Preparation containing microorganisms that form mycelial masses and method for producing the same

Abstract

To provide a high-hardness formulation used as a material containing microorganisms, and a method for producing the same. [Solution] The formulation according to the present disclosure comprises a solid culture containing a microorganism that forms mycelial masses and a solid medium on which the microorganisms are cultured. The formulation has a water content of 1 to 15% by weight. The formulation also has a hardness of 30 to 200 N. The manufacturing method of the formulation according to the present disclosure comprises step (1): drying the culture containing a microorganism that forms mycelial masses and a solid medium on which the microorganisms are cultured so that the water content is 1 to 15% by weight. The manufacturing method also comprises step (2): solidifying the dried culture.

Description

[Technical Field]

[0001] The present invention relates to a preparation containing microorganisms that form mycelial masses and a method for producing the same. [Background technology]

[0002] In recent years, agriculture has seen increasing efforts to utilize beneficial microorganisms found in nature. For example, microorganisms called endophytes (also known as "endophytes") are known to live symbiotically within plant hosts, providing benefits to the host such as promoting growth and increasing stress tolerance (e.g., conferring pathogenicity), and various types are being developed.

[0003] For example, Patent Document 1 discloses that the xsd08001 strain can be used as an endophyte in the cultivation of highly functional vegetable components. A powdered material has been developed by cultivating this endophyte in wheat bran. By adding this powdered material near the roots of plants, plant growth can be promoted.

[0004] Furthermore, Patent Document 2 discloses a biofertilizer obtained by inducing spore formation of bacteria of the genus Bacillus and immersing them in porous materials such as diatomaceous earth, zeolite, and silica gel.

[0005] Patent Document 3 discloses organic fertilizer pellets made by combining fermented and composted organic waste with a carrier on which actinomycetes are supported, and then adding water to compress and solidify the mixture. It is disclosed that these organic fertilizer pellets not only promote plant growth but can also control plant diseases.

[0006] Furthermore, Patent Document 4 discloses that a material containing the filamentous fungus Trichoderma can function as a pesticide against white root rot and purple root rot in fruit trees such as apples, and potato scab. The document also discloses that tap water is added and mixed when the material is formed into pellet-shaped microbial material.

[0007] On the other hand, Patent Document 5 does not disclose solid materials containing microorganisms such as endophytes, but it discloses a method for producing fertilizer by blending inorganic salts, specifically inorganic salts of ligninsulfonic acid, inorganic salts of stearic acid, and 3 to 10% by weight of talc in the granular fertilizer components with organic matter, and then compressing the mixture into tablets. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2018-174708 [Patent Document 2] Japanese Patent Publication No. 2015-113274 [Patent Document 3] Japanese Patent Publication No. 2004-189545 [Patent Document 4] Japanese Patent Publication No. 2006-199601 [Patent Document 5] Japanese Patent Publication No. 2001-146495 [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide a high-hardness formulation used as a material containing microorganisms and a method for producing the same. [Means for solving the problem]

[0010] As a result of diligent research, the present inventors have discovered that a culture containing microorganisms that form mycelial masses can be molded into a high-hardness formulation even with a low water content, and have completed the present invention. That is, this disclosure includes the following embodiments.

[0011] [1] Microorganisms that form mycelial masses and The solid culture medium in which the above microorganisms were cultured and A preparation comprising a culture. The water content of the above preparation is 1 to 15% by weight. Also, the hardness of the above preparation is 30 to 200 N. [2] In the preparation according to [1], the microorganism forming the mycelial mass is a filamentous fungus having a cell wall composed of polysaccharides. [3] In the preparation according to [2], the filamentous fungus is at least one selected from the group consisting of Ascodesmidaceae family fungi, Trichoderma genus fungi, Talaromyces genus fungi, Beauveria genus fungi, Metarhizium genus fungi, Paecilomyces genus fungi, Neotyphodium genus fungi, Epichloe genus fungi, Aspergillus genus fungi, Monacrosporium genus fungi, Arthrobotrys genus fungi, Paecilomyces genus fungi, Cephaliophora genus fungi, Fusarium genus fungi, Lecanicillium genus fungi, and mycorrhizal fungi. [4] In the preparation according to any one of [1] to [3], the microorganism forming the mycelial mass is the xsd08001 strain (accession number NITE P-02438). [5] In the preparation according to any one of [1] to [4], the bacterial concentration is 1×10 4 ~9×10 7 cfu / g. [6] In the preparation according to any one of [1] to [5], it further contains a magnesium compound. [7] In the preparation according to any one of [1] to [6], the magnesium compound contains magnesium oxide. [8] In the preparation according to any one of [1] to [7], the solid medium is selected from one or more of the group consisting of bran, okara, bamboo powder, sawdust, pine bark, bagasse, cellulose powder, cellobiose, coffee grounds, and starch.

[0012] [9] The method for producing the preparation according to [1], comprising: Step (1): drying a solid culture containing a microorganism forming a mycelial mass and a solid medium on which the microorganism has been cultured so that the water content becomes 1 to 15% by weight. The production method further includes Step (2): subjecting the dried solid culture to a consolidation treatment.

[10] In the production method according to [9], the microorganism forming the mycelial mass is a filamentous fungus having a cell wall composed of polysaccharides.

[11] In the production method according to

[10] , the filamentous fungus includes at least one selected from the group consisting of fungi of the family Ascodesmidaceae, fungi of the genus Trichoderma, fungi of the genus Talaromyces, fungi of the genus Beauveria, fungi of the genus Metarhizium, fungi of the genus Paecilomyces, fungi of the genus Neotyphodium, fungi of the genus Epichloe, fungi of the genus Aspergillus, fungi of the genus Monacrosporium, fungi of the genus Arthrobotrys, fungi of the genus Paecilomyces, fungi of the genus Cephaliophora, fungi of the genus Fusarium, fungi of the genus Lecanicillium, and mycorrhizal fungi.

[12] In the production method according to any one of [9] to

[11] , Step (2) is performed by molding at a pressure of 10 to 20N / mm 2 ^2.

[13] In the production method according to any one of [9] to

[12] , Step (2) further contains a magnesium compound. [[ID=(14]]

[14] In the production method according to

[13] , the magnesium compound contains magnesium oxide. It should be noted that there seems to be an error in the original text where "10~200N / mm" in [9] should probably be "10~200N / mm²" as guessed in the translation to make more sense. Also, the "菌糸塊" is translated as "mycelial mass" here. You can adjust according to the actual situation.In the manufacturing method described in any one of items

[15] [9] to

[14] , the solid culture medium is selected from the group consisting of wheat bran, okara, bamboo powder, sawdust, rice hulls, bagasse, cellulose powder, cellobiose, coffee grounds, and starch.

[0013]

[16] A method for promoting plant growth, comprising applying a preparation described in any one of items [1] to [8] to the rhizosphere of a plant or the surrounding environment thereof.

[0014]

[17] A method for producing crops obtained from plants, comprising applying a preparation described in any one of items [1] to [8] to the rhizosphere of a plant or the surrounding environment thereof, and growing the plant. The method comprises harvesting the crops obtained from the plant. [Effects of the Invention]

[0015] According to this disclosure, it is possible to provide formulations with high hardness (e.g., 30-200N) that contain microorganisms that form mycelial masses. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 shows the hardness of the microbial material or bran compressed into tablets in Experiment 1. [Figure 2] Figure 2 shows the thickness of the microbial material or bran compressed into tablets in Experiment 1. [Figure 3] Figure 3 shows the appearance of tablets produced in Experiment 1 by compressing microbial materials or wheat bran. [Figure 4] Figure 4 shows the hardness of tablets made using the magnesium powder formulation material compressed in Experiment 2. [Figure 5] Figure 5 is a photograph showing the state of the microbial material cultured on the fourth day in Experiment 7. [Figure 6] Figure 6 shows the hardness of tablets produced in Experiment 8 by compressing microbial materials with different particle sizes. [Figure 7] Figure 7 shows the thickness of tablets compressed with microbial materials of different particle sizes in Experiment 8. [Figure 8] Figure 8 shows the results (harvested weight) of strawberry cultivation conducted in Experiment 9, with and without compressed microbial material. [Figure 9] Figure 9 shows the results (harvested weight) of strawberry cultivation when compressed microbial material or powdered microbial material was applied in Experiment 10. [Figure 10] Figure 10 shows the results (harvested weight) of strawberry cultivation when compressed microbial material or powdered microbial material was applied during seedling cultivation or transplanting, as conducted in Experiment 11. [Figure 11] Figure 11 shows the results (harvested weight) of strawberry cultivation in Experiment 12, with and without sterilization treatment of compressed or powdered microbial materials. [Modes for carrying out the invention]

[0017] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments and can be implemented in any form without departing from the spirit of the invention.

[0018] One aspect of this embodiment is Microorganisms that form mycelial masses and A solid culture medium in which the aforementioned microorganisms were cultured and Includes solid cultures containing, The water content is 1-15% by weight. This invention relates to a pharmaceutical formulation characterized by having a hardness of 30 to 200 N.

[0019] Furthermore, one aspect of this embodiment is a method for manufacturing the above-described preparation, (1) Dry a solid culture containing microorganisms that form mycelial masses and a solid culture medium on which the microorganisms are cultured, so that the water content is 1 to 15% by weight. (2) The dried solid culture is subjected to a solidification treatment. This relates to the manufacturing method, including the manufacturing method.

[0020] The formulation provided in one embodiment has a low water content (e.g., 1-15% by weight) but high hardness (e.g., 30-200N). As a result, the formulation provided has improved moldability even without containing a binder. Furthermore, because the formulation provided in one embodiment has high hardness, it is easier to handle when applying fertilizer. Also, because the formulation provided in one embodiment can be provided as a high-hardness formulation, it is less susceptible to the effects of wind and other elements. Furthermore, the formulation provided in one embodiment can be provided as a material containing a desired amount of microorganisms that form mycelial masses, reducing the effort required for weighing when using it. Furthermore, the formulation provided in one embodiment can be provided even if the microorganisms do not produce spores or spores. Also, because the formulation provided in one embodiment has a low water content (e.g., 1-15% by weight), it is possible to prevent the growth of other bacteria in the formulation during the manufacturing and / or storage process.

[0021] [Microorganisms that form mycelial masses] In this disclosure, "microorganisms that form mycelial masses" refers to microorganisms that, when cultured under any culture conditions, produce hyphae and have the ability to adhere and aggregate to form mycelial masses. Microorganisms that form mycelial masses are preferably filamentous fungi whose cell walls are composed of polysaccharides. Examples of polysaccharides that constitute the cell wall of microorganisms that form mycelial masses include α-1,3-glucan, β-1,3-glucan, β-1,6-glucan, chitin, galactosaminogalactan, and galactomannan. The components that constitute the cell wall of microorganisms that form mycelial masses may also include components other than the polysaccharides mentioned above.

[0022] In one embodiment of this present invention, filamentous fungi whose cell walls are composed of polysaccharides include, for example, Ascodesmidaceae, Trichoderma, Talaromyces, Beauveria, Metarhizium, Paecilomyces, Neotyphodium, and Epiclo e) May include at least one species selected from the group consisting of fungi of the genera Aspergillus, Monacrosporium, Arthrobotrys, Paecilomyces, Cephaliophora, Fusarium, Lecanicillium, and mycorrhizal fungi.

[0023] In one embodiment of this embodiment, the microorganism that forms the mycelial mass is preferably an endophyte. In this disclosure, "endophyte" also refers to a microorganism that lives in symbiosis within the body of a plant host and brings benefits to the host, such as promoting growth and increasing stress tolerance. In one embodiment of this embodiment, if the microorganism that forms the mycelial mass is an endophyte, it may be, for example, a fungus of the family Ascodesmidaceae, a fungus of the genus Cephaliophora, or a fungus of the genus Lecanicillium.

[0024] Furthermore, in one embodiment of this invention, the microorganism that forms the mycelial mass is preferably strain xsd08001. Strain xsd08001, which can be used in this embodiment, was deposited with the National Institute of Technology and Evaluation (NITE) on March 6, 2017, and is available under accession number NITE P-02438.

[0025] [Solid culture medium] In this specification, "solid culture medium" means a culture medium capable of culturing microorganisms that form the above-mentioned mycelial masses. In one embodiment, the solid culture medium can be, for example, wheat bran, okara (soybean pulp), bamboo powder, sawdust, rice hulls, bagasse, cellulose powder, cellobiose, coffee grounds, or starch.

[0026] [Solid culture] In this specification, "solid culture" refers to culturing a strain using a solid culture medium containing a solid culture medium and water. The process of performing such solid culture may be referred to as the "solid culture process." In this specification, "solid culture product" refers to the culture obtained as a result of solid culture, in which the strain has been cultured in a solid culture medium. The water used in solid culture may contain, for example, various additives that contribute to the growth of the bacterial strain. Examples of additives include antibiotics that can suppress the growth of bacteria other than the target microorganism.

[0027] Solid culture media can be prepared by adding water to a solid medium and mixing. The water content of the solid culture medium is not limited, but for example, the upper limit of the water content is usually 85% by weight or less, more preferably 80% by weight or less, even more preferably 75% by weight or less, and especially preferably 70% by weight or less. The lower limit of the water content is usually 30% by weight or more, more preferably 40% by weight or more, even more preferably 45% by weight or more, and especially preferably 50% by weight or more. If the water content of the solid culture medium is too high, there will be excess moisture and insufficient space will be created in the medium, so growth will not be promoted. On the other hand, if the water content of the solid culture medium is too low, it will not be possible to secure sufficient moisture for cultivation. For example, in one embodiment, if the water content of the solid culture medium is 50 to 70% by weight, for example 60% by weight, a solid culture with an appropriate mycelial concentration can be obtained.

[0028] Solid culture in this embodiment can be carried out, for example, by inoculating a liquid culture obtained by the liquid culture described later into a solid culture medium and then using a predetermined method. For inoculation, for example, inoculation can be carried out using a micropipette or other instruments, or a method of directly inoculating by decanting after sterilization by at least one of the methods such as burning the mouth of the container containing the strain with a burner can be adopted. Whichever method is used, by inoculating the liquid culture into the solid culture medium, it becomes possible to spread the liquid culture throughout the solid culture medium. As a result, the mycelium propagates so as to grow more uniformly throughout the solid culture medium, and a more uniform culture can be obtained, and it is considered that particles of the culture with more uniform particle size can be obtained.

[0029] Solid culture can be carried out by appropriate culture means. For example, solid culture can be carried out by using a container such as a bag made of plastic or the like and allowing the container to stand still. Also, for solid culture, it is preferable to adjust the culture conditions using a temperature control device, a humidity measurement device, etc. If necessary, one or more devices selected from a stirring device, a vibration device, a pH adjustment device, a turbidity measurement device, a light control device, a specific gas concentration measurement device, and a pressure measurement device may be used. The specific gas concentration measurement device only needs to be able to measure, for example, O2 and CO2 as specific gases. From the viewpoint of preventing contamination, it is preferable to use a disposable container for solid culture, and measures such as arbitrarily closing the mouth of the bag may be taken. Appropriate stirring culture, shaking culture, or static culture may be carried out. For example, in one embodiment, a disposable bag equipped with an air filter is used, the mouth of the bag is closed, and static culture is carried out.

[0030] The period of solid culture is not limited, but for a desired concentration, for example, the average number of colonies per 1 g of the solid culture is usually 1×10 4 ~9×10 8 cfu / g, especially 5×10 4 ~5×10 7 cfu / g, further 1×10 5 ~5×10 7 cfu / g, particularly preferably 5×10 5 ~1×107 cfu / g, for example, approximately 1 × 10⁻⁶ 6 Culturing can be completed when the cfu / g is reached. For example, the upper limit of the solid culture period is usually 20 days or less, preferably 18 days or less, even more preferably 15 days or less, and especially preferably 12 days or less. The lower limit of the solid culture period is usually 5 days or more, preferably 6 days or more, even more preferably 7 days or more, and especially preferably 8 days or more. If the culture period of solid culture is too long, the risk of contamination by unwanted bacteria increases. Also, if the culture period of solid culture is too long, it hinders the efficient production of microorganisms that form mycelial masses. On the other hand, if the culture period of solid culture is too short, the mycelium does not grow sufficiently and mycelial masses cannot be formed. For example, in one embodiment, if solid culture is performed for 8 to 12 days, for example 10 days, a solid culture with an appropriate bacterial concentration for formulation can be obtained.

[0031] In this specification, "liquid culture" refers to culturing a strain using a liquid medium. The process of performing such liquid culture may be referred to as the "liquid culture process." To subject a microorganism that forms mycelial masses to liquid culture means to inoculate the microorganism into a liquid medium and cultivate it in the liquid medium. The microorganism that forms mycelial masses may be in the form of a seed culture carried in a medium. Agar is an example of a medium. To avoid contamination by unwanted bacteria, inoculation is preferably performed under sterile conditions.

[0032] In this specification, "liquid culture" refers to a liquid culture obtained as a result of liquid culture. A liquid culture medium can be prepared, for example, by adding various additives to a liquid to adjust its composition to one suitable for culture. Examples of liquids include water. Examples of additives to be added to a liquid culture medium include sugars, minerals, nitrogen sources, vitamins, organic acids, inorganic acids, organic bases, and inorganic bases. Preferably, the liquid culture medium contains sugars and a nitrogen source. An example of a nitrogen source contained in a liquid culture medium is peptone.

[0033] [Humidity] While there are no restrictions on humidity during solid-state culture, the upper limit is usually 100%RH or less, more preferably 95%RH or less, and more preferably 90%RH or less. The lower limit for solid-state culture humidity is usually 55%RH or higher, more preferably 60%RH or higher, and more preferably 65%RH or higher. In some embodiments, it can be carried out at a humidity of 60-80%RH, for example, 70%RH. On the other hand, there are no particular restrictions on humidity during liquid culture, and any humidity can be used.

[0034] [temperature] In both solid-state and liquid-state culture, the upper temperature limit is usually 40°C or lower, more preferably 35°C or lower, more preferably 30°C or lower, and especially preferably 25°C or lower. The lower temperature limit for both liquid-state and solid-state culture is usually 5°C or higher, more preferably 10°C or higher, more preferably 15°C or higher, and especially preferably 20°C or higher. In one embodiment, the process can be carried out at 20-25°C, for example, 25°C ± 1°C.

[0035] [Sterilization] Furthermore, from the viewpoint of preventing contamination, it is preferable to sterilize solid culture media and / or solid culture media, liquid culture media, by any known means such as filtration sterilization, autoclave sterilization, boiling sterilization, and radiation sterilization, sodium hypochlorite, or ozone treatment. It is preferable to perform each operation, such as inoculation, under a sterile atmosphere. For example, in the case of solid culture media, an appropriate amount of water can be added to the solid media before sterilization.

[0036] [Drying and grinding] In one embodiment, the method for producing the above-mentioned formulation includes drying a solid culture containing a microorganism that forms mycelial masses and a solid medium on which the microorganisms are cultured, so that the water content is 1 to 15% by weight. The lower limit of the water content is 1% by weight or more, more preferably 2% by weight or more, and more preferably 3% by weight or more. On the other hand, the upper limit of the water content is 15% by weight or less, more preferably 9% by weight or less, and more preferably 8% by weight or less.

[0037] Drying can be done using general-purpose equipment such as air conditioners and dehumidifiers. Alternatively, it can be done by leaving the product in an environment of 25°C and 50% RH for three days.

[0038] Dried solid cultures can be ground using general-purpose grinders such as food processors, coffee grinders, and pepper mills.

[0039] [Particle size] The particle size of the solid culture used in the manufacturing method of this embodiment is not limited, but for example, a solid culture prepared to have a particle size of 5 mm or less, preferably 3 mm or less, and more preferably 2 mm or less may be used. For example, in one embodiment, the cumulative 50% particle size (D) in the volume particle size distribution may be further adjusted. 50 ) is 100-900 μm and has a cumulative 90% particle size (D 90 This may include adjusting the particle size so that it is between 1000 and 5000 μm. Particle size can be measured by any method such as laser diffraction, scattering, imaging, light transmission centrifugal sedimentation, sedimentation, electrical resistance, specific surface area, or sieve passage. Particle size can be measured using a suitable particle size analyzer, such as a commercially available instrument like the LMS-2000e or LMS-3000 (Seishin Corporation). Since the solid culture is dried before grinding, it is preferable to use a dry measurement method for particle size measurement.

[0040] [Consolidation treatment] In this specification, "consolidation treatment" refers to a treatment in which pressure is applied to the solid culture produced above to consolidate the solid culture and form it into a formulation. From the viewpoint of achieving the effects of the present invention, it is preferable to granulate the dried and pulverized solid culture by dry granulation. It is also preferable to use dry tableting when performing tableting. Formulations prepared by dry granulation and / or dry tableting may be referred to as "dry formulations". For the granulator and tablet press, for example, commercially available dry granulators such as manual tabletop tablet presses, small high-speed rotary tablet presses, or roll-type granulators may be used, but manual tabletop tablet presses or small high-speed rotary tablet presses are preferred. The pressure during molding should be 10 to 200 N / mm from the viewpoint of moldability of the formulation and the viability of the microorganisms that form the mycelial masses contained in the formulation. 2 Preferably, the load is 30-150 N / mm². 2 It is more preferable that the load is 50-120 N / mm 2 It is even more preferable that this be the case. The shape of the pestle of the dry granulator may be any of the following: standard R, two-stage R, sugar-coated R, corner R, corner flat, rounded flat, etc.

[0041] [Water content] The water content in the formulation obtained in this embodiment may be 1 to 15% by weight relative to the weight of the solid culture. From the viewpoint of achieving the effects of the present invention, the lower limit of the water content is 1% by weight or more, more preferably 2% by weight or more, and more preferably 3% by weight or more. On the other hand, the upper limit of the water content is 15% by weight or less, more preferably 9% by weight or less, and more preferably 8% by weight or less. The water content may be adjusted as appropriate after the solidification treatment.

[0042] [hardness] In this specification, "hardness" is an indicator of the hardness of a formulation and can be measured with a commercially available tablet hardness tester. Specifically, it can be determined by measuring the tablet hardness in the diametrical direction using a tablet hardness tester such as the DC-50 (Okada Seikou Co., Ltd.). A formulation provided in one embodiment has a hardness of 30 to 200 N. From the viewpoint of achieving the effects of the present invention, if the hardness is too low, the degree of abrasion increases, so a lower limit of 30 N or more is preferable, more preferably 40 N or more, and even more preferably 50 N or more. On the other hand, from the viewpoint of handling, a higher hardness is desirable, but in one embodiment, formulations with hardness of 200 N or less, 180 N or less, or 150 N or less can be obtained.

[0043] It is known that the water content in a formulation greatly affects its hardness. Generally, a higher water content results in higher hardness, while a lower water content results in lower hardness. The water that contributes to moldability is considered to be adhering water, and in particular, when the particle size is coarse, such as in the solid culture medium contained in the formulation obtained in this embodiment, the surface area is small, and therefore the amount of water that can adhere is small. Consequently, a larger amount of water is required to obtain a suitable hardness for good moldability and to avoid tableting problems. However, the formulation obtained in this embodiment can achieve a high hardness of 30 to 200 N despite having a low water content of 1 to 15% by weight.

[0044] [Bacterial concentration of the preparation] The bacterial concentration in the formulation obtained in this embodiment is typically 1 × 10¹⁶ colonies per gram of the formulation, for example. 4 ~9×10 7 cfu / g, especially 5×10 4 ~5×10 7 cfu / g, and furthermore, 1 × 10⁻⁶ 5 ~1 × 10 7 cfu / g, particularly preferably 5 × 10 5 ~5×10 6 cfu / g, for example, approximately 1 × 10⁻⁶ 6 It is cfu / g.

[0045] [Magnesium compounds] In one embodiment, the formulation provided preferably further contains a magnesium compound. The inclusion of a magnesium compound in the formulation provides an effect of further increasing hardness. Examples of magnesium compounds include magnesium oxide (MgO), magnesium chloride (MgCl2), magnesium sulfate (MgSO4), magnesium nitrate (Mg(NO3)2), magnesium carbonate (MgCO3), and magnesium acetate (Mg(CH3COO)2), but magnesium oxide (MgO) is preferred.

[0046] [Plants to be cultivated and production methods] The formulation provided by this embodiment may be applied to trees, flowers, vegetables, and fruit trees, for example. For example, if the plant is a vegetable grown for fresh produce, that is, for market sale or consumption, it may be a leafy vegetable such as cabbage, Chinese cabbage, or spinach, where the leaves are edible. Alternatively, it may be a root vegetable such as radish, turnip, carrot, or leek, where the rhizome is edible. It may also be a fruit vegetable such as cucumber, bell pepper, or eggplant, grown for fruit production.

[0047] Furthermore, in one embodiment, examples of vegetables or fruit trees to which the formulation provided by this embodiment is applied include: Chenopodiaceae crops such as spinach, Swiss chard, and sugar beet; Asteraceae crops such as lettuce, salad greens, garland chrysanthemum, and burdock; Brassicaceae crops such as cabbage, broccoli, Chinese cabbage, radish, and turnip; Liliaceae crops such as onions and leeks; Apiaceae crops such as carrots, celery, and Japanese parsley; Solanaceae crops such as tomatoes, eggplants, bell peppers, chili peppers, torbam, red soybeans, and tobacco; Cucurbitaceae crops such as cucumbers, melons, watermelons, pumpkins, and dried gourd strips; Poaceae crops such as sweet corn; Fabaceae crops such as peas, broad beans, green beans, and soybeans; and Rosaceae crops such as strawberries, apples, loquats, plums, cherries, peaches, and pears. Among these, the application of this embodiment to Rosaceae crops, Asteraceae crops, or Chenopodiaceae crops is preferred, and strawberries are particularly preferred.

[0048] Furthermore, in one embodiment, the plants to which the formulation provided by this embodiment is applied include, for example, plants of the Rosaceae family such as roses, plants of the Asteraceae family such as chrysanthemums, dandelions, and thistles, and plants of the Liliaceae family such as lilies and tulips, which are used as horticultural or ornamental plants.

[0049] The formulation provided by this embodiment may be applied to a method for producing plants. Furthermore, the formulation provided by this embodiment may be applied to a method for promoting plant growth. Furthermore, the formulation provided by this embodiment may be applied to a method for producing crops obtained from plants. Any of the above methods for applying the formulation of this embodiment involves applying the formulation to the rhizosphere of the plant or its surrounding environment, such as the soil and water in which the plant is cultivated, and cultivating it according to conventional farming methods. The cultivated plant or crop obtained from the plant to which the formulation has been applied may then be harvested. In this disclosure, "rhizosphere" means the soil space influenced by plant root secretions and soil organisms.

[0050] Furthermore, the formulation provided by this embodiment may be dissolved in water or the like and applied to plant structures such as leaves, stems, flowers, fruits, seedlings, and seeds.

[0051] The timing of application of the formulation provided by this embodiment to plants is not particularly limited, but it can be particularly effective when applied at the time of planting.

[0052] The method of storing the formulation provided by this embodiment is not particularly limited, but from the viewpoint of suppressing the effects of moisture, storage in airtight packaging materials or containers is preferable, and a desiccant may be further added to such packaging materials or containers. Alternatively, the formulation provided by this embodiment may be packed into a packaging sheet such as a PTP sheet and sealed for storage. [Examples]

[0053] The present invention will now be described in more detail with reference to examples. However, the present invention is not limited thereto.

[0054] <Materials and methods used> Preparation of microbial materials The microbial strain used was strain xsd08001, which was deposited with the National Institute of Technology and Evaluation (NITE) under accession number NITE P-02438. Microbial materials were prepared using strain xsd08001 according to the following procedure.

[0055] (1) The xsd08001 strain, which had been cryopreserved at -80°C, was pre-cultured in glucose peptone medium at 25°C for 3 days. (2) The mixed bran was packed into the mushroom substrate bag and autoclaved at 121°C for 60 minutes. (3) After cooling, the pre-cultured bacterial culture solution was inoculated and incubated at 25°C for 10 days. (4) After culturing, the material was dried at room temperature or in a dryer until the moisture content reached 3-8% by weight, and then ground in a pulverizer. (5) Bran containing crushed microbial cultures was used as a microbial material.

[0056] <Experiment 1: Tabletization of microbial materials> (1) Microbial materials with a particle size distribution of 50 μm to 5000 μm were sieved to a particle size of 2 mm or less. The same procedure was performed for wheat bran. (2) 250 mg or 1000 mg of the sieved material was weighed out. (3) Tablets were compressed using a manual tabletop tablet molding machine (HANDTAB-200) according to the conditions in Table 1. No water was added during tablet compression.

[0057] [Table 1]

[0058] (4) The hardness of the tablets was measured using a load cell type bench hardness tester, and the thickness was measured using a digital caliper (n=8). The results are shown in Figures 1 and 2.

[0059] As shown in Figure 1, Examples 1 and 2 demonstrated higher hardness than Comparative Examples 1 and 2, which had the same filling volume. Furthermore, as shown in Figure 2, measurements of the thickness of the prepared tablets revealed that the thickness of Example 2 was smaller than that of Comparative Example 2. The results from Figures 1 and 2 suggest that a smaller tablet thickness is associated with higher hardness.

[0060] Furthermore, 250 mg, 500 mg, 1000 mg, or 1500 mg of the microbial material after (1) above, or 1000 mg of wheat bran, were weighed out and compressed into tablets using a manual tabletop tablet molding machine at a compression pressure of 7 to 20 kN. No water was added during tableting. The appearance of the tablets is shown in Figure 3.

[0061] <Experiment 2: Effect of mixing with magnesium powder on tablet hardness> (1) Microbial materials with a particle size distribution of 50 μm to 5000 μm were sieved to a particle size of 2 mm or less. The same procedure was performed for wheat bran. (2) The magnesium powder used was subjected to a component analysis by the Japan Fertilizer Inspection Association. The results are shown in Table 2.

[0062] [Table 2]

[0063] (3) Each sample was prepared according to Table 3. [Table 3] (4) Each prepared sample was compressed into tablets using a manual tabletop tablet molding machine (n=4). No water was added during tableting.

[0064] As shown in Figure 4, Examples 3, 4, 6, and 7, which contained magnesium powder, showed even higher hardness than Examples 5 and 8, which did not contain magnesium powder. A similar trend was observed in Comparative Examples 3 and 4, which contained bran and magnesium powder, but their hardness was lower compared to Examples 3, 4, 6, and 7. These results suggest that adding magnesium powder to microbial materials significantly increases their hardness.

[0065] <Experiment 3: Changes in bacterial count due to tablet compression> (1) Microbial material with a particle size distribution of 50 μm to 5000 μm was sieved to a particle size of 2 mm or less. (2) Tablets (filling amount 1000 mg, diameter 15 mm, hardness approximately 30 N) were manufactured using a small, high-speed rotary tablet press (manufactured by Kikusui Seisakusho Co., Ltd.). No water was added during tableting. (3) 1 g each of the prepared tablets (Example 9) and the powdered microbial material (Comparative Example 6) was weighed out. (4) Add 9 mL of sterile water and stir vigorously. (5) Let it stand for 15 minutes. (6) Take 1 mL of supernatant from the suspension and add 9 mL of sterile water to dilute it 100-fold. (7) 100 μL of the diluted sample was added to Rose Bengal medium and spread over the medium using a convex stick. (8) The cells were incubated at 25°C and 70% RH for 3 days. (9) After culturing, the number of colonies was counted and the CFU (colony forming unit) was calculated. [Table 4]

[0066] As shown in Table 4, it was demonstrated that when powdered microbial materials are compressed into tablets, they can be granulated while retaining their live bacteria.

[0067] Furthermore, when the number of colonies was calculated for the tablets of Example 2 in the same manner, it was the same as the number of colonies in Example 9.

[0068] <Experiment 4: Granulation using a roll-type granulator> (1) Microbial material with a particle size distribution of 50 μm to 5000 μm was sieved to a particle size of 2 mm or less. The moisture content of the microbial material was 3%. (2) Granulation was performed using a roll-type granulator (manufactured by Shinto Kogyo Co., Ltd.) under the conditions shown in Table 5. No water was added during granulation. [Table 5] (3) The granulated material, which was molded in a sheet form, was separated from the sheet to obtain granulated material with a diameter of φ4.8 mm and flattened. (4) The number of colonies in the powder material before granulation (Comparative Example 7), the granules immediately after granulation (Example 10), and the granules after one month (Example 11) was measured using rose bengal medium. [Table 6]

[0069] As shown in Table 6, no colonies could be detected in Examples 10 and 11, which were granulated using a roll-type granulator. Colonies were detected in Comparative Example 7 before granulation, indicating that granulation using a roll-type granulator significantly reduces the number of colonies.

[0070] <Experiment 5: Granulation of microbial materials with different moisture content using a roll granulator> (1) The moisture content of the microbial material was adjusted to 8% (Example 12) and 12% (Example 13), respectively. (2) Granulation was carried out in the same manner as in <Experiment 4>, according to the conditions in Table 7. [Table 7] (3) The granulated material was separated and the number of colonies was measured after one month. [Table 8]

[0071] In Examples 10 and 11 of Experiment 4, no microbial colonies were detected, so the moisture content of the microbial material was adjusted and the granulation conditions were investigated. As a result, no colonies were detected in Examples 12 and 13, even after the moisture content was adjusted.

[0072] <Experiment 6: Disintegration of tablets in aqueous solution> (1) Microbial material was compressed into 250 mg and 1000 mg tablets under the same conditions as in Experiment 1. (2) Add 50 ml of pure water to a beaker and drop in one tablet. (3) The time it took for the 250 mg (Example 14) and 1000 mg (Example 15) tablets to completely disintegrate was measured (n=5). [Table 9]

[0073] Example 14 completely disintegrated in 27.4 seconds, and Example 15 disintegrated in 101.8 seconds. Disintegration began immediately upon dropping each tablet into the aqueous solution, indicating good disintegration properties.

[0074] <Experiment 7: Heat resistance of microbial materials> (1) Microbial material with a particle size distribution of 50 μm to 5000 μm was sieved to a particle size of 2 mm or less. (2) The materials were placed in a glass container and heated at 100°C for 60 minutes. (3) After heating, the material was placed on glucose peptone medium and cultured at 25°C and 70% RH. (4) A photograph was taken on the fourth day of culture (Example 16).

[0075] As shown in Figure 5, mycelial growth was observed even when the dry microbial material was heated at 100°C for 1 hour.

[0076] <Experiment 8: Tablet compression using materials with different particle sizes> (1) Microbial materials with a particle size distribution of 50 μm to 5000 μm were sieved to a particle size of 500 μm (Example 17) or 2 mm (Example 18) or less. (2) The experimental procedure from this point onward is the same as in Experiment 1.

[0077] As shown in Figures 6 and 7, no significant difference was observed in the hardness and thickness of the tablets even when the particle size of the microbial material differed.

[0078] <Comparison of pressure generated in each granulator> Table 10 summarizes the approximate pressures generated by each granulator used in Experiment 1, Experiment 3, and Experiment 4. [Table 10]

[0079] The manual tabletop tablet press and the small, high-speed rotary tablet press used in Experiments 1 and 3 were shown to generate lower pressure per unit area compared to the roll-type granulator used in Experiment 4. From these results, it is expected that the number of colonies will decrease during granulation when a certain pressure is applied.

[0080] <Experiment 9: Strawberry cultivation test> (1) Microbial material with a particle size distribution of 50 μm to 5000 μm was sieved to a particle size of 2 mm or less. (2) Tablets (filling amount 1000 mg, diameter 15 mm, hardness approximately 30 N) were manufactured using a small, high-speed rotary tablet press. No water was added during tableting. (3) Using the prepared tablets, cultivation tests were conducted on strawberries (30 plants per test plot). (4) When transplanting the grown strawberry seedlings, two tablets (2g) were placed in the planting hole for each seedling. (5) The test group with added tablets was designated as Example 19, and the test group without added tablets was designated as Comparative Example 8. (6) Harvested two months after planting, and the harvested weight was measured.

[0081] As shown in Figure 8, in Example 19, 2,000g of strawberries were harvested, nearly double the yield compared to Comparative Example 8.

[0082] <Experiment 10: Verification of the effects of tablet and powder application rates on strawberry cultivation> (1) Tablet compression was performed under the same conditions as in Experiment 9. (2) Cultivation trials were conducted in which 0.1 g or 2.0 g of microbial materials in tablet and powder form were applied at the time of planting, according to Table 11 (10 plants per test plot, 18 replicates). [Table 11]

[0083] As shown in Figure 9, Example 20 showed a higher and more stable yield compared to Comparative Example 9. On the other hand, as also shown in Figure 9, Example 21 showed a similar yield to Comparative Example 10. These results indicate that tablets have a greater effect on increasing yield at low application doses. It is presumed that tablets can be applied locally near the roots, and that even a small application dose of 0.1g resulted in a stable yield.

[0084] <Experiment 11: Verification of the effects of tablet and powder application timing on strawberry cultivation> (1) Tablet compression was performed under the same conditions as in Experiment 9. (2) Cultivation trials were conducted in which microbial materials in tablet and powder form were applied during seedling cultivation or transplanting, according to Table 12 (10 plants per test plot, 18 replicates). [Table 12]

[0085] As shown in Figure 10, when applied during seedling cultivation, the yields of Example 22 and Comparative Example 11 were similar. On the other hand, as shown in Figure 10, when applied at transplanting, Example 23 showed a higher and more stable yield compared to Comparative Example 12. From these results, it was found that the effect of increasing yield when applied at transplanting is greater with tablets. When applied at transplanting, the soil space is larger because it is applied to the main field, but powder is easily washed away by wind and water and may not be distributed uniformly in the soil, making it difficult to apply locally near the roots. On the other hand, tablets can be applied locally near the roots, and it is presumed that they showed a stable yield even when applied at transplanting.

[0086] <Experiment 12: Verification of the effect of sterilization treatment on strawberry cultivation with and without tablets and powder> (1) Tablet compression was performed under the same conditions as in Experiment 9. (2) Cultivation tests were conducted using tablet and powder microbial materials, both with and without sterilization, according to Table 13 (10 strains per test plot, 18 replicates). Sterilization was performed using an autoclave. [Table 13]

[0087] As shown in Figure 11, Example 24 yielded a similar amount to Comparative Example 13. On the other hand, as also shown in Figure 11, Example 25 yielded a higher and more stable amount compared to Comparative Example 14. These results indicate that the effect of sterilization on yield is greater in tablets. Powder is easily washed away by wind and water and may not be uniformly distributed in the soil, but tablets can potentially provide nutrients from bran decomposition products in a specific location, and it is presumed that they showed a stable yield even in the absence of the xsd08001 strain.

[0088] <Consideration of the action and function of the tableted microbial material (formulation) according to the embodiment> 1. Sustained release of ingredients (continuous supply) The tableted microbial material according to this embodiment may have the potential to supply nutrients and growth-promoting factors to the soil over a long period of time, as the components are slowly released due to the tablet form. On the other hand, powdered microbial material is more soluble in water than tableted microbial material, so it may be released rapidly all at once, which may not match the timing of plant absorption. By tableting the microbial material, the components are gradually released and supplied in accordance with the growth period required by the plant, which may allow for sustained effectiveness.

[0089] 2. Uniform improvement of the soil environment While microbial materials in powder form are easily washed away by wind and water and may not be uniformly distributed in the soil, tablet form provides a stable supply of components to specific locations, making it easier to sustain soil improvement near the plant's root zone. In particular, if the microbial material is in the form of a sustained-release tablet, microbial activity in the soil can be maintained for a long period, potentially allowing plants to absorb nutrients more efficiently through their roots.

[0090] 3. Improved stability of ingredients Fermented products are susceptible to deterioration due to moisture and oxygen, making long-term storage difficult when microbial materials are in powder form. By encapsulating microbial materials, the components become more stable internally, and the active ingredients produced by fermentation (e.g., plant hormones and enzymes) are preserved without degradation, potentially allowing for more stable effects on plants.

[0091] 4. Ensuring appropriate application rates. By packaging microbial materials into tablets, the application rate becomes consistent, making it easy to ensure the optimal amount for plant growth. When microbial materials are in powder form, the application rate tends to vary, potentially leading to differences in effectiveness for each plant. However, with tablets, the required amount can be supplied uniformly, potentially resulting in more stable effects.

[0092] 5. Strengthening synergistic effects with soil microorganisms In the embodiment, the tableted microbial material may have components leached from the surface of the tablet that readily interact with beneficial microorganisms in the rhizosphere, potentially promoting microbial activation and colonization. Increased microbial activity further enhances nutrient supply in the soil, improving the soil environment for plant growth.

[0093] 6. Improvement of parasitic effects on plants through the decomposition of solid culture media by filamentous fungi and the promotion of filamentous fungal growth. The tabletized microbial material according to this embodiment has improved stability as a habitat for filamentous fungi, increasing their growth capacity, which may also promote the decomposition of solid culture media such as wheat bran. The decomposed solid culture media itself becomes nutrients for plants. In addition, it may be able to exist locally near the roots, improving the efficiency of root parasitism, and promoting plant growth through nutrient supply to the plant and production of useful plant hormones.

[0094] (reference) • Filamentous fungi have been reported to exhibit an important ability to decompose wheat bran and promote plant growth (Mittermeier et al. 2024). Specifically, filamentous fungi secrete hydrolytic enzymes (such as cellulase and xylanase) to decompose recalcitrant organic matter in wheat bran, breaking it down into monosaccharides and organic acids that are easily absorbed by plants. This not only increases the utilization rate of nutrients in the soil but also promotes the activation of soil microorganisms and reduces dependence on chemical fertilizers (Argumedo-Delira et al. 2022; Mittermeier et al. 2024).

[0095] References: [Table 14]

Claims

1. Microorganisms that form mycelial masses and A solid culture medium in which the aforementioned microorganisms were cultured and Includes solid cultures containing, The water content is 1 to 15% by weight. A pharmaceutical formulation characterized by having a hardness of 30 to 200 N.

2. The formulation according to claim 1, wherein the microorganism that forms the mycelial mass is a filamentous fungus whose cell wall is composed of polysaccharides.

3. The aforementioned filamentous fungi include Ascodesmidaceae, Trichoderma, Talaromyces, Beauveria, Metallidium, Paeciliomyces, Neotyphodium, Epicloe, and Aspergillus (As The formulation according to claim 2, comprising at least one selected from the group consisting of fungi of the genus Pergillus, Monacrosporium, Arthrobotrys, Paeciliomyces, Cephaliophora, Fusarium, Lecanicillium, and mycorrhizal fungi.

4. The formulation according to claim 1, wherein the microorganism that forms the mycelial mass is strain xsd08001 (accession number NITE P-02438).

5. Bacterial concentration is 1 x 10 ４ ~9 x 10 ７ The formulation according to claim 1, wherein the concentration is cfu / g.

6. Furthermore, the formulation according to claim 1, further containing a magnesium compound.

7. The formulation according to claim 6, wherein the magnesium compound comprises magnesium oxide.

8. The formulation according to claim 1, wherein the solid culture medium is selected from one or more of the group consisting of wheat bran, okara (soy pulp), bamboo powder, sawdust, rice hulls, bagasse, cellulose powder, cellobiose, coffee grounds, and starch.

9. A method for producing the pharmaceutical product according to claim 1, Step (1): A solid culture containing microorganisms that form mycelial masses and a solid culture medium on which the microorganisms are cultured is dried so that the water content is 1 to 15% by weight. Step (2): The dried solid culture is subjected to a solidification treatment. A manufacturing method that includes this.

10. The method for producing the mycelial mass according to claim 9, wherein the microorganism that forms the mycelial mass is a filamentous fungus whose cell wall is composed of polysaccharides.

11. The aforementioned filamentous fungi include Ascodesmidaceae, Trichoderma, Talaromyces, Beauveria, Metallidium, Paecilomyces, Neotyphodium, Epicloe, and Aspergillus. The method for producing the product according to claim 10, comprising at least one selected from the group consisting of fungi of the genus Rgillus, Monacrosporium, Arthrobotrys, Paeciliomyces, Cephaliophora, Fusarium, Lecanicillium, and mycorrhizal fungi.

12. In step (2) above, 10 to 200 N / mm ２ The manufacturing method according to claim 9, wherein the molding is performed under pressure.

13. The manufacturing method according to claim 9, wherein the magnesium compound is further included in step (2) above.

14. The manufacturing method according to claim 13, wherein the magnesium compound includes magnesium oxide.

15. The manufacturing method according to claim 9, wherein the solid culture medium is selected from one or more of the group consisting of wheat bran, okara (soybean pulp), bamboo powder, sawdust, rice hulls, bagasse, cellulose powder, cellobiose, coffee grounds, and starch.

16. A method for promoting plant growth, Applying the formulation described in claim 1 to the rhizosphere of a plant or its surrounding environment. Methods that include...

17. A method for producing crops obtained from plants, Applying the formulation described in claim 1 to the rhizosphere of a plant or its surrounding environment, and growing the plant, Harvesting crops obtained from the aforementioned plants. Methods that include...

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