JP4831898B2 - Method for producing a preparation containing filamentous fungi as active ingredients - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
TECHNICAL FIELD The present invention relates to a method for producing a preparation containing as active ingredients spores of a solid-cultured filamentous fungus culture, and more particularly to a method for producing a preparation which is a microbial pesticide or a soil modifier.
[0002]
[Prior art]
For the control of pests in the agricultural field, chemically synthesized pesticides are mainly used for reasons such as immediate effect, stability of effects, superiority in cost effectiveness, and ease of use.
[0003]
On the other hand, however, problems such as a decrease in efficacy due to the emergence of drug-resistant bacteria, adverse effects on useful organisms, and the environment of drugs or residues in crops have become apparent.
[0004]
In recent years, the idea of comprehensive control (a method that uses a variety of control systems by refining chemo-synthetic pesticide-dependent agriculture) has been adopted as a method to compensate for these problems. .
[0005]
The first reason for this is that the organisms used in biological pesticides or their active ingredients are inherently in nature, and the target pests are limited. This is because it can be expected to compensate for the drawbacks.
[0006]
In particular, in the field of plant soil diseases (diseases caused by diseased filamentous fungi growing in soil), there are many diseases that cannot be expected to have a stable effect even with chemical pesticides.
[0007]
For controlling soil fungi, filamentous fungi (fungi) that have an antagonistic ability to the fungus are usually used. Known are Dilium berens, Gliocladium catenuratam, Penicillium niglycans, Penicillium chrysosynum, Penicillium oxalicum. These filamentous fungi inhibit the growth of diseased bacteria by disrupting the host cell membrane, degrading the host cell wall by lytic enzymes such as glucanase and chitinase, and producing antibiotics.
[0008]
Usually, biopesticides utilizing the ability of filamentous fungi are commercialized in the state of conidia because spores are preferred over mycelia from the viewpoint of easy handling and durability against environmental changes.
[0009]
Therefore, for the purpose of increasing the production of conidia, various studies have been conducted on liquid culture using a liquid substance or solid medium dissolved or dispersed in water and solid culture using a solid medium. .
[0010]
In liquid culture, mycelium easily grows, and thick film spores and sprouting spores can be generated by adjusting the culture conditions. Generally, conidia spores formed at the interface between the medium and the gas phase are liquid cultures. Will hardly generate.
[0011]
Thick film spores or budding spores produced in liquid culture have a spore concentration of about 1/100 or less compared to conidial spores that are easily produced by solid culture methods. In general, production of conidia by a solid culture method is employed.
[0012]
This solid culture is a method of culturing at a suitable temperature for growth of microorganisms while maintaining a certain moisture content after adjusting the growth environment of microorganisms by adding water to solid components derived from natural products, and representative examples include: Mention of soy sauce used in the production of sake, miso and soy sauce.
[0013]
As solid media for cultivating filamentous fungi, those containing organic matter such as bran, rice bran, rice bran, wood flour, humus, peat moss, compost, mushroom waste, and pulverized products thereof alone or with other nutrients added may be used. Is called.
[0014]
Incubation is performed by inoculating the above-conditioned medium into a fermentor or a metal tray so that the layer thickness is constant and inoculating the inoculum. When the culture starts, the solid medium generates heat as the mycelium grows, and the medium is solidified by the medium mycelia, so the operation of loosening the medium to make the culture temperature uniform and improve the medium utilization rate is necessary. Do.
[0015]
After completion of mycelium growth, spore growth proceeds and the culture is generally completed in about 1 week to 1 month. Although the moisture content of the cultured product at this time varies depending on the medium composition, it is in the range of about 20 to 80% (w / w). In the solid culture after culturing, it is possible to prevent contamination with bacteria and improve storage stability. For the purpose, the water content is adjusted to 40% (w / w) or less by a method such as freeze drying or blow drying.
[0016]
The obtained culture is uniformly mixed with excipients that are substances for imparting various properties as a formulation, such as organic nutrients, micronutrients, binders, hardness modifiers, etc., and for further formulation Of water is added to form a formulation.
[0017]
Organic nutrients include bran, rice bran, rice bran, wood flour, humus, peat moss, compost, mushroom waste, or pulverized products thereof, which serve as nutrients for the desired fungus.
[0018]
Micronutrients are substances that activate the growth of microorganisms such as inorganic substances including iron, magnesium, potassium, etc., or organic substances such as crab, oil cake, blood meal, meat meal, and wood vinegar.
[0019]
The binder is a so-called formulation binder, and carboxymethyl cellulose, methyl cellulose, polyvinyl alcohol, clay, organic polymer latex, starch and the like are used.
[0020]
The hardness adjusting agent is an auxiliary agent that adjusts the strength, hardness, disintegration degree in water, etc. of the preparation itself, and the above-mentioned binders are used in the same manner.
[0021]
Of course, these excipients are appropriately selected according to the bacterial species to be used, the method of using the intended preparation, and the like.
[0022]
Mixing of these excipients and the culture becomes difficult because the water content of the culture end product is usually 40% or more.
[0023]
In addition, if a mechanically high shearing force is applied to mix and disperse in order to achieve uniform mixing, the spores will be killed because the spores are vulnerable to heat and mechanical shearing.
[0024]
Therefore, it is conceivable to dry the culture once and mix and agitate, but in this case as well, the dried product becomes blocked during the drying process and must be crushed. The force will cause the spores to die.
[0025]
In order to prevent the death of spores, it is only necessary to omit the mechanical shearing and the pulverization process that causes the temperature increase. For example, the particle diameter of the bran used as a medium is as wide as about 100 μm to 5 mm, about 200 μm. In order to prepare a mixture by uniformly mixing with a bulking agent (for example, wheat flour) having a function as a nutrient component having an average particle size and a function as an excipient, a pulverization treatment was essential.
[0026]
In addition, a culture method in which the culture medium is pulverized to about 200 μm in advance to perform solid culture is conceivable. However, since the air permeability of the culture medium is remarkably lowered, a problem occurs in solid culture of filamentous fungi.
[0027]
[Problems to be solved by the invention]
The problem to be solved by the present invention is to provide a method for producing a preparation that can be formulated without reducing the survival rate (or recovery rate) of conidia of filamentous fungi obtained by solid culture.
[0028]
[Means for Solving the Problems]
In order to solve the above problems, the culture after solid culture has the property of being easily adapted to moisture, the spores are easily dispersed in moisture and oil, and the culture and the size of the spore. The present invention has been completed by paying attention to the fact that it can be easily separated because of its remarkably large size.
[0029]
That is, the present invention provides (1) the culture after completion solid culture of filamentous fungi using a solid medium, a medium which can disperse the spores of filamentous fungi, water, and inorganic or organic salts in water Separated by adding a solution or natural fats and oils , (2) separating the culture residue and spore dispersion from the suspension obtained in the mixing step, and (3) separating. The present invention provides a method for producing a preparation comprising the steps of mixing a spore dispersion and an excipient, and (4) granulating the mixture obtained in the mixing step.
[0030]
The medium added to the culture after completion of the solid culture is a solution containing water such as distilled water, ion-exchanged water, tap water, or a solution containing an inorganic or organic salt such as a buffer solution or physiological saline. Alternatively, natural fats and oils such as soybean oil and olive oil may be used as long as spores are not killed.
[0031]
The amount of medium added depends on the type of solid medium and the type of medium, but from the viewpoint of the state of dispersion of the culture in suspension and the state of dispersion of the spores, It is preferable to make it 5 to 5000 times (in terms of weight).
[0032]
Further, a mixed solution in which the above medium is further mixed with a solution that is a nutrient component for spores may be used.
[0033]
As the solid medium, various media as described above can be mentioned. It is easy to cultivate mycelium, a large number of spores can be obtained, the uniformity of quality is excellent, and the suspension of (2) From the viewpoint of easy separation in the step of separating the residue and the spore dispersion, a bran medium is preferable.
[0034]
For bran medium, Trichoderma harzinam, Trichoderma hamatum, Trichoderma bilide, Trichoderma lignoram, Gliocladium viloclamens, Gliocladium catenuratam, Penicillium penicillium niglycans, Penicillium penicillium It can use suitably with respect to various filamentous fungi.
[0035]
For example, in the case of a bran medium, the size of the residue in the suspension thus obtained is usually 500 to 1000 μm in a dry state, but is 2000 to 5000 μm due to swelling.
[0036]
On the other hand, since the size of the spore in the suspension is 10 μm or less, there is a difference of 2 orders or more in the size between the two.
[0037]
Therefore, when separating the spore dispersion from the suspension, various separation means such as filter paper, filter cloth, sieve, and hollow paper can be applied as long as they can be separated with respect to the respective particle sizes. However, in view of separation efficiency such as separation speed and clogging, it is preferable to use a sieve having a diameter of 50 to 1000 μm.
[0038]
This diameter is preferably changed depending on the type of preparation to be produced. That is, when preparing a granule wettable powder, 50-200 micrometers is preferable, and when preparing a granule, 200-1000 micrometers is preferable, and 300-500 micrometers is especially preferable.
[0039]
This separation means may have a plurality of stages. For example, when producing a granulated wettable powder, when a two-stage separation process is used, coarse filtration is performed using a large-diameter sieve (for example, a 200-1000 μm sieve) as the first stage, and a small-diameter is used as the second stage. It is good also as a structure which carries out dense filtration using a sieve (for example, 50-200 micrometers).
[0040]
Similarly, when a two-stage separation process is used for granule production, coarse filtration is performed using a large-diameter sieve (for example, a 500-1000 μm sieve) as the first stage, and a small-diameter is used as the second stage. It is good also as a structure which carries out dense filtration using a sieve (for example, 200-500 micrometers).
[0041]
These may be appropriately selected with respect to the total amount of suspension, and in a production line where batch processing is performed in a mixing tank, a plurality of stages of the above sieves may be arranged in the circulation line. When configuring a continuous production line, it is good also as a structure which arrange | positions the said sieve in multiple steps to piping, and collect | recovers only filtrates in a filtrate collection tank.
[0042]
Spores in the culture easily disperse in water, for example, by adding water and stirring, but the retention capacity of the filtration residue is usually very large, so the spore recovery efficiency is solely the recovery rate of the filtrate in the filtration residue. Will depend on.
[0043]
Therefore, in order to increase the spore recovery rate, a method such as vacuum filtration, pressure filtration, or squeezing filtration residue may be used as a separation step, and it is also effective to use a combination of these methods. is there.
It is also effective to increase the filtration efficiency by using a surfactant or the like that does not kill spores.
[0044]
The spore dispersion thus obtained can be used as it is in the granulation step for preparation.
[0045]
The above spore dispersion is added to the raw material in which the above-mentioned various excipients are uniformly mixed by a method such as spraying, and the mixture is kneaded and granulated by extrusion granulation to obtain a preparation.
[0046]
Of course, it is also preferable to improve the efficiency of the preparation process by simultaneously kneading the raw material and spraying the spore dispersion and further granulating the material at the same time. It is preferred to use a granulator.
[0047]
The rolling granulator includes, for example, a rotating container type, a vibration type, a pan type, a drum type, a mixing machine type and the like, and the mixing granulator includes various types such as a blade type granulator and a pin granulator. Although there is a granulator, any method may be adopted.
[0048]
【Example】
Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples.
Example 1
Inoculated Trichoderma harzianum ATCC 74058 conidia cultivated in potato dextrose agar medium (Difco) at a culture temperature of 28 ° C. for 1 week into a sterilized bran medium (manufactured by Chiba Flour Milling Co., Ltd.) for 60 minutes. The cells were cultured at a moisture content of 68% and a culture temperature of 28 ° C. for 1 week.
[0049]
Next, according to the process shown in FIG. 1, 9 times by volume of tap water 2 was added to the culture 1 after the completion of the solid culture and stirred to prepare a suspension. Next, this suspension was passed through a sieve 3 having an opening of 1000 μm and subsequently a sieve 4 of 300 μm, and only the bran used as a medium was removed.
[0050]
2 kg (double weight equivalent) of wheat flour (Nisshin Flour Milling Co., Ltd.) is mixed in advance with 1 kg of the spore-containing filtrate. ), The filtrate was sprayed at 1 L / min while stirring at a spindle rotation speed of 230 rpm, granulated for 20 minutes, and then dried at 50 ° C. for 4 hours to obtain a granulated product 6.
[0051]
The granulated product 7 obtained by the above production method showed an extremely high numerical value of 80% in terms of the conidia survival rate compared to the culture after solid culture.
[0052]
(Example 2)
Blastoma medium (produced by Chiba Flour Milling Co., Ltd.) sterilized for 60 minutes at 121 ° C. with conidia of Gliocladium bilens IFO6355 cultured on potato dextrose agar medium (Difco) at a culture temperature of 25 ° C. for 1 week. And inoculated for 1 week at a water content of 68% and a culture temperature of 25 ° C.
[0053]
A 9-fold amount of tap water in a weight ratio was added to the culture after the completion of the solid culture and mixed by stirring to prepare a suspension. Next, this suspension was passed through a sieve having an opening of 1000 μm and then 300 μm, and only the bran used as a medium was removed.
[0054]
200 g (double weight equivalent amount) of wheat flour (Nisshin Flour Milling Co., Ltd.) is charged in a rolling granulator laboratory matrix (LMA5) (Nara Machinery Co., Ltd.) with respect to 100 g of the spore-containing filtrate. Then, while stirring at 600 rpm, the filtrate was dripped at a rate of 10 g / min to thoroughly blend the flour and filtrate, and then the spindle rotation speed was changed to 100 rpm for 10 minutes of granulation. went. The granulated product was blown and dried at 30 ° C. for 24 hours to obtain a granulated product.
[0055]
The granulated product obtained by the above production method showed an extremely high numerical value of 72% in terms of the conidial spore survival rate compared to the culture after solid culture.
[0056]
(Comparative Example 1)
Inoculated Trichoderma harzianum ATCC 74058 conidia cultivated in potato dextrose agar medium (Difco) at a culture temperature of 28 ° C. for 1 week into a sterilized bran medium (manufactured by Chiba Flour Milling Co., Ltd.) for 60 minutes. The cells were cultured at a moisture content of 68% and a culture temperature of 28 ° C. for 1 week.
[0057]
Next, according to the process shown in FIG. 2, the culture after completion of the solid culture was dried at 30 ° C. for 24 hours using a dehumidifying dryer 7T-5F type (manufactured by Tanaka Chemical Machinery Co., Ltd.).
[0058]
Next, this dried product was pulverized to an average particle size of 300 μm with an EK sample mill 8KII-1 type (produced by Fuji Paudal Co., Ltd .: currently Dalton Co., Ltd.), and used as a raw material for granules.
[0059]
1 kg and 2 kg (double weight equivalent) of wheat flour (Nisshin Flour Milling Co., Ltd.) are mixed in advance with this granulated granulator 5 Ladige Mixer M-20 type (total selling company) Pinewood), 1.5L of tap water is sprayed at 1L / min while stirring at 230 rpm for the spindle rotation speed, granulated for 20 minutes, and then dried at 50 ° C for 4 hours. It was set to 9.
[0060]
The granulated product 9 obtained by the above production method had a conidia spore survival rate of 12% compared to the culture after solid culture, which was greatly reduced.
[0061]
(Comparative Example 2)
Blastoma medium sterilized for 60 minutes at 121 ° C. after cultivating conidia of Glyocladium bilens IFO 6355 on a potato dextrose agar medium (manufactured by Difco) at a culture temperature of 28 ° C. for 1 week (manufactured by Chiba Flour Milling Co., Ltd.) And inoculated for 1 week at a water content of 68% and a culture temperature of 25 ° C.
[0062]
The culture after the completion of the solid culture was dried at 30 ° C. for 24 hours using a dehumidifying dryer T-5F type (manufactured by Tanaka Chemical Machinery Co., Ltd.).
[0063]
Next, this dried product was pulverized to an average particle size of 300 μm using EK sample mill KII-1 type (Fuji Paudal Co., Ltd .: present Dalton Co., Ltd.), and used as a raw material for granules.
[0064]
200 g (twice weight equivalent) of wheat flour (Nisshin Flour Milling Co., Ltd.) is pre-rolled with a rolling granulator laboratory matrix (LMA5) (manufactured by Nara Machinery Co., Ltd.). After adding 150 g of tap water at a rate of 10 g / min with stirring at 600 rpm, the spindle and the filtrate were mixed well, and then the spindle speed was changed to 100 rpm for 10 minutes. A granulation process was performed. The granulated product was blown and dried at 30 ° C. for 24 hours to obtain a granulated product.
[0065]
The granulated product obtained by the above production method had a conidial spore survival rate of 8% compared to the culture after solid culture, which was greatly reduced.
[0066]
【The invention's effect】
As described above in detail, the residue of the culture and the spore dispersion are separated from the suspension obtained by adding a medium that can disperse the filamentous fungus spores to the culture after the completion of the solid culture, By mixing and granulating the separated spore dispersion and the excipient, it can be formulated without reducing the survival rate of filamentous fungal conidia.
[Brief description of the drawings]
FIG. 1 is a process diagram showing an example of a method for producing a preparation according to the present invention.
FIG. 2 is a process chart showing an example of a method for producing a preparation by a conventional method.
[Explanation of symbols]
1 Culture 2 Tap Water 3 Sieve 4 Sieve 5 Mixing / Granulating / Drying Machine 6 Granulated Product 7 Dryer 8 Crusher 9 Granulated Product

Claims (3)

(1) to the culture after completion solid culture of filamentous fungi using a solid medium, a medium which can disperse the spores of filamentous fungi, water and the solution was allowed to contain an inorganic or organic salt in water, or natural fat And (2) separating the culture residue and spore dispersion from the suspension obtained in the mixing step, and (3) the spores separated in the separating step. A method for producing a preparation, comprising: a step of mixing a dispersion and an excipient; and (4) a step of granulating the mixture obtained in the step of mixing. The method for producing a preparation according to claim 1, wherein the solid medium is a bran medium. (3) The method for producing a preparation according to claim 1 or 2, wherein the separating step is a step of separating using a sieve having a diameter of 50 to 1000 µm.

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