JPH07111887A - Preservative for microorganism - Google Patents

Abstract

PURPOSE:To provide a preservative for microorganism capable of easily preserving a large amount of microorganism over a long period (for example several months) at room temperature to enable easy transportation of a large amount of microorganism. CONSTITUTION:Microorganisms are held on a surface of a plurality of gelatinized water-absorbing resin particles. The water-absorbing resin particles are gelatinized and swollen by water absorption to keep a closely contacting state. The microorganisms are encircled by the gelatinized and swollen water- absorbing resin particles and, accordingly, shielded from oxygen in air and protected from drying.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a microbial preservative for preserving microorganisms while maintaining their activity.

[0002]

2. Description of the Related Art Conventionally, as a method for preserving microorganisms, there have been known methods for preserving at low temperature such as freeze-drying, frozen preservation, and refrigeration preservation. However, although these methods are suitable for storing a small amount of microorganisms, the energy cost for keeping a low temperature is large for storing a large amount of microorganisms, and the process and operation are complicated. Further, since it must be handled in a low temperature state at all times, it is inconvenient to carry it, for example. Therefore, the above storage method is not suitable for carrying out on an industrial as well as a commercial scale.

Therefore, in recent years, various microbial preservatives for preserving a large amount of microorganisms at room temperature have been investigated. For example, Japanese Examined Patent Publication No. 1-30476 discloses a method in which an aqueous dispersion in which microorganisms are dispersed and a water-absorbent resin are mixed by stirring to absorb the above-mentioned aqueous dispersion containing microorganisms in the water-absorbent resin. A microbial preservative (referred to as immobilized microorganism in the above publication) obtained by contacting a resin with a solution of a polyvalent metal salt to cause release of water and a crosslinking reaction is shown. Since the above-mentioned microorganism preservative is immobilized by confining the microorganisms in the pores inside the water absorbent resin, it is possible to preserve the microorganisms at room temperature.

[0004]

However, the size of the pores inside the water-absorbent resin that traps the microorganisms is usually 1 or less.
Therefore, the conventional microorganism preservatives have a problem that the size of microorganisms that can be preserved is limited to 1 micron or less. Further, the conventional microbial preservative, when confining the microorganisms in the pores inside the water-absorbent resin, contact the water-absorbent resin with the polyvalent metal salt solution, it has to perform the release of water and the crosslinking reaction, Further, since it is necessary to wash the water-absorbent resin with water in order to remove the polyvalent metal salt after the reaction, there are problems that the manufacturing process and operation become complicated and that the manufacturing takes time.

The object of the present invention is to solve the above-mentioned problems, to enable simple production at low cost in a short time, and to maintain the activity of microorganisms at room temperature simply and easily. It is to provide a microbial preservative capable of preserving a large amount.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies as to a microbial preservative capable of easily storing a large amount of a microorganism at room temperature while maintaining its activity. The microorganisms are retained on the surface of the water-absorbent resin particles, and by adhering these water-absorbent resin particles to each other, it is found that the microorganisms are stored for a long period of time, and the entire water-absorbent resin particles are protected from oxygen by a blocking material. It was confirmed that the microorganisms are more stably preserved by the blocking, and the present invention has been completed.

That is, the present invention is characterized in that microorganisms are held on the surfaces of a plurality of gelled water-absorbent resin particles, and the water-absorbent resin particles are in close contact with each other. Further, it is characterized in that it is provided with a barrier material that shields the entire water-absorbent resin particles from oxygen. Also,
It is characterized in that the size of the microorganism is 1 micron or more.

The present invention will be described in detail below.

The water-absorbent resin used in the present invention is
It is not particularly limited as long as it has a water absorption capacity (= weight of water absorbed at maximum / weight of water-absorbent resin) of absorbing 10 to 1000 times its own weight of pure water. Therefore, in the present invention, a hydrophilic crosslinked polymer having a hydrophilic functional group such as anionic, nonionic or cationic can be used. Specifically, for example, a crosslinked product of carboxymethyl cellulose, a hydrolyzate of a starch-acrylonitrile graft copolymer, a neutralized product of a starch-acrylic acid graft copolymer, a crosslinked product of a poly (meth) acrylic acid salt polymer. , Cross-linked product of partially neutralized polyacrylic acid, cross-linked product of isobutylene-maleic anhydride copolymer, saponification product of vinyl acetate-acrylic acid ester copolymer, hydrolysis of acrylonitrile copolymer or acrylamide copolymer Examples thereof include crosslinked products thereof, crosslinked products of sulfo group-containing polymers, and crosslinked products of polyethylene oxide and polyethyleneimine. Among these water-absorbent resins, a neutralized product of a starch-acrylic acid graft copolymer, a crosslinked product of a partially neutralized polyacrylic acid, and a crosslinked product of a sulfone group-containing polymer are preferable. Of course, it is also possible to use a commercially available product of the water absorbent resin. The water-absorbent resin gelates and swells when it absorbs water.

The water-absorbent resin can be used in any of the commonly available shapes such as spherical shape, flake shape, granular shape, lump shape, film shape, fibrous shape, web shape, sheet shape and the like. Further, its size is not particularly limited. Furthermore, a non-woven fabric or the like to which the water absorbent resin is integrally fixed may be used. For convenience of explanation, in the present invention, the above-mentioned shapes are collectively referred to as “particles” and hereinafter referred to as water-absorbent resin particles as necessary.

The microorganisms that can be stored in the present invention are not particularly limited, but for example, they have been widely used in various fields such as various aerobic microorganisms capable of producing useful products and water treatment. The various aerobic bacteria described above are preferred. Specifically, for example, the genus Acinetobacter (Ac
inetobacter), Candida, Nocardia (N
ocardia), Streptomyces, Nitrosomonas, Arthrobacter (Arthr
bacterium), Aspergillus, Sporotrichum, Rhodococ
cus), Fusarium, Achromobacter
(Achromobacter), Chromobacter
and aerobic microorganisms such as Mycobacterium. These microorganisms may be used alone or, of course, a mixed microorganism in which two or more kinds of microorganisms are mixed may be used. In addition, the size of the storable microorganisms is not particularly limited, but as described below, the size of the microorganisms should be set so that the microorganisms are retained on the surface of the water absorbent resin particles.
Micron or more is preferable.

In general, microorganisms have, as their biological characteristics, a high metabolic capacity not found in higher organisms and an extremely unstable cellular tissue, and they are strongly influenced by the surrounding environment. It For example, aerobic microorganisms grow and become active in the presence of oxygen and a large amount of water at room temperature, but die in a short period of time without nutrients. Therefore, in order to preserve the activity of aerobic microorganisms at room temperature for a long period of time (for example, for several months), oxygen in the air and excess water other than the water necessary for protection from drying should be used. It has to be removed.

The barrier material used in the present invention is not particularly limited as long as it can shield the entire water-absorbent resin particles from oxygen in the air. Specific examples include containers and bags made of various synthetic resins that are impermeable to oxygen, films and the like, and containers made of various metals. Then, in order to further improve the barrier property, the air inside these containers and bags may be replaced with an inert gas such as nitrogen and sealed, and a so-called oxygen scavenger together with a water absorbent resin may be used. It may be enclosed. Furthermore, the upper portion of the water-absorbent resin filled in a container or the like may be covered with an organic substance such as liquid paraffin that has no toxicity to aerobic microorganisms. Of course, it is also possible to use these three means together.

An example of the production method for producing the microbial preservative according to the present invention will be shown below.

First, an aqueous dispersion is prepared by dispersing, for example, an aerobic microorganism desired to be preserved in water. This aqueous dispersion can be obtained by using a so-called liquid culture method which has been conventionally performed. Incidentally, when an aqueous dispersion is obtained by using the liquid culture method as described above, various components used in culturing the microorganism remain in the aqueous dispersion, but these various components are absorbed by the water-absorbent resin. At that time, since it is taken into the inside of the resin, there is no possibility of adversely affecting the preservation of microorganisms.

The number of microorganisms per unit volume of the above aqueous dispersion is not particularly limited, but per 1 ml
10 ³ or more is preferable. When the number of microorganisms per 1 ml of the aqueous dispersion is less than 10 ^{3, the} number of microorganisms per unit area retained on the surface of the water-absorbent resin particles is reduced, and the storage efficiency is reduced, which is not preferable. . further,
When the number of microorganisms per 1 ml of the aqueous dispersion is 10 ³ or more, the microorganisms are retained at a high density on the surface of the water-absorbent resin particles. Therefore, after the preservation, the microorganisms need not be pre-cultured before they are used. It is suitable because it can be used immediately.

Next, water-absorbent resin particles are mixed with the aqueous dispersion thus prepared. At this time, the weight of the water-absorbent resin relative to 1 part by weight of the aqueous dispersion is such that the water-absorbent resin particles are gelated and swollen in consideration of the storage stability of microorganisms, and
The weight ratio of the aqueous dispersion and the water-absorbent resin is 1/1 or more and less than 100/1, preferably 9/1 or more and less than 100/1 so that the swollen water-absorbing resin particles adhere to each other. Is preferred.

As described above, a simple operation of preparing an aqueous dispersion liquid in which the microorganisms are dispersed and mixing the water-absorbent resin particles with the aqueous dispersion liquid is carried out, and the microorganisms can be produced at a low cost at a low cost. A preservative is obtained. Then, the microorganisms retained on the surface of the water-absorbent resin particles are gelled and swollen by absorbing water so that the water-absorbent resin particles are brought into close contact with each other, so that the periphery thereof is surrounded by these water-absorbent resin particles. Therefore, the microorganisms are shielded from oxygen in the air and protected from drying, and are stored in, for example, an asphyxia state.

Since the microorganisms are retained on the surface of the water-absorbent resin particles, when the microorganisms are taken out of the microbial preservative, for example, the microorganisms and the water-absorbent resin particles can be separated only by washing the microbial preservative with water. it can.

The microbial preservative of the present invention can be used for various applications such as oil decomposition, water treatment, environmental measures such as deodorization, food industry such as alcohol fermentation and amino acid fermentation, chemical industry such as citric acid fermentation and itaconic acid fermentation. It becomes possible to store a large amount of the microorganisms used at room temperature while maintaining its activity. Moreover, a large amount of microorganisms can be easily transported by using the microbial preservative of the present invention.

[0021]

According to the above construction, the microorganisms retained on the surface of the water-absorbent resin particles are in contact with each other because the water-absorbent resin particles swollen and gelled and swelled by absorbing water. Surrounded by resin particles. Therefore, the microorganisms are shielded from oxygen in the air and protected from drying. Therefore, it becomes possible to easily and in large quantities store microorganisms at room temperature for a long period of time (for example, several months) while maintaining their activity. Further, it becomes possible to easily carry a large amount of microorganisms.

Further, according to the above constitution, since the water-absorbent resin particles are provided with the blocking material for blocking the whole from oxygen, it becomes possible to more stably store the microorganisms.

Further, according to the above-mentioned constitution, it becomes possible to easily store a large amount of microorganisms having a size of 1 micron or more at room temperature for a long period of time while maintaining their activity.

The microbial preservative having the above-mentioned constitution is, for example,
A simple operation of preparing an aqueous dispersion liquid in which microorganisms are dispersed and mixing the water-absorbent resin particles with the aqueous dispersion liquid can be obtained at low cost without taking time.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto. In the following description, "parts" means "parts by weight" and "%" means "% by weight" unless otherwise specified.

[0026]

【Example】

[Example 1] Acinetobacter calcoaceticu (Acinetobacter calcoaceticu) was used as a microorganism desired to be preserved in an aqueous solution containing the following components using a liquid culture method.
s) was dispersed to prepare an aqueous dispersion having a number of bacteria of 3 × 10 ⁷ cells / ml. The above ingredients are olive oil 2.0%, ammonium sulfate 0.6%, disodium phosphate dodecahydrate 0.6%, monopotassium phosphate 0.4%, magnesium sulfate heptahydrate 0.001%, ferrous sulfate 0.001%, calcium chloride 2 Hydrate 0.001%, malt exudate 0.1%,
And the yeast extract was 0.1%, and the pH of the aqueous solution was 6.8.

A microbial preservative was obtained by mixing 3 parts of a water absorbent resin (trade name: Aquaric CA, manufactured by Nippon Shokubai Co., Ltd.) with 100 parts of the aqueous dispersion prepared as described above.

The obtained microbial preservative was sealed in a polypropylene container and stored at room temperature for 2 months. afterwards,
When the oil and fat decomposition test was carried out using the stored Acinetobacter calcoaceticus, the oil and fat decomposition rate of the above-mentioned preserved bacteria showed the same value as the oil and fat decomposition rate of the same bacteria before storage.

The obtained microbial preservative was placed in a polypropylene container, the air inside was replaced with nitrogen, and the container was sealed and stored at room temperature for 4 months. Then, when an oil and fat decomposition test was conducted using the stored Acinetobacter calcoaceticus, the oil and fat decomposition rate of the above-mentioned preserved bacteria showed a value equivalent to the oil and fat decomposition rate of the same bacteria before storage.

Example 2 Using the liquid culture method, 3 × 10 ⁷ cells / ml were dispersed by dispersing Toyulopusis candida as a microorganism desired to be preserved in an aqueous solution containing the components shown below. An aqueous dispersion having the number of bacteria was prepared. The above ingredients are olive oil 2.0
%, Ammonium sulfate 0.6%, disodium phosphate dodecahydrate 0.03%, monopotassium phosphate 0.47%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate 0.001%, calcium chloride dihydrate 0.001%, malts Leachate 0.1%, yeast extract
0.1%, polypeptone 0.1%, and manganese sulfate tetrahydrate 0.001%, and the pH of the aqueous solution was 5.5.

A microbial preservative was obtained by mixing 3 parts of a water-absorbent resin (trade name: Aquaric CA, manufactured by Nippon Shokubai Co., Ltd.) with 100 parts of the aqueous dispersion prepared as described above.

The obtained microbial preservative was sealed in a polypropylene container and stored at room temperature for 2 months. afterwards,
When the oil and fat decomposition test was carried out using the stored B. aureus, the oil and fat decomposition rate of the above-mentioned preserved bacteria showed a value equivalent to the oil and fat decomposition rate of the same bacteria before storage.

The obtained microbial preservative was placed in a polypropylene container, the air inside was replaced with nitrogen, and the container was sealed and stored at room temperature for 4 months. Then, when the oil and fat decomposition test was carried out using the preserved Toulopsis candida, the oil and fat decomposition rate of the preserved bacteria showed a value equivalent to the oil and fat decomposition rate of the same bacteria before storage.

[Example 3] 25 parts of the aqueous dispersion prepared in Example 1 and 75 parts of the aqueous dispersion prepared in Example 2 were mixed with 100 parts of a mixed solution to absorb water. A microbial preservative was obtained by mixing 3 parts of a functional resin (trade name: Aquaric CA, manufactured by Nippon Shokubai Co., Ltd.).

The obtained microbial preservative was sealed in a polypropylene container and stored at room temperature for 2 months. afterwards,
When a fat and oil decomposition test was conducted using a mixed strain of stored Acinetobacter calcoaceticus and Toulopsis candida, the oil and fat decomposition rate of the above stored mixed strain was equal to the oil and fat decomposition rate of the same mixed strain before storage. showed that.

The obtained microbial preservative was placed in a polypropylene container, the air inside was replaced with nitrogen, and the container was sealed and stored at room temperature for 4 months. Then, when a fat and oil decomposition test was performed using a mixed bacterium of the stored Acinetobacter calcoaceticus and Toulopsis candida,
The oil and fat decomposition rate of the above-mentioned preserved mixed bacterium showed a value equivalent to the oil and fat decomposition rate of the same mixed bacterium before preservation.

Comparative Example 1 The water dispersion prepared in Example 1 and the water dispersion prepared in Example 2 were each sealed in a polypropylene container and stored at room temperature. On the second day after the start, a precipitate considered to be a microbial carcass was formed at the bottom of these containers, and Acinetobacter calcoaceticus and Toulopsis candida were killed.

[Comparative Example 2] The aqueous dispersion prepared in Example 1 and the aqueous dispersion prepared in Example 2 were placed in polypropylene containers, and the air inside was replaced with nitrogen. It was sealed and stored at room temperature.
On the day, a precipitate that appeared to be a microbial carcass was formed on the bottom of these containers, and Acinetobacter calcoaceticus and Toulopsis candida were killed.

The above-mentioned Examples 1 and 2 and Comparative Examples 1 and 2
As is clear from the above results, the microbial preservative according to the present invention can easily store microorganisms at room temperature for a long period of time while maintaining their activity.

[0040]

According to the above structure, the microorganisms retained on the surface of the water-absorbent resin particles are in contact with each other because the water-absorbent resin particles swollen into gel and swollen by absorbing water. It is surrounded by water-absorbent resin particles. Therefore, the microorganisms are shielded from oxygen in the air and protected from drying. Therefore, it becomes possible to easily and in large quantities store microorganisms at room temperature for a long period of time (for example, several months) while maintaining their activity. Further, it becomes possible to easily carry a large amount of microorganisms.

Further, according to the above constitution, since the water-absorbent resin particles are provided with the blocking material for blocking the whole from oxygen, it becomes possible to more stably store the microorganisms.

Further, according to the above construction, it becomes possible to easily store a large amount of microorganisms having a size of 1 micron or more at room temperature for a long period of time while maintaining their activity.

The microbial preservative having the above-mentioned constitution is, for example,
A simple operation of preparing an aqueous dispersion liquid in which microorganisms are dispersed and mixing the water-absorbent resin particles with the aqueous dispersion liquid can be obtained at low cost without taking time.

Therefore, the microbial preservative having the above-mentioned constitution has an effect that it can be suitably used for preserving a large amount at room temperature for a long period of time while maintaining the activity of microorganisms.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tanehiko Higashi Tanehiko 4-7-22-611, Minamikochu, Suminoe-ku, Osaka-shi, Osaka (72) Inventor Nobuyuki Harada Nishioki, Okihama, Aboshi-ku, Himeji-shi, Hyogo 992-1 Japan Catalysis Himeji Laboratory (72) Inventor Koichi Sakano 1-2-2 Uchisaiwaicho, Chiyoda-ku, Tokyo Incorporated company Nippon Shokubai

Claims (3)

[Claims] 1. A microbial preservative characterized in that microorganisms are retained on the surface of a plurality of gelled water-absorbent resin particles, and the water-absorbent resin particles are in close contact with each other. 2. The microbial preservative according to claim 1, further comprising a barrier material that shields the entire water-absorbent resin particles from oxygen. 3. The microbial preservative according to claim 1, wherein the size of the microorganism is 1 micron or more.