JP2004275111A - Method for producing sporangia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a sporangium of a fungus belonging to Bacillus popilliae including a spore and a parasporal body in which the sporangium is efficiently cultured and the sporangium not containing an absorbent can readily be recovered, and to provide a production apparatus used for the production method. <P>SOLUTION: This method for producing the sporangium of the fungus belonging to Bacillus popilliae including the spore and the parasporal body comprises culturing Bacillus popilliae in a liquid culture medium containing the absorbent and a culture liquid and producing the sporangium including the spore and the parasporal body. In the production method, the absorbent is contained in a container through which the absorbent is not passed but the culture liquid is passed, and the absorbent is brought into contact with the culture liquid. The apparatus for producing the sporangium is equipped with a culturing tank containing the culture liquid and the container through which the absorbent is not passed, but the culture liquid is passed. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing a sporangium of a bacterium belonging to the genus Bacillus popier that contains a spore and a parasporal body, and an apparatus for producing a sporangium used in the production method.
[0002]
[Prior art]
Scarabaeidae larvae feed on and damage the roots of a wide range of plants, such as turf, agricultural and horticultural crops and trees. Since these larvae live in the ground, chemical pesticides sprayed from the ground are difficult to work, and their habitats are difficult to identify, so in order to obtain a sufficient insecticidal effect, a large amount of pesticides must be used in a wide range. It had to be sprayed and penetrated into the ground. Furthermore, as a result, there is a possibility that environmental pollution may be caused.
[0003]
On the other hand, bacteria belonging to Bacillus popiliae use these larvae of Scarabaeidae as parasitic hosts, and usually infect them orally in the form of sporangia, proliferate in large quantities in hemolymph, eventually cause emulsification disease, It is known to cause death. For this reason, attempts to utilize bacteria belonging to Bacillus popiliae for controlling insects of the order Scarabaeidae, for which chemical pesticides are difficult to work, have been made for a long time. This method is considered to be a promising alternative to chemical pesticides as a biological control method.
[0004]
As a method for cultivating a bacterium belonging to Bacillus popiliae, a method for producing a sporangia by growing the bacterium using a live larva of a scarab beetle, which is a parasitic host, has been conventionally known.
That is, the sporangium of the bacterium belonging to Bacillus popirie is sprayed on the breeding medium in which the larva of Scarabaeid insects are present, or the sporangium of the bacterium belonging to Bacillus popirie is orally ingested by the larva of Scarabaeid insect, Alternatively, after injection into body fluids by injection, larvae are bred for 3 to 4 weeks, Bacillus populiae is grown in the larvae, and then larvae are pierced or incised to collect bodily fluids. This is a method of obtaining sporangia increased in the larva by centrifuging or filtering the body fluid.
However, according to this method, it is necessary to breed a large number of larvae of Scarabaeidae, and as a result, it has been difficult to obtain a large number of sporangia in a short time and economically. For this reason, many studies have been conducted on a culture method using an artificial medium that does not use larvae of Scarabaeidae.
[0005]
By the way, in the conventional literature on a culture method using an artificial medium of Bacillus popirie, sporangia and spores are often used without distinction, and the term spore in the literature means only spores In many cases, it is unclear whether the term refers to a sporangium containing spores but not a parasporal body, or a sporangium containing spores and a parasporal body.
On the other hand, it has been reported that a spore sac of a bacterium belonging to Bacillus popillier, which contains a spore and a parasporal body, has an excellent control effect (see Patent Document 1). Therefore, there is a demand for a more efficient method for producing a sporangia of a bacterium belonging to Bacillus popiliae that contains a spore and a parasporal body.
[0006]
As a culture method using an artificial medium without using the larva of a beetle, there are a solid culture and a liquid culture. However, the production of sporangia by solid culture is not efficient, and the production of sporangia by liquid culture is necessary.
When a sporangia is obtained by liquid culture, there is a culture example in which activated carbon is added to a medium as an adsorbent for removing a growth inhibitor, a peroxide, etc., and a sporangium is obtained by liquid culture (for example, See Non-Patent Document 1 and Patent Documents 1 to 3.) Among these, examples of producing a sporangia containing a spore and a parasporal body are only those described in Patent Documents 1 to 3.
[0007]
All of these methods add activated carbon to the medium, but have major problems in the following points.
First, when powdered activated carbon is used, sporangia and powdered activated carbon are mixed in the culture solution.Furthermore, sporangium and powdered activated carbon are similar in particle size and weight, so centrifugation, It is difficult to separate sporangia from powdered activated carbon by ordinary means such as filtration. In such a case, it is difficult to obtain only the sporangium, and the sporangium must be collected in a form containing activated carbon. In addition, for example, a sporangia containing activated carbon produced by the method described in Patent Document 3 contains 2.3 to 23 times activated carbon with respect to the weight of the sporangia, and contains an adsorbent such as activated carbon. Spore sac is greatly increased in volume during the process of concentration adjustment, filling, etc. during drying or commercialization, and also when formulated, especially when using activated carbon, etc. There is a high risk of inhaling or inducing a dust explosion, and there is a problem that handling is troublesome. There was also a problem that the final product was colored and had poor appearance.
[0008]
On the other hand, when granular activated carbon is simply added to the culture tank, not only is a separation step between the sporangia and the activated carbon necessary, but also the adsorption efficiency is reduced due to the activated carbon settling down at the time of culture. Inhibitors cannot be removed sufficiently. In the usual culturing method, the inside of the culturing device is stirred, so that a method of dispersing the granular activated carbon by increasing the number of rotations of the stirring blades can be considered. A large amount of sporangia cannot be obtained.
[0009]
[Non-patent document 1]
WC Haynes et al., Journal of Bacteriology, (USA), June 1966, Vol. 91, No. 6, p. 2270-2274.
[Patent Document 1]
JP 2002-291467 A
[Patent Document 2]
JP-A-2002-291468
[Patent Document 3]
JP-A-2002-355030
[0010]
[Problems to be solved by the invention]
The problem to be solved by the present invention is to efficiently culture a sporangium of a bacterium belonging to Bacillus popiriae containing a spore and a parasporal body, and to easily collect a sporangium containing no adsorbent. It is an object of the present invention to provide a method for producing a sporangia capable of being produced and a production apparatus used for the method.
[0011]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above-described problems, and as a result of including the adsorbent in a container through which the adsorbent is not allowed to pass but the culture solution is allowed to pass, by bringing the adsorbent into contact with the culture solution, The inventors have found that the above-mentioned problems can be solved, and have completed the present invention.
That is, the present invention is a method for producing a sporangia containing spores and a parasporal body by culturing Bacillus popirie in a liquid medium containing an adsorbent and a culture solution, wherein the adsorbent is adsorbed. This is a method for producing a sporangia of a bacterium belonging to Bacillus popiliae, which contains a spore in contact with the culture solution and a parasporal body, wherein the spores are contained in a container through which the agent does not pass but the culture solution does.
The present invention also relates to an apparatus for producing a sporangia used in the production method, wherein the spores and the parasporal spores each include a culture tank containing a culture solution, and a container that does not allow the adsorbent to pass but allows the culture solution to pass. This is an apparatus for producing a sporangia of a bacterium belonging to Bacillus popiliae that contains a body.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
The method of the present invention for producing a sporangia of a bacterium belonging to Bacillus popillier which contains a spore and a parasporal body comprises culturing Bacillus popilier in a liquid medium containing an adsorbent and a culture solution, and then forming the spore and the parasporial. A method for producing a sporangia containing a body, wherein the adsorbent is contained in a container through which the adsorbent does not pass but a culture solution does pass, and is in contact with the culture solution. Is what you do.
[0013]
In the present invention, a sporangia is a sporangia in which a spore and a body called a parasporal body (or parasporium) are included in a sporangia sac wall, and is produced by a bacterium belonging to Bacillus populiae.
[0014]
The bacterium belonging to Bacillus popilliae used in the present invention (hereinafter, may be simply referred to as "bacillus of Bacillus popilliae") is a bacterium belonging to Bacillus popilliae. According to the Bergeys 'Manual of Determinative Bacteriology, the bacteriological properties of the Bacillus spp. Species are 1.3-5.2 μm in length, according to the Bergeys' Manual of Determinative Bacteriology. A gram-negative bacillus with a width of 0.5 to 0.8 μm, a growth temperature of 20 to 35 ° C., and a parasporal body in the sporangia.
Examples of the bacteria belonging to the genus Bacillus popilliae include Bacillus popilliae var. ), A strain of Bacillus popilliae var. Popilliae Hime (FERM P-17660), a strain of Bacillus popilliae sakura (Bacillus popilliae var. (Bacillus popilliae var. Popilli) e DIC-2001) (FERM P-18250), Bacillus popilliae-Deyutoki strain (Bacillus popilliae Dutky, include the American Type Culture Collection No. 14706) and the like.
[0015]
In recent years, the theoretical opinion that "Bacillus spp., Including past strains" should be reclassified as "Paenibacillus popillia" (Bertil Pettersson et al., 1999, Int. J. Syst. Bacteriol., Vol. 49, pp. 531-540), and the handling of both is not clear at this stage. Therefore, in the present invention, “a bacterium belonging to Bacillus spp. Species” includes “a bacterium belonging to Paenibacillus spp. Species”.
[0016]
The adsorbent used in the present invention is used for the purpose of removing substances that inhibit the growth of bacteria belonging to Bacillus populiae. Examples of the adsorbent include activated carbon and a synthetic adsorbent. It is important that any of these is a porous material that adsorbs a fine substance and that can efficiently adsorb an inhibitor in an aqueous solution due to a pore structure developed to the inside of the particle.
[0017]
The shape of the activated carbon used in the present invention may be in the form of powder, granules, sheets, or the like, and is not particularly limited. However, granular activated carbon is particularly preferred because particles are not scattered and are easy to handle.
Further, the synthetic adsorbent used in the present invention means a porous polymer that adsorbs fine substances, and is, for example, a crosslinkable porous polymer formed into particles. Specific examples include SEPABEADS SP825, SEPABEADS SP850, DIAION HP20, and DIAION HP21 manufactured by Mitsubishi Chemical Corporation.
[0018]
Since these adsorbents need to be held in a container through which the adsorbent does not pass but through which the culture solution passes (hereinafter, referred to as an “adsorbent-containing container”), the average particle size is 0.3 mm to 10 mm. And more preferably 2.0 mm to 6.0 mm.
If the average particle size is smaller than 0.3 mm, clogging or the like will occur in the adsorbent-containing container, and the liquid permeability of the container will be significantly impaired, and the adsorption efficiency of the inhibitor will be significantly reduced, which is not preferable. On the other hand, when the average particle size exceeds 10 mm, the liquid permeability is good, but the amount of the adsorbent used increases because the surface area required for adsorption of the inhibitor decreases, and as a result, the adsorbent-containing container becomes large. It is not efficient.
[0019]
The amount of the adsorbent used depends on its adsorptive power and is not particularly limited. However, it is necessary that the inhibitor in the culture solution can be sufficiently removed. In addition, the adsorbent must be sufficiently sterilized and not contaminated by other microorganisms, and a pre-sterilized adsorbent may be used, or the adsorbent may be contained in an adsorbent-containing container and contacted with the culture medium. It may be used after being sterilized together with the medium in a state where it has been used.
[0020]
In the method for producing a sporangia of the present invention, the culture conditions of Bacillus populiae bacteria may be performed according to conventionally known culture conditions for obtaining sporangia, and are not particularly limited. It is necessary to adjust the temperature and pH appropriately. For example, in a Bacillus popirie semada strain, the culture temperature is preferably 25 to 30 ° C, and the pH is preferably 7 to 8. The pH can be adjusted with a commonly used acid such as hydrochloric acid or sulfuric acid or an alkali such as sodium hydroxide, potassium hydroxide or ammonia.
[0021]
The culture solution used in the present invention may be a known and commonly used liquid medium used for producing a sporangia containing Bacillus populiae spores and a parasporal body.For example, a nitrogen source or a carbon source may be used. What is added to water such as distilled water is used. Examples of the nitrogen source include organic nitrogen sources such as peptone, meat extract, fish meat extract, lactalbumin hydrolyzate or yeast extract, which are usually used for culturing microorganisms. Replenish the kind. Other nitrogen sources include inorganic nitrogen sources such as ammonia, nitric acid and their salts. The concentration of the nitrogen source used in the present invention is not particularly limited as long as the sporangia can be produced. However, the concentration is preferably 5% by mass or less based on the culture solution, and more excellent bacteria. Is preferably in the range of 0.2 to 4% by mass in order to exhibit the growth promoting effect.
Although various amino acids are contained in the nitrogen source, specific amino acids can also be added in order to produce sporangia more effectively. Glutamic acid is preferred as the amino acid to be added, and proline is particularly preferred. Either one or both can be added. The medium of the present invention preferably contains glutamic acid in an amount of 0.2 to 4% by mass of the medium by adding glutamic acid, and further adds proline to the medium in an amount of 0.1 to 0. More preferably, it contains 7% by mass. Further, the medium of the present invention is preferably prepared such that the ratio of glutamic acid to all amino acids in the medium is 35 to 90% by mass, and more preferably added so that proline is 10 to 65% by mass. . However, the term "all amino acids" as used herein generally means alanine, arginine, aspartic acid, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, which are known to be contained in a nitrogen source. , Serine, threonine, histidine, tyrosine, or valine. The total amount of all amino acids, that is, the total amount of the 16 types of free amino acids described above is used as a general indicator of the amount of all free amino acids contained in peptone, yeast extract, and the like.
[0022]
Further, examples of the carbon source include carbon sources used for culturing ordinary microorganisms, and include, for example, saccharides such as trehalose and sucrose, molasses, starch decomposition products, and agricultural wastes such as cheese whey. The addition concentration of these carbon sources is not particularly limited as long as the effects of the present invention are achieved. preferable. However, in order to form a sporangia containing a spore and a parasporal body, the presence of glucose is not preferable, and the concentration of glucose contained in the medium should be 0.01% by mass or less based on the culture solution. preferable.
[0023]
The culture solution used in the present invention may optionally contain a phosphate such as potassium dihydrogen phosphate or dipotassium hydrogen phosphate or an inorganic salt such as a sodium salt thereof. The concentration of the inorganic salt is not particularly limited as long as the effect of the present invention is achieved, but is preferably 1% by mass or less based on the culture solution.
[0024]
By further adding pyruvic acid and a physiologically acceptable salt thereof to the culture solution used in the present invention in addition to the above-mentioned components, more excellent bacterial growth and sporangialization are exhibited. Physiologically acceptable salts of pyruvate include sodium pyruvate, potassium pyruvate and the like. The concentration of pyruvic acid or a physiologically acceptable salt thereof is 0.05 to 0.5% by mass based on the culture solution. 0.1 to 0.3% by mass. The added pyruvic acid and its salt may be sterilized together with the medium components, or may be sterilized separately from the medium components and added at the start of the culture.
[0025]
Examples of the culture method suitably used for the above culture conditions include batch culture, continuous culture, semi-batch culture, and fed-batch culture. The culturing time varies depending on the culturing method, culturing temperature, culturing pH, amount of inoculated cells and the like, but is 7 to 10 days in the case of batch culturing.
[0026]
In the method for producing a sporangia of the present invention, the adsorbent is stored in an adsorbent-containing container, and the culture solution is passed through the adsorbent-containing container and brought into contact with the adsorbent. There is no need to increase the rotation speed and stir to disperse in the culture tank, and it is possible to obtain a spore sac of Bacillus popiliae containing a spore and a parasporal body at a high concentration in the culture solution. .
When the sporangia obtained by the above-mentioned method is separated from the culture solution and collected, the method may be carried out by an easy method such as generally performed centrifugation and filtration.
[0027]
The sporangia obtained by the production method of the present invention contains spores and a parasporal body, has insecticidal properties against various Scarabaeidae insects, and has a controlling effect. Therefore, the sporangia is used as a controlling agent for controlling scarabidae insects, and can be applied to turf, agricultural and horticultural crops, trees, and the like.
[0028]
The Scarabaeidae insects, cupreous chafer (Anomala cuprea), Semadarakogane (Blitopertha orientalis), Japanese beetle (Popillia japonica), Usuchakogane (Phyllopertha diversa), Chairokogane (Adoretus tenuimaculatus), rufocuprea (Anomala rufocuprea), and the like. The pesticidal agent of the present invention has an excellent insecticidal property against them, all of which show an excellent pesticidal effect, and have an excellent pesticidal property against larvae of Douganebuui and Semaedaragane, which are relatively large beetles. Show the effect.
[0029]
Next, an apparatus for producing a sporangium of a bacterium belonging to Bacillus popillier which contains a spore and a parasporal body of the present invention is an apparatus for producing a sporangium used in the production method, and includes a culture solution. It is characterized by comprising a culture tank and a container (adsorbent-containing container) that does not allow the adsorbent to pass but allows the culture solution to pass.
The above-mentioned adsorbent is accommodated in an adsorbent-containing container and comes into contact with the culture solution. Examples of the structure of a portion that does not allow the adsorbent provided in the adsorbent-containing container to pass but allows the culture solution to pass include a structure having pores having a diameter smaller than the average particle diameter of the adsorbent, a network structure, and the like. By making the mesh interval or pore diameter of such a network structure smaller than the average particle size of the adsorbent, the adsorbent stays in the adsorbent-encapsulated container and can be cultured without flowing out into the culture solution. Can be.
[0030]
Preferable examples of the culturing apparatus including the above-described culture tank and the adsorbent-containing container include, for example, a device in which the adsorbent-containing container is installed inside the culture tank and an apparatus in which the adsorbent-containing container is installed outside the culture tank. In this case, part or all of the adsorbent-containing container is constituted by the structure of the portion that does not allow the adsorbent to pass but allows the culture solution to pass.
When the adsorbent-containing container is placed inside the culture tank, the adsorbent-containing container is cultured so that at least a part of the structure of the portion through which the adsorbent does not pass but the culture solution does pass is located below the liquid level of the culture solution. It is preferable to perform predetermined culturing while being fixed inside the tank. That is, by performing aeration, agitation, or the like inside the culture tank, the culture solution circulates through the adsorbent-containing container, contacts the adsorbent, removes the growth-inhibiting substances, and can efficiently obtain sporangia. If the adsorbent-containing container is not fixed as described above, the container is taken in by the bubbles generated during the culturing, and floats out of the liquid surface together with the bubbles, does not come into contact with the culture solution, and does not exhibit sufficient growth inhibitor removing ability. There are cases.
[0031]
When the adsorbent-containing container is installed outside the culture tank, it is preferable to install the adsorbent-containing container outside the culture tank and connect the adsorbent-containing container to the culture tank using a pipe or the like through which the culture solution can pass. Then, the structure of the portion of the adsorbent-containing container that connects and connects the pipes and the like to introduce and discharge the culture solution has a structure that does not allow the adsorbent to pass but allows the culture solution to pass. Furthermore, by connecting a circulation pump, the culture solution circulates through the adsorbent-containing container from the culture tank via pipes and the like, comes into contact with the adsorbent, the growth inhibitor is removed, and the sporangia is efficiently obtained. Can be.
[0032]
The sporangia obtained by the above-mentioned production method may be used as a liquid in which the sporangia is suspended, or may be used after drying as a powder as a control agent for Scarabaeidae. After drying, it may be used as a suspension of water or a buffer solution. Further, these sporangia can be used together with known and commonly used excipients, carriers, nutrients, and other various components used in pesticides, in accordance with a general method for producing microbial pesticides, in the form of powders, granules, wettable powders, emulsions, solutions, flowables. It may be formulated into a coating agent or the like. It is also possible to use the sporangia of the present invention in combination with other microbial preparations.
[0033]
As the optional component, as a solid carrier, mineral powder such as carion clay, bentonite, montmorillonite, diatomaceous earth, acid clay, talc, perlite, vermiculite, etc., inorganic salts such as ammonium sulfate, urea, ammonium chloride, ammonium nitrate, bran, Organic fine powders such as chitin, polysaccharides, rice bran, flour, etc., and adjuvants such as casein, gelatin, gum arabic, alginic acid, sugars, synthetic polymers (polyvinyl alcohol, polyacrylic acids, etc.), bentonite, etc. Examples of agents, dispersants and other components include antifreezing agents such as propylene glycol and ethylene glycol, and thickeners such as natural polysaccharides such as xanthan gum and polyacrylic acids.
[0034]
The weight content of the sporangia contained in the above-mentioned control agent is not particularly limited as long as it is within the range of exhibiting a control effect on Scarabaeidae insects, but is preferably 0.0001 to 100%.
The method of application is appropriately selected depending on the form of use such as the dosage form, crops, etc., for example, above-ground liquid application, above-ground solid application, aerial liquid application, aerial solid application, in-plant application, soil admix application, soil irrigation application, etc. Can be mentioned.
[0035]
In addition, other drugs, such as insecticides, nematicides, acaricides, herbicides, fungicides, plant growth regulators, fertilizers, or soil improvement materials (peat, humic acid materials, polyvinyl alcohol-based materials, etc.), etc. It is also possible to mix and apply, or alternately without mixing or simultaneous application.
[0036]
The application rate of the above-mentioned control agent cannot be unconditionally defined because it varies depending on the type of scarabidae insect, the type of applied plant, the dosage form, and the like. ¹⁰ -10 ^Fifteen Pcs / a, preferably 10 ¹¹ -10 ¹⁴ The number may be about one piece / a.
[0037]
【Example】
Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited to these examples.
FIG. 1 is a schematic cross-sectional view of one embodiment of a production apparatus suitably used in the method for producing a sporangia of the present invention.
As shown in FIG. 1, the culturing apparatus 10 has a schematic configuration in which two adsorbent-containing containers 3 and stirring blades 2 are installed inside a culturing tank 1.
[0038]
As the culture tank 1, a conventionally known one can be used, and it is not particularly limited. The capacity of the culture tank 1 is also not particularly limited, and may be determined depending on the amount of Bacillus populiae bacteria to be cultured.
As the stirring blade 2, a stainless steel stirring blade is preferably used, but the material is not limited.
[0039]
The adsorbent-containing container 3 is a cylindrical container made of stainless steel, and has a mesh network structure 4 at the lower part of the container in contact with the culture solution.
The material of the adsorbent-containing container 3 is arbitrary, but is preferably a material having heat resistance because it is heated during culturing and sterilization. Examples of the material include metals such as stainless steel, aluminum, copper, and titanium; Inorganic materials such as ceramics are preferred. The outer shape of the adsorbent-containing container 3 is not particularly limited, and may be any of a cylindrical shape, a rectangular parallelepiped shape, and the like. For example, it is preferably a cylindrical shape as in this embodiment. It should be noted that the adsorbent-containing container 3 may be provided alone or in plurals.
[0040]
In the present embodiment, a mesh structure 4 is adopted as a structure that does not allow the adsorbent of the adsorbent-containing container 3 to pass but allows the culture solution to pass. In addition, a structure such as a pore can be adopted, and there is no particular limitation. The mesh spacing or the diameter of the pores is not limited as long as it is smaller than the average particle size of the contained adsorbent. Such a pore or a network structure may be provided on the entire container or on a part thereof. However, it is preferable to be provided on a wider container surface because the efficiency of circulating the culture solution in the container is increased and the growth inhibitor of bacteria can be removed more efficiently.
By using the manufacturing apparatus of the present embodiment, the culture solution is circulated in the adsorbent-containing container by performing aeration, stirring, and the like inside the culture tank, and the growth inhibitor is removed by contact with the adsorbent. A sporangia can be obtained.
[0041]
Next, as a second embodiment, FIG. 2 shows a schematic cross-sectional view of an example of a production apparatus suitably used in the method for producing a sporangia of the present invention.
In the sporangia manufacturing apparatus 20 according to the present embodiment, the adsorbent-containing container 3 is installed outside the culture tank 1 and connected to the culture tank using a pipe or the like through which the culture solution can pass. The circulation pump 5 is installed in the middle of the pipe. Further, the structure of a portion of the adsorbent-containing container 3 for introducing and discharging the culture solution by connecting a pipe or the like is a mesh filter 4. Except for the above, the configuration is the same as that of the manufacturing apparatus 10 described above.
[0042]
In this embodiment, as in the case of the first embodiment, the material and outer shape of the adsorbent-containing container 3 are not limited. For example, in this embodiment, a glass cylindrical container is employed. In addition, this adsorbent-containing container 3 may be provided alone or in plurals. When a plurality are installed, they may be connected in series or in parallel. As the circulation pump 5, a commonly used pump can be used.
When the production apparatus 20 of the present embodiment is used, by using the circulation pump 5, the culture solution circulates through the adsorbent-containing container 3 from the culture tank 1 via a pipe or the like, and comes into contact with the adsorbent, whereby the growth inhibitory substance is removed. It is removed and sporangia can be obtained efficiently.
[0043]
Hereinafter, more specific examples of the present invention will be described.
(Example 1)
Spore sac was produced using the culture apparatus 10 shown in FIG.
In the present embodiment, in this culture apparatus 10, the adsorbent-containing container (3) is used in a culture tank (1) “MDL-5L” having a total volume of 5 L manufactured by Marubishi Bio-Engineering Co., Ltd. 1) Two were installed inside. The adsorbent-containing container (3) was a stainless-steel cylindrical shape having an inner diameter of 2.0 cm, and the lower portion of the container in contact with the culture solution had a mesh network structure (4) with a mesh interval of 0.5 mm. Further, a stirring blade (2) was attached to the culture tank (1).
30 g of granular activated carbon manufactured by Kanto Chemical Co., Ltd. having a particle size of 3.35 mm to 4.75 mm was added to each of the adsorbent-containing containers (3), for a total of 60 g.
[0044]
As a medium component, 3 L of distilled water, 0.75% by mass of peptone “Polypeptone S” manufactured by Nippon Pharmaceutical Co., 0.8% by mass of lactalbumin hydrate manufactured by Wako Pure Chemical Industries, 0.75% by mass of yeast manufactured by Oxoid Co., Ltd. 0.5% by mass of trehalose manufactured by Kako Pure Chemical Industries, 0.64% by mass of sodium glutamate manufactured by Wako Pure Chemical Industries, 0.5% by mass of proline manufactured by Wako Pure Chemical Industries, 0.05% by mass of sodium pyruvate manufactured by Wako Pure Chemical Industries, After adding and dissolving 0.33 g / l of an antifoaming agent “DISFORM CA-123” manufactured by NOF CORPORATION, it was put into the culture tank (1).
Next, the culture tank (1) in which the above-mentioned adsorbent-containing container (3) is installed is subjected to autoclave sterilization at 121 ° C. for 20 minutes, and then aseptically inoculated with a spore of the Bacillus popirie semadala strain and cultured. Started. During the culture, the culture temperature was controlled at 30 ° C., the pH was controlled at 7.6, the stirring speed was controlled at 100 rotations / minute, and the aeration rate was controlled at 0.8 vvm.
After culturing for 7 days, 7.0 × 10 ⁸ A sporangia of a bacterium belonging to Bacillus popiliae containing a spore and a parasporal body was obtained at a concentration of individual (spore sac) / ml. The culture solution obtained after the completion of the culture did not contain activated carbon, and sporangia was easily collected by centrifugation at 8000 rpm for 10 minutes.
[0045]
(Example 2)
In this example, cultivation was performed using an ion exchange resin "SEPABEADS SP825" manufactured by Mitsubishi Chemical Corporation as an adsorbent. The same culture apparatus 10 as in Example 1 was used, except that the mesh interval of the network structure (4) below the adsorbent-containing container (3) was 250 μm, and the amount of adsorbent added was set to each adsorbent-containing container (3). A total of 30 g was added in 15 g portions. The amount of medium, medium components, and culture conditions are the same as in Example 1.
After culturing for 7 days, 7.3 × 10 ⁸ A sporangia of a bacterium belonging to Bacillus popiliae containing a spore and a parasporal body was obtained at a concentration of individual (spore sac) / ml. The culture solution obtained after completion of the culture did not contain an ion exchange resin, and sporangia was easily collected by centrifugation at 8,000 rpm for 10 minutes.
[0046]
(Example 3)
Using the culture device 20 shown in FIG. 2, sporangia was produced.
In the present embodiment, in this culture apparatus 20, one adsorbent-containing container (3) is provided outside the culture tank using a 5 L total volume culture tank (1) “MDL-5L” manufactured by Maruzen Bioengineering Co., Ltd. Attached. The adsorbent-containing container (3) was a glass cylinder having an inner diameter of 4 cm, and the entrance and exit of the container were provided with a mesh filter (4) having a mesh interval of 0.5 mm, and connected to the culture tank (1) by piping. Further, a circulation pump (5) was installed in the middle of the pipe. Further, a stirring blade (2) was attached to the culture tank (1).
In the adsorbent-containing container (3), 60 g of granular activated carbon was added in the same manner as in Example 1. The amount of the medium and the components of the medium are the same as in Example 1.
[0047]
After autoclaving the culture tank (1) and the adsorbent-containing container (3) containing activated carbon at 121 ° C. for 20 minutes, the spores of Bacillus popirie semadala strain are aseptically inoculated and culture is started. did. During the culture, the pump (5) was operated to circulate the culture solution, and was brought into contact with the activated carbon in the adsorbent-containing container (3). The culture temperature was 30 ° C., the pH was 7.6, the stirring speed was 100 rpm, and the aeration rate was 0.8 vvm.
After culturing for 7 days, 6.9 × 10 ⁸ A sporangia of a bacterium belonging to Bacillus popiliae containing a spore and a parasporal body was obtained at a concentration of individual (spore sac) / ml. The recovered culture solution did not contain activated carbon, and sporangia was easily recovered by centrifugation at 8,000 rpm for 10 minutes.
[0048]
(Comparative Example 1)
In this comparative example, powdered activated carbon was used as an adsorbent, and cultivation was performed without using an adsorbent-containing container.
9 g of powdered activated carbon manufactured by Kanto Chemical Co., Ltd. having an average particle diameter of 20 μm was added to the liquid medium, and Bacillus popiriae was cultured under the same amount of medium, medium components and culture conditions as in Example 1.
After culturing for 7 days, 7.1 × 10 ⁸ A sporangia of a bacterium belonging to Bacillus popiliae containing a spore and a parasporal body was obtained at a concentration of individual (spore sac) / ml. The recovered culture solution contained activated carbon, and sporangia and activated carbon could not be separated by centrifugation.
[0049]
(Comparative Example 2)
In this comparative example, granular activated carbon was used as an adsorbent, and culturing was performed without using an adsorbent-containing container.
60 g of granular activated carbon manufactured by Kanto Chemical Co., Ltd. having a particle size of 3.35 mm to 4.75 mm was added to the liquid medium, and Bacillus popiriae was cultured under the same amount of medium, medium components, and culture conditions as in Example 1. . However, the rotation speed of the stirring blade was set to 500 rotations / minute so that the granular activated carbon could be sufficiently mixed.
After culturing for 7 days, 1.3 × 10 ⁸ A sporangia of a bacterium belonging to Bacillus popiliae containing a spore and a parasporal body was obtained at a concentration of individual (spore sac) / ml. In the collected culture solution, activated carbon and sporangia could be separated by filtration, but the total number of spores obtained was small.
[0050]
【The invention's effect】
INDUSTRIAL APPLICABILITY According to the present invention, a sporangium capable of efficiently culturing a sporangium of a bacterium belonging to Bacillus popillier encapsulating a spore and a parasporal body and easily recovering the sporangia from an adsorbent-free culture solution And a manufacturing apparatus used in the manufacturing method. In addition, it is possible to provide a control agent for Scarabaeidae insects using the sporangia as an active ingredient, and a method for controlling Scarabaeidae insects using the sporangia.
[Brief description of the drawings]
FIG. 1 is an example in which a container containing an adsorbent is installed in a culture tank.
FIG. 2 is an example in which a container containing an adsorbent is installed outside a culture tank.
[Explanation of symbols]
10,20 Culture device
1 Culture tank
2 stirring blade
3 Containment container for adsorbent
4 Network structure
5 Circulation pump

Claims (5)

A method for producing a sporangia containing a spore and a parasporal body by culturing Bacillus populiae in a liquid medium containing an adsorbent and a culture solution, wherein the adsorbent does not allow the adsorbent to pass therethrough but is cultured. A method for producing a sporangia of a bacterium belonging to the genus Bacillus popirie, which contains a spore and a parasporal body, wherein the spore is contained in a container through which the liquid passes and is in contact with the culture solution. The Bacillus. Containing the spores and the parasporal body according to claim 1, wherein the adsorbent has an average particle diameter of 0.3 mm to 10 mm and is at least one selected from the group consisting of activated carbon and a synthetic adsorbent. A method for producing a sporangia of a bacterium belonging to Popiriae. An apparatus for producing a sporangia used in the production method according to claim 1 or 2, comprising a culture tank containing a culture solution, and a container through which the adsorbent does not pass but the culture solution passes. For producing a sporangium of a bacterium belonging to the genus Bacillus popillier, which contains a spore and a parasporal body. 4. A sporangium of a bacterium belonging to Bacillus popiliae that contains the spore and the parasporal body according to claim 3, wherein a container that does not allow the adsorbent to pass therethrough but allows the culture solution to pass therethrough is provided inside the culture tank. Manufacturing equipment. A container that does not allow the adsorbent to pass therethrough but allows the culture solution to pass therethrough is installed outside the culture tank and is connected to the culture tank, and the culture solution does not allow the adsorbent to pass through the culture tank but the culture solution An apparatus for producing a spore sac of Bacillus popiliae, which contains the spores and the parasporal body according to claim 3, wherein the spores circulate in a container through which the spores pass.

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