqueous solution preservation method, which are characterized by ease of operation, but in both cases the storage temperature is limited to about 5°C. If these methods are used to preserve the tissue at lower temperatures for longer periods of time with a higher survival rate, tissue destruction may occur due to the formation of ice crystals. However, when storing a certain amount of microbial strains, problems arise such as the need to dissolve the microbial strain-containing material each time to obtain a uniform state. The goal is usually not achieved.

[Summary of the Invention] In view of the above-mentioned circumstances, the present inventors have conducted intensive research to preserve microbial strains with a high survival rate for a long period of time using simple operations and without using expensive equipment. , completed the invention. - That is, the present invention involves immersing a microbial strain in a liquid containing MP and a microbial strain preservative characterized by comprising a protein modified with a higher alcohol, a fatty acid, or an amino acid ester (hereinafter referred to as MP). This invention relates to a method for preserving microbial strains characterized by storing them at 10°C.

[Embodiments of the invention] Examples of proteins used in the present invention include casein, gelatin, albumin, lactalbumin, soybean protein,
Although fish meat protein and the like can be suitably used, the present invention is not limited thereto. If these proteins are difficult to dissolve in water, such as casein, water.

For the purpose of increasing solubility, it may be used after being acylated or succinylated.

In the present invention, the protein is modified with a higher alcohol, fatty acid, or amino acid ester using an enzyme or by a conventional chemical method to produce MP used as a preservative for microbial strains.

Examples of the higher alcohol include aliphatic alcohols having 6 to 30 carbon atoms, and specific examples thereof include octyl alcohol, lauryl alcohol, oleyl alcohol,
Examples include stearyl alcohol.

Examples of the fatty acid include fatty acids having 4 to 30 carbon atoms, and specific examples thereof include octylic acid, lauric acid, valmitic acid, oleic acid, and stearic acid.

As the amino acid constituting the amino acid ester, hydrophobic amino acids such as leucine, norleucine, and alanine are preferably used, but the amino acid is not limited thereto.

The alcohol constituting the amino acid ester is
Aliphatic alcohols are preferred, but not limited thereto. Among aliphatic alcohols, carbon number is 4-1
6 is more preferred; as the number of carbon atoms decreases, the foaming power of the produced MP increases, and as the number of carbon atoms increases, the emulsifying power tends to increase.

Specific examples of MP in the present invention include succinylcasein leucine dodecyl ester and leucine dodecyl ester, for example, with a molecular weight of 200
It is from 0 to 40,000 Daltons.

When enzymes are used to modify proteins with amino acid esters, thiol proteases such as papain, bromelain, and ficin are preferably used.

As a specific example of modifying a protein with an amino acid ester using an enzyme, see Watanabe et al., J. et al. Foo
d Sci. , Li 1467-1469 (1981), it can be obtained by enzymatically reacting a protein and an amino acid ester in an aqueous solvent. Examples of aqueous solvents used in this case include water-acetone, water-acetone, and water-acetone.
It is preferable to use an ethanol-based compound and a thiol protecting agent such as 2-mercaptoethanol or cysteine in order to make the reaction system uniform and to proceed with the enzyme reaction efficiently. Since such a method is easy to carry out, it is possible to produce large quantities on an industrial scale.

Reaction conditions may be appropriately selected depending on the type of protein, type of enzyme, type of amino acid ester, etc., but usually the reaction system is adjusted to a pH of 7 to 10, and a pH of 0.25 to 50°C at 5 to 50°C.
It is sufficient to react for 24 hours.

Specific examples of producing HP by chemical methods include ultrasonication of a mixture of proteins and higher alcohols, fatty acids, or amino acid esters, or covalent bonding of fatty acids to proteins by transesterification in the form of esters. Examples of methods include, but the method is not limited to these methods.

Microbial strains for which the microbial strain preservative of the present invention can be used include, for example, spore-bearing yeasts such as the genus Satucharomyces, genus Hansenula, and the genus Pichia, nonspore-bearing yeasts such as the genus Candida, genus Rhodotorula, and the genus Torulopsis, and spore-bearing yeasts such as the genus Penicillium and the genus Aspergillus. Examples include, but are not limited to, filamentous fungi such as Actinomyces, actinomycetes such as Actinomyces, and bacteria such as Lactobacillus and Brevibacterium. Any microbial strain that can be preserved by immersion in a liquid such as liquid can be applied.

Next, existing methods using the microbial strain preservative of the present invention will be explained.

For example, the desired base, preferably 0.1 to 1
MPo
, 03 to 10 parts, preferably 0.05 to 5 parts,
Store at 2 to -10°C, preferably 0 to -8°C, more preferably 0 to -5°C.

If you try to store a solution containing microbial strains that does not contain MP at a temperature below about 5°C, the problems mentioned above will occur.
Although the mechanism is not clearly elucidated, the addition of MP can lower the freezing point to a considerably lower temperature, at least to below -5° C., which is below the maximum ice formation zone.

Therefore, when using MP, the microbial strain-containing solution can be reduced by 5 to -1
Even when stored at temperatures as low as 0°C, problems that occur at temperatures below 5°C do not occur without the use of MP, and microorganisms can be stored for long periods of time with a high survival rate using simple operations and without the use of expensive equipment. Bacterial strains can now be stored. Note) If the amount of IP added is less than 0.03 parts per 100 parts of water, the freezing point of the solution containing microbial strains will be around 1°C, and the effect of using MP will not be sufficiently obtained. Even if you use it more than that, the effect will not change.

The microbial strain preservative of the present invention may be used alone, but when used in combination with salts such as common salt, sugars such as sucrose, glucose, and fructose, and sugar alcohols such as sorbitol and maltitol, it may be used alone. When used, the effect of lowering the freezing point is further increased, which is preferable.

Next, the microbial strain preservative of the present invention and the preservation method using the same will be explained based on Examples.

Production Example 1 Commercially available gelatin was added to a 1H carbonate M solution (pl(9)) containing 10 mH cysteine and 20% (wt%) ethanol to a concentration of 33%, and L-leucine decyl ester was added. Added 0.1 mol of to 1009 gelatin, and added 1 mole of papain to 1009 gelatin.
g added. After that, after reacting at 37°C for 15 minutes,
The enzyme reaction was stopped by adding 1N hydrochloric acid to lower the pH to 1, and the non-dialyzable fraction was collected by running water dialysis. The lyophilized product of the non-dialyzable fraction was washed with dichloromethane and acetone to obtain purified enzyme-modified gelatin (hereinafter referred to as
EHG-10) was obtained.

Example 1 EHG-10 obtained in Production Example 1 was dissolved in distilled water to prepare a 0.05% ERG-10 aqueous solution, 10 m of it was dispensed into test tubes, and sterilized at 1'20°C for 10 minutes. did.

Saccharomyces cerevisiae
) One slant culture of TFO2043 cells was suspended in the test tube and stored for 3 months at the temperature shown in Table 1.
The survival rate of Satucharomyces cerevisiae IFO2043 was measured based on the following method. The results are shown in Table 1.

(Survival rate) Dilute the suspension, measure the number of viable bacteria by plate culture method, and -
Calculate the survival rate using the following formula.

Viable cell count survival rate after storage - x 100 Comparative example 1 of viable cell count before storage The survival rate of bacterial cells was measured in the same manner as in Example 1 except that EHG-10 was not used. The results are shown in Table 1.

Comparative Example 2 10% skim milk and 1% sodium glutamate were added as protective agents to a microbial strain-containing solution prepared in the same manner as Comparative Example 1.
The mixture was freeze-dried, stored at -5°C for 3 months, and then suspended in sterile water. The survival rate was measured in the same manner as in Example 1 and found to be 93%.

Example 2 and Comparative Example 3 The survival rate was measured in the same manner as in Example 1 and Comparative Example 1, respectively, except that the bacterial cells were changed to Saccharomyces rosei AHU 3975. The results are shown in Table 1.

Comparative Example 4 A microbial strain-containing solution prepared in the same manner as in Comparative Example 3 was freeze-dried in the same manner as in Comparative Example 2, stored for 3 months, and the survival rate was measured and found to be 95%.

Example 3 The microbial strain-containing solutions prepared in Example 1 and Example 2 did not freeze even if they were left at -7°C for 90 days.

From the above results, according to one method of the present invention using the microbial strain preservation solution of the present invention, the microbial strain-containing solution does not freeze even at -7°C and can be stored in liquid form at such low temperatures. Therefore, it is easy to operate, does not require expensive equipment, has low utility costs, and can preserve bacterial strains with a high survival rate.

Claims (1)

[Claims] 1. A microbial strain preservative characterized by comprising a protein modified with a higher alcohol, fatty acid or amino acid ester. 2 Immerse a microbial strain in a liquid containing a protein modified with a higher alcohol, fatty acid or amino acid ester,
A method for preserving microbial strains, characterized by preserving them at 2 to -10°C.