JPH0439993B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a lichen cell mass, particularly to a lichen cell mass suitable for propagating lichen cells and substantially free from contamination with various bacteria, and a method for collecting and propagating the same. Lichens are symbionts made up of certain fungi and algae, and are a group of plants that occupy a botanically unique position. When you observe a lichen under a microscope, you can see that its internal structure consists of a cortical layer (the outermost tissue of the lichen that protects it, made up of fungal systems that aggregate and fuse with each other), an algal layer (lichen A tissue in which hyphae surround and hold the algae that make up the body), a pulp layer (a tissue in which hyphae are loosely intertwined and form the basis of the lichen body), a pseudoroot (a tissue that protrudes from the cortex at the lower edge, and forms the basis of the lichen body) It can be seen that the cells are differentiated into a tissue that fixes the cells to the substrate. (However, in some cases, false roots do not arise from the lower cortex.) The so-called lichen components, which are the metabolic products of such lichen plants, are completely different from those of many higher and lower plants, and are chemically (Asahina and Shibata, "Chemistry of Lichen Components," Kawade Shobo (1948)). Regarding the physiological significance of lichen components, it is thought that because lichens grow slowly, they serve as a defense against attack by microorganisms and from being eaten by small animals, and because they grow in the sun, they serve as a defense against ultraviolet rays. . Therefore, lichen components have been used for purposes derived from these functions since ancient times. for example,
These include dyes, antibiotics, and fragrances. However, in addition to slow growth, lichen plants are subject to constraints from the natural environment such as season, climate, temperature, and latitude, as well as from the human environment such as sulfur dioxide concentration and smoke concentration, making it extremely difficult to cultivate them naturally. ,
Not successful. In addition, many lichens have completely different components even though they have similar external shapes, so skill is required to select materials, which makes it difficult to collect them in large quantities from nature. In view of the remarkable development of biotechnology in recent years, the present inventors have undertaken research on an efficient artificial propagation method for lichen plants, with the ultimate aim of effectively utilizing lichen components. To do this, we first thought it necessary to isolate lichen cells and lichen algae cells in a vertical frame, and we applied methods conventionally used for isolation of higher plant cells and pure isolation of microorganisms. We attempted to isolate pure such cells. However, pure separation of such cells has been extremely difficult using these conventional methods. For example, in the method of removing bacteria attached to lichen tissue using a cleaning disinfectant, the lichen tissue is water-absorbing, so it easily absorbs and retains the disinfectant, and both the bacterial cells and algae cells are Because they are similar to bacterial cells, if you try to completely kill the bacterial cells, the lichen cells will also die. Furthermore, even if one tries to extract only lichen cells by growing lichen tissue on a suitable medium while sterilizing it, it is practically impossible because the growth of lichen cells is significantly slower than that of bacterial cells. If the lichen is a type that produces seedlings, there should be no contamination by bacteria if the spores are extracted from it. However, to achieve this,
Extremely precise technology is required. Furthermore, the growth of spores cannot necessarily be carried out smoothly. In addition, not all lichens produce seed organs, and the period of their formation is limited, so the above method cannot be universally applied. As a result of various researches, the present inventors have found that by micronizing lichen tissue into a mass of multiple lichen cells, and passing this state through an appropriate filter, the lichen cell mass can be separated from undesirable bacterial cells. Through separation, we succeeded in obtaining a pure lichen cell mass that was substantially free from contamination with contaminants.
Advantageously, such cell clusters can grow and proliferate significantly faster than single bacterial or algal cells. In the past, attempts have been made to culture lichen tissue, but it has been difficult to collect tissue of an appropriate size,
It is common to culture this tissue as it is, and such tissues are not micronized before culturing. This is because such miniaturization would kill the lichen cells, which was considered undesirable in view of the purpose of propagation. One of the reasons why the present inventors succeeded in purifying the lichen cell mass is that they dared to adopt such a micronization method that goes against common sense. On the other hand, in the cultivation of lichen plant cells, except for the cultivation of plant tissue as mentioned above, it is common to use bacterial cells or algae cells as single cells, and it is not done to use them as cell clusters. I wasn't there. Therefore, the fact that lichen cell clusters grow significantly faster than single cells has never been confirmed, and can be said to be a finding confirmed for the first time by the present invention. The present invention is significantly superior to the above-mentioned method of collecting spores from offspring organs in that it is easy to operate, grows quickly, and can be universally applied regardless of the type. The gist of the present invention is as follows: (1) A lichen cell body contaminated with contaminants is separated into at least two lichen cells with a maximum diameter of 10 mm.
The lichen cell mass is micronized so that at least one of the following lichen cell masses remains, and the lichen cell mass remains in the aqueous dispersion of this micronized product, but lichen cells and miscellaneous bacterial cells with smaller diameters pass through. consisting of two or more lichen cells and having a maximum diameter not exceeding 10 mm, characterized in that the lichen cell mass remaining on the filter is collected through at least one filter such as
Lichen cell mass with virtually no contamination by bacteria. The present invention is a universal invention that can be applied to various lichen plants, as is clear from the Examples described below. That is, the present invention is generally applicable to each of the following lichen families: Teloscystetheceae, Centipedeaceae, Myriataceae, Myriataceae, Antigulaceae, Amenaceae, Candleliaceae, and Myriataceae. Family, Atricotaceae, Prunustaceae, Prunustaceae, Prunustaceae, Prunustaceae,
Helitoridae, Saragosaceae, Asterotiliaceae, Asterotiliaceae, Aceridae, Amaranthidae, Acarinaceae, Agaricaceae, Asteroidaceae, Alocatidae, Ilanaceae, Lichinaceae, Asteraceae, Alocatidae, Alocatidae, Ananigaceae, Asteraceae, Alocatidae , Coral moss, Pingolaceae, Pygmyaceae, Pingolaceae, Pygmyaceae, Pygmyaceae, Pyrolaceae, Pyrolaceae, Pylophyllaceae, Matsutakeceae, etc. Next, using as an example Akase saru Ogase, which belongs to the order Lecanora and family Salogamaceae, a specific operating procedure will be explained regarding a method for separating and culturing Akase saru Ogase cells from undesirable bacterial cells. Pure lichen cells can be obtained from other lichen plants by substantially similar operations. First, cut the natural lichen of Akasarina lichen into appropriate sizes, wash the small pieces thoroughly with running water, and then
A collection of small cell clusters is formed by physical or chemical miniaturization methods. To physically perform the micronization, conventional means of mechanically dividing the organized structure into smaller structures may be employed. For example, as a crushing means, a mortar, pestle, or homogenizer may be used. Further, as a cutting means, a scalpel, scissors, razor, microtome, etc. may be used. These devices may be used in appropriate combinations. For scientific purposes, reagents that block intercellular connections may be used, and an oxygen agent containing cellulase is a typical example of a reagent suitable for this purpose. Examples of enzyme agents include Driselase (manufactured by Kyowa Hakko Co., Ltd.) and Cellulase Onozuka (manufactured by Kinki Yakult Co., Ltd.).
Examples include. The obtained cell mass is a mixture of single-celled bacteria, A. chinensis cells, and a. It is necessary to create a system consisting only of cell clusters with a maximum diameter of 10 mm or less. Preferably, a system consisting only of cell clusters of 10 cells or more and a maximum diameter of 1 mm or less is preferable, judging from the efficient elimination of contaminants and the certainty of subsequent survival and growth. The maximum diameter here refers to the longest dimension of the cell cluster. Methods for obtaining cell clusters of a predetermined size by eliminating single cells include a method of selecting only cell clusters of a predetermined size using a manipulator, a method of using a cell sorter, and a method of using centrifugation. However, the method of passing through one or more filters having a specific size is advantageous and preferred from an industrial perspective because a large amount of cell aggregates can be obtained quickly. The material of the filter is not particularly limited as long as it does not affect lichen cells, but stainless steel and nylon are preferable, for example. The obtained population of small cell clusters is first filtered through a mesh having a size of 1000 μm or less, and then filtered through a mesh of 10 μm or larger, and the cell clusters remaining on the mesh are also washed several times with sterile water. Next, place the cell mass in an appropriate medium,
Culture under constant temperature conditions of 0 to 40°C. By such culturing, the cell culture of A. chinensis grows to the point where it can be observed with the naked eye in about one month. Cultivation may be carried out by any method such as shaking culture, static culture, or stirring culture. As a culture medium, a natural or synthetic, organic or inorganic medium is used. For example, it may be based on various known inorganic or organic synthetic media, to which organic substances such as nutrients, carbon sources, and various naturally extracted substances are added. Examples of the above inorganic synthetic medium include White medium, Hildebrand medium, Linsmeyer-Skoog medium, Murashige-Skoog medium, Lily-Barnett medium, Hamada No. 118 medium, etc., and organic synthetic medium such as malt/yeast extract medium, soil extract. Examples include culture medium. Other improved versions of these media can also be used. As the nutritional source, vitamins such as thiamine hydrochloride, pyridoxine hydrochloride, and nicotinic acid, amino acids such as glycine and asparagine, and hexahydric alcohols such as inosyte and sorbitol may be used. The above carbon sources include carbohydrates (sucrose, glucose,
(maltose, etc.), organic acids (acetic acid, etc.), and alcohols (methanol, glycerin, etc.) can be used. Examples of the above-mentioned various natural extracts include casein hydrolyzate, coconut milk, soil extract, etc., which may be used alone or in appropriate combinations. Furthermore, by adding an antibiotic to the culture medium, a desired cell mass of either lichen cells or lichen algae cells can be obtained. As for antibiotics, if you want to obtain only lichen cells, use anti-algal agents such as kanamycin (manufactured by Meiji Seika Co., Ltd.) or Sorcillin (manufactured by Takeda Pharmaceutical Company Limited), and if you want to obtain only lichen cells, use anti-algal agents. For example, a benzimidazole-based substance such as Courtside (manufactured by Takeda Pharmaceutical Co., Ltd.) may be used. In addition, the pure bacterial cell mass of Lecanorae, Acerophataceae, Acanthophyllaceae (Example 2 described later) obtained in this way was given accession number to the Institute of Microbial Technology (FEI), Agency of Industrial Science and Technology. As No. 6932,
It has been deposited with the American Type Culture Collection (ATCC), Maryland, USA, under deposit number ATCC20668. Also,
The corresponding algae cell mass (Example 2, described later) was not accepted by the Institute of Fine Technology as it was not subject to consignment, but it has been deposited with the ATCC under deposit number ATCC20667. EXAMPLES The present invention will be explained in more detail with reference to Examples below. Example 1 A large amount of Lecanora, Psyllidaceae (no reproductive organs) collected in Kyoto City, Kyoto Prefecture.
It was cut into small pieces of about 0.5 cm ² , thoroughly washed with water, and then placed in 10 ml of an oxygen solution having the following composition in a clean bench. The oxygen solution was Driselase (manufactured by Kyowa Hakko) 4% (w/v), 0.7M mannitol,
20mM morpholineethanesulfonic acid and 5mM magnesium chloride were dissolved in distilled water, centrifuged at 2500 rpm, and the supernatant was further sterile filtered through a 0.22μ Millipore filter. This at 25℃
The mixture was shaken at 70c/s with a width of 5cm for 6 hours. After the reaction, the oxygen solution is filtered through a 150μ filter, and the filtrate is further filtered.
It was filtered through a 62μ filter, and the lichen cell mass remaining on the filter was washed several times with sterile water. 100 cells were appropriately selected from the cell mass on the filter and placed one by one in the test tube medium described below. As the medium, malt extract (Difco) 2% (w/v), yeast extract (Difco) 0.2% (w/v), and agar 2% (w/v) were added, and 5 ml of each medium was sterilized as usual and tested. The solution was dispensed into tubes and used. The cell mass of P. moss placed in such a medium was cultured in the dark at a culture temperature of 20°C. At about the first month, the lichen cell culture had grown to the point where it could be clearly observed with the naked eye. The non-contamination rate (number of non-contaminated samples/number of total samples) was 41%, and the survival rate (number of viable samples/number of non-contaminated samples) was 100%. The surviving samples were of three types: one consisting only of bacterial cells, one consisting only of algae cells, and one consisting of both cells. Example 2 A Lecanora, Aphthalidae, Aphragidae (without reproductive organs) collected in Kyoto City, Kyoto Prefecture was cut into small pieces of approximately 1 cm in length, which were thoroughly washed with water and then ground in a mortar in a clean bench. did. The pulverized liquid was filtered through a 500μ filter, the filtrate was filtered through a 150μ filter, the cell mass remaining on the filter was washed several times with sterile water, and then placed in a test tube medium in the same manner as in Example 1. The same medium as in Example 1 was used. Cultivation was also carried out in the same manner as in Example 1. It grew to the point where it could be observed with the naked eye around the first month. The non-contamination rate was 30% and the survival rate was 100%. As in Example 1, three types of surviving samples were observed. Example 3 A Moegitorihadago moss (no reproductive organ) collected in Hirakata City, Osaka Prefecture and belonging to the order Lecanora, family Aconitidae, was cut into small pieces of approximately 0.5 cm ² in size.
The cells were placed in a test tube culture medium in the same manner as described above. The medium is the medium used in Example 1 plus 0.1% coat side.
(w/v) was used. Cultivation was carried out in the same manner as in Example 1. It grew to the point where it could be observed with the naked eye around the first month. Non-contamination rate is 69%, survival rate is
It was 97%. Each surviving sample consisted solely of algae cells. Example 4 Kikubagomodoki, a member of the order Lecanora, family Prunusidae, collected in Hirakata City, Osaka Prefecture (no reproductive organs)
The mixture was cut into small pieces approximately 0.5 cm ² in width, thoroughly washed with water, and then finely cut using a sterile scalpel in a clean bench. After dispersing the crushed material in sterile water,
The mixture was filtered using a filter in the same manner as in Example 2, and placed in a test tube culture medium. The medium used was the same as in Example 1 to which 50 ppm of salicillin and 50 ppm of kanamycin were added. Cultivation was carried out in the same manner as in Example 1. It grew to the point where it could be observed with the naked eye around the first month. The non-contamination rate was 23% and the survival rate was 77%. Each surviving sample consisted solely of algae cells. Example 5 50 mg of the cell culture obtained in the same manner as in Example 2 was ground in a mortar in a clean bench, and placed in a test tube medium in the same manner as in Example 2. The same medium as in Example 1 was used. Cultivation was carried out in the same manner as in Example 1. It grew to the point where it could be observed with the naked eye around the first month. The survival rate was 100%. As in Example 2, three types of surviving samples were observed. Examples 6 to 29 In the same manner as in Example 1, small pieces obtained from the following lichen plants were treated to produce pure bacterial cell masses, algal cell masses, and cells consisting of bacterial cells and algal cells. Got a lump.

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Claims (1)

[Scope of Claims] 1. A lichen cell body contaminated with germs is micronized so that at least one lichen cell mass consisting of at least two lichen cells and having a maximum diameter of 10 mm or less remains, This aqueous dispersion of the fine particles is passed through at least one filter that allows the lichen cell mass to remain, but lichen cells with a smaller diameter and miscellaneous bacteria cells to pass through. A lichen cell mass consisting of two or more lichen cells, having a maximum diameter not exceeding 10 mm, and substantially free from contamination by bacteria, characterized by collecting a plant cell mass. 2. The lichen cell mass according to item 1, wherein the micronization is performed by a physical method and/or a chemical method.