JPH10243781A - Long-term storage of microorganisms - Google Patents

Abstract

(57) [Summary] [Object] To provide a method for preserving microorganisms for a long time by freeze-drying method, which facilitates resuscitation of the preserved microorganisms. A cellulose-based biopolymer is added to a medium for culturing microorganisms.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preserving microorganisms for a long time, and more particularly to a method for preserving microorganisms for a long time by freeze-drying. In the present invention, what is referred to as microorganisms is aerobic, anaerobic, most bacteria, viruses, yeasts, molds, and other typical microorganisms, as well as spirulina, diatoms, green algae, brown algae, and red algae. It includes microalgae such as algae and cyanobacteria and sperm of humans, horses, cows, rats and the like.

[0002]

2. Description of the Related Art In recent years, the importance of the preservation of biological resources such as microorganisms has been deepened with the situation where the Convention on Biological Diversity has been concluded. For long-term storage of microorganisms, a freeze-drying storage method has conventionally been used. In this preservation method, a microorganism to be preserved is collected or cultured and multiplied is placed in a preservation ampule tube and rapidly frozen, and the frozen water generated by the freezing is reduced under aseptic pressure. This is a method in which microorganisms are dried by evaporating them under, and then stored in a sealed state. However, in such a method, the external water surrounding the preserved organism is reduced by freeze-drying, and as a result, the water in the organism rapidly oozes out of the cell through the cell membrane due to osmotic pressure, and the organism to be preserved is reduced. The phenomenon of death occurs. If such a phenomenon occurs due to freeze-drying, the preservation of microorganisms cannot be expected. Therefore, in order to alleviate the movement of water in an organism due to the osmotic pressure, skim milk, sucrose, etc. are generally added to the medium in which the microorganisms are cultured. , Preserved organisms such as bouillon, glucose, sodium glutamate, cysteine, and gelatin are added, and the preserved organisms are not killed during lyophilization or when reviving preserved organisms by adding water to preservation ampules. I have to. However, even in this case, the water in the environment of the preserved organism suddenly decreases, and the moisture in the cells may go out or the cells may be destroyed, which may make it difficult to revive the preserved organism. there were.

[0003]

Therefore, it has been desired to develop a method for preserving microorganisms or the like by freeze-drying for a long period of time without causing the above-mentioned inconvenience in reviving preserved organisms.

[0004]

The present inventor has conducted various studies in view of the above situation, and as a result, in the above-mentioned conventional method for long-term storage of microorganisms by freeze-drying in which skim milk or sucrose is added to a medium, If a cellulosic biopolymer is added to the medium, the cellulosic biopolymer is frozen in a water-retaining state, and since it is a very hydrophilic solid substance, a mild drying environment is created. From this, it has been found that resuscitation of microorganisms is facilitated. The present invention has been made based on such findings, and aims to provide a method for long-term preservation of microorganisms by freeze-drying, which provides a method that facilitates resuscitation of the preserved microorganisms. This is achieved by the present invention having the following configuration. (1) Microorganisms to be preserved are centrifuged, the culture solution is discarded, and the microorganism is suspended in a dispersion medium to which a dispersion medium such as skim milk, sucrose, or bouillon is added. In a preservation ampoule and rapidly frozen.Frozen water generated by the freezing is evaporated under reduced pressure when aseptic, thereby drying the microorganisms together with the culture medium. A long-term preservation method for microorganisms in which water is added to the dispersion medium containing the microorganisms at the time of resuscitation of the stored microorganisms to resuscitate the microorganisms, wherein a cellulosic biopolymer is added to the dispersion medium. Long-term preservation method for microorganisms. (2) The microorganism according to (1), wherein the dispersion medium is one or more of skim milk, sucrose, bouillon, horse serum, glucose, sodium glutamate, cysteine, and gelatin. Long-term storage method. (3) Cellulose-based biopolymer is contained in a medium at 0.05%.
The method for long-term storage of microorganisms according to the above (1) or (2), wherein the microorganism is added in an amount of up to 5% by weight.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganisms to be preserved in the present invention include those generally referred to as microorganisms, particularly microorganisms cultivated in a medium, and include, for example, aerobic and anaerobic. In addition to those treated as typical microorganisms, such as most bacteria, viruses, yeasts, and molds, spirulina, diatoms, green algae, brown algae, red algae, and cyanobacteria, microalgae, humans, horses, cattle, It contains spermatozoa such as rats. As a medium for storing microorganisms preserved by the present invention, a medium generally used for culturing and preserving microorganisms can be used.For example, a medium to which a dispersion medium such as skim milk, sucrose, and bouillon is added can be used. This microorganism storage medium can be used as a dispersion and osmotic pressure adjusting material in sucrose, bouillon, skim milk, horse serum,
One or more of glucose, sodium glutamate, cysteine, and gelatin are added. These additives are usually contained in the microorganism storage medium in an amount of 0.01 to 5.0.
% By weight. Culture of the microorganisms can be performed under well-known conditions suitable for the growth of each microorganism.
Freeze-drying performed for long-term storage of microorganisms can be performed according to a conventional freeze-drying method.Specifically, for example, after centrifugation of a culture solution, sterilization is performed aseptically in the centrifuge. Treated main solution (for example, 5% glucose, 5%
% Skim milk, 1% biopolymer), and mixed and suspended so that the number of bacteria becomes 10 ⁸ to 10 ¹⁰ cells / ml. It can be immersed in a liquid to freeze the water in the ampoule, aseptically put into a freeze dryer vacuum system, and dried.

The microorganisms placed in the preservative ampoule and freeze-dried as described above are
The glass of the ampoule is kept sealed by cutting the glass of the ampule with a burner while sucking the vacuum in a vacuum system, such as a refrigerator controlled at 2-5 ° C or a low-temperature room controlled at 10 ° C or less. Stored under conditions for long periods. In order to revive the preserved microorganisms using water, it is possible to use a conventionally adopted method, for example, open an ampoule containing the preserved microorganisms in a sterile box with a file or a glass cutter, and open it. The microorganism can be revived by adding 5 ml of a culture medium or physiological saline suitable for the microorganism and shaking the culture at an appropriate temperature. In this resuscitation, the cellulosic biopolymer is added to the medium, so that the cells of the preserved microorganisms are prevented from abruptly moving the given water into the cells by osmotic pressure, and this biopolymer is cushioned. As a result, the advantage that resuscitation of preserved microorganisms is facilitated is obtained. When certain microorganisms such as acetic acid bacteria are cultured under special conditions, extremely fine fibrous polymer materials are biosynthesized using sugar as a nutrient source. The cellulose-based biopolymer used in the present invention refers to such a polymer material produced in the body of a microorganism, and this biopolymer is an ultrafine cellulose having a high purity.
The crystallinity is high, the crystal structure is unique,
It has water absorption and water retention properties, and has properties such as higher polymerization degree than natural cellulose derived from plants, is harmless to the growth and resuscitation of microorganisms, and is sterilized and sterilized It is chemically stable without causing hydrolysis or the like even under high pressure and temperature conditions under which the reaction is carried out. Cellulose-based biopolymers are, for example, HiroshiToyosakiet
al, BiosciBiotecBiochem, 5
9 (8), 1498-1502.1995, Fumih
iroYosinaga, et al, Biosci
Biotec Biochem 61 (2), 219-2
24. A product manufactured by the method described in 1997 may be used, or a commercially available product such as biocellulose manufactured by Biopolymer Research Co., Ltd., or biocellulose manufactured by Ajinomoto Co., Ltd. may be used. Good.
This biopolymer is preferably added to the medium in an amount of 0.05 to 5% by weight.

[0007]

Next, the present invention will be described with reference to examples, but the present invention is of course not limited to such examples. Example 1: Escherichia coli K-1
2 was inoculated into a sterilized medium in 20 ml of a 100 ml Erlenmeyer flask containing 10 g of Peptone, 5 g of NaCl, and 1000 ml of distilled water.
Shaking culture was performed at 280 rpm for a day. Separately, Pepto
ne 1% by weight, Meat extract 1% by weight,
0.5% by weight of NaCl and 1.0% by weight of biopolymer were mixed with distilled water to prepare a sterilized storage medium having 1.0% by weight of cellulosic biopolymer added to the medium. Subsequently, the cells were cultured in the medium thus prepared for 24 hours, and the cultured medium was centrifuged aseptically, and the sedimented cells were separately prepared, and sterilized 3% by weight of the skim milk, biopolymer 1 .
The mixture was dispersed in a 0% by weight mixed solution, and adjusted so as to have the same volume and the same weight of 5 g each in an ampoule tube for cryopreservation.
Thereafter, the ampoule was immersed and frozen in a methanol dry ice solution kept at -40 ° C, and the ampoule was burned and sealed with a burner under reduced pressure for a whole day and night using Freez DryerVO-15 manufactured by Itec. Ten lyophilized ampules were prepared and stored in a low-temperature room at 5 ° C. for 45 days. The stored ampules were opened aseptically, and 5 ml of distilled water was dropped into each ampule and mixed well. When the viable cell concentration of each of the stored ampules was measured, four of them were 5 × 10 ⁸ , three were 4 × 10 ⁸ ,
The number of books was 6 × 10 ⁸ , the number of 3 × 1 was 10 ⁸ , and the average viable cell count was 4.7 × 10 ⁸ cells / ml. For comparison, Escherichia coli was used in the same manner as above, except that no biopolymer was added to the medium.
After lyophilizing the liK-12 strain and storing it for the same day,
When the cells were revived in the same manner, the number of viable cells in the ten stored media was 5.7 × 10 cells / ml on average.

Example 2: Pseudomonas ae
rugimosa in 1% by weight of Peptone, Mea
The cells were cultured overnight at 38 ° C. on a shaking incubator in a 20 ml Erlenmeyer flask containing 1% by weight of extract and 0.5% by weight of NaCl in distilled water. This was centrifuged aseptically, and 30 ml of a medium in which 0.7% by weight of a cellulosic biopolymer was added to the same medium as the precipitated bacteria was prepared. Then, the medium thus prepared was mixed well, and 3 ml was dispensed evenly into a lyophilization ampoule tube by a conventional method, and freeze-dried all day and night in the same manner as in Example 1. The cell concentration before the freeze-drying was 8 × 10 ⁹ cells / ml. The lyophilized ampule was sealed under reduced pressure aseptic and stored in a refrigerator at 5 ° C. for 30 days. After storage for 30 days, 3 ml of distilled water was added to each ampoule, and the concentration of viable cells in each ampoule was measured by a conventional method. The average viable cell count was 5.3 × 10 ^8.
Pcs / ml. For comparison, Ps was prepared in the same manner as described above, except that no biopolymer was added to the medium.
Eudomonas aerugimosa was lyophilized, stored for the same day, and then revived in the same manner. The viable cell count was 3.3 × 10 ² cells / ml.

[0009]

As is apparent from the above description, according to the present invention, in the method for long-term preservation of microorganisms by freeze-drying method, when the freeze-dried and preserved microorganisms are revived using water. In addition, the biopolymer serves as a medium to regulate the osmotic pressure of the cells, thereby facilitating resuscitation.

Claims (3)

[Claims] 1. A microorganism to be preserved is centrifuged, the culture solution is discarded, and the microorganism is suspended in a dispersion medium to which a dispersion medium such as skim milk, sucrose, bouillon or the like is added. Was dispensed into a storage ampoule tube, rapidly frozen, and the frozen water produced by the freezing was evaporated under reduced pressure under aseptic conditions to dry the microorganisms together with the culture medium. In a method for long-term preservation of microorganisms in which water is added to a dispersion medium containing the microorganisms at the time of resuscitation of the preserved microorganisms, the cellulose-based biopolymer is added to the dispersion medium. A method for long-term storage of microorganisms, characterized in that: 2. The microorganism according to claim 1, wherein the dispersion medium is one or more of skim milk, sucrose, bouillon, horse serum, glucose, sodium glutamate, cysteine, and gelatin. Long-term storage method. 3. The method for long-term storage of microorganisms according to claim 1, wherein 0.05 to 5% by weight of a cellulose biopolymer is added to the medium.