JP6918312B2 - Spore germination inhibitor - Google Patents

Description

The present invention relates to a substance that suppresses germination of bacterial spores.

Gram-positive spore-forming bacteria are highly resistant to the environment and difficult to control their growth. Spore-forming bacteria have long been known to cause food spoilage. In recent years, it has been reported that spores are formed in a biofilm, and that spores derived from floating cells and spores derived from a biofilm have different properties such as heat resistance. Since most bacteria in the environment are considered to exist in a biofilm state, it is important to control spores not only in floating cells but also in biofilms in order to suppress the growth of bacteria.

For bacteria that are not easy to sterilize by heating or sterilizing agents due to the presence of spores, a technique has been proposed in which the growth of the spores is suppressed by controlling the spores. For example, a method of rapidly germinating all spores to make them into vegetative cells and sterilizing them, or a method of suppressing germination and suppressing proliferation for a long period of time has been attempted.

Conventionally, amino acids are often used to promote germination and inhibit germination of spores. For example, L-aspartic acid is used as a germination promoter for Bacillus (Non-Patent Document 1), while it is used as a germination inhibitor for Clostridium (Clostridium). Non-Patent Document 2). Glycine is used as a germination inhibitor for the genus Bacillus (Non-Patent Document 3), while it is used as a germination promoter for the genus Clostridium (Non-Patent Document 4).

The genus Paenibacillus is a genus of bacteria reclassified from the genus Bacillus and is distributed in soil, plant rhizosphere, water, insects, foods, and the like. Bacteria of the genus Paenibacillus are spore-forming bacteria and have been attracting attention in recent years as food spoilage hazards. The spores of Paenibacillus bacteria have higher resistance to peracetic acid, which is a powerful bactericidal agent, and can grow even at low temperatures, as compared with spore-forming bacteria of other species (Non-Patent Document 5). In the field of various beverages and foods, the problem of resistant bacteria such as Paenibacillus bacteria, which show strong resistance to peracetic acid, has become apparent. In addition, the spoilage bacteria Paenibacillus sp. And Paenibacillus odorifer were isolated from cooked foods, these bacteria were able to grow at 4 ° C, and Paenibacillus bacteria have spore-forming ability and low-temperature growth ability, which is harmful to food. It has been reported that this can be caused (Non-Patent Document 6).

On the other hand, in the fields of horticulture and agriculture, Paenibacillus bacteria are utilized as organic biofertilizers or microbial preparations for the purpose of promoting plant growth and controlling organisms. In these organic biofertilizers or microbial preparations, spore formation of Paenibacillus bacteria is considered to be effective for improving the survival rate of the bacteria in the fertilizers and preparations, promoting the plant growth and maintaining the biological control effect. (See Non-Patent Document 7).

Applied and Environmental Microbiology, 2016, doi: 10.1128 / AEM-00594-16. Journal of Bacteriology, 2010, 192 (2): 418-425 Microbiology and Immunology, 1985, 29 (3): 229-241 Journal of Bacteriology, 2016, 198 (20): 2767-2775 Japan Society for Bioscience and Biotechnology 2016 Annual Meeting Abstract, Presentation No. 2F043 Biocontrol science, 2006, 11 (1): 43-47 Bioresource Technology, 2012, 108: 190-195

The present invention provides a substance capable of suppressing germination of spores of spore-forming bacteria.

The present inventors have found that aspartic acid has an action of suppressing germination of spores of Paenibacillus spp.

Therefore, in one embodiment, the present invention provides a germination inhibitor for Paenibacillus bacterial spores containing aspartic acid as an active ingredient.
In another embodiment, the present invention provides an agent for suppressing the growth of Paenibacillus bacteria containing aspartic acid as an active ingredient.
In another embodiment, the present invention provides an agent containing aspartic acid as an active ingredient to prevent microbial contamination by Paenibacillus bacteria.
In another embodiment, the present invention provides a stability improver for fertilizers or microbial preparations containing Paenibacillus bacteria containing aspartic acid as an active ingredient.

According to the present invention, germination of spores of Paenibacillus bacteria can be effectively suppressed regardless of the presence or absence of bacteria such as floating cells and biofilms. Further, according to the present invention, by suppressing the germination of spores of Paenibacillus bacterium, the growth of the bacterium can be suppressed. The present invention can be applied to suppress germination of Paenibacillus bacterial spores in foods, fertilizers, microbial preparations and the like. According to the present invention, it is possible to prevent food spoilage and deterioration due to Paenibacillus bacterial spores contaminated in the manufacturing process or remaining after the manufacturing process without performing heat sterilization or chemical treatment that impairs the flavor of the food. .. Further, according to the present invention, the Paenibacillus bacterium contained in the fertilizer or the microbial preparation can be kept as a spore to improve the survival rate and maintain the effect of the fertilizer or the microbial preparation.

Phase-contrast microscopic images of biofilm (BF) -derived Paenibacillus polymyxa ATCC39564 spores cultured in the presence (30 mM) and in the absence (0 mM) of L-Asp. After 30 minutes of culturing. Phase-contrast microscopic images of biofilm (BF) -derived Paenibacillus polymyxa ATCC39564 spores cultured in the presence (30 mM) and in the absence (0 mM) of L-Asp. After 28 hours of culturing. Phase-contrast microscopic image of Paenibacillus polymyxa ATCC39564 spores derived from suspension culture cultured in the presence (3 to 30 mM) and in the absence (0 mM) of L-Asp. After 30 minutes of culturing. Phase-contrast microscopic images of Paenibacillus polymyxa ATCC39564 spores from suspension culture cultured in the presence (30 mM) and in the absence (0 mM) of L-Asp. After 28 hours of culturing. Phase-contrast microscopic images of Paenibacillus polymyxa ATCC39564 spores from suspension culture cultured in the presence (30 mM) and in the absence (0 mM) of D-Asp. After 30 minutes of culturing. Phase-contrast microscopic images of Paenibacillus sp. KMC150 spores derived from suspension culture cultured in the presence (30 mM) and in the absence (0 mM) of L-Asp. After 24 hours of culturing. Phase-contrast microscopic images of Paenibacillus sp. KMC150 spores derived from suspension culture cultured in the presence (30 mM) and in the absence (0 mM) of D-Asp. 16 hours after culturing. Phase-contrast microscopic images of Paenibacillus glucanolyticus KMC129 spores derived from suspension culture cultured in the presence (30 mM) and in the absence (0 mM) of L-Asp. After 24 hours of culturing. Phase-contrast microscopic images of Paenibacillus glucanolyticus KMC129 spores derived from suspension culture cultured in the presence (30 mM) and in the absence (0 mM) of D-Asp. 16 hours after culturing.

In the present invention, aspartic acid is used as an active ingredient for suppressing germination of spores of Paenibacillus spp.

Furthermore, in the present invention, by suppressing the germination of spores of Paenibacillus bacteria with aspartic acid, it is possible to suppress the growth of the bacterium and further prevent microbial contamination by the bacterium. Therefore, in the present invention, aspartic acid can also be used as an active ingredient for suppressing the growth of Paenibacillus bacteria or for preventing microbial contamination by Paenibacillus bacteria. These aspects of the present invention are useful in fields such as food production.

Further, in the present invention, aspartic acid can be used as an active ingredient for improving the stability of fertilizers or microbial preparations containing Paenibacillus bacteria. In a fertilizer or microbial preparation containing the Paenibacillus bacterium, by suppressing the spore germination of the bacterium with aspartic acid, the viability of the bacterium in the fertilizer or the preparation is improved, and the fertilizer or the preparation is used. It is possible to maintain the effect of promoting plant growth or controlling organisms. Preferably, the improvement in the stability of the fertilizer or the microbial preparation in the present invention means the survival rate of Paenivatilus bacteria in the fertilizer or the preparation as compared with the case where aspartic acid is not added, or the quality of the fertilizer or the preparation ( For example, it refers to the improvement or prevention of deterioration of the plant growth promotion or biological control effect).

In the present invention, the species of the genus Paenibacillus is not particularly limited, but preferred examples include Paenibacillus polymyxa, Paenibacillus chibensis, Paenibacillus glucanolyticus, and Paenibacillus glucanolyticus. Paenibacillus massiliensis, Paenibacillus popilliae, Paenibacillus alvei, Paenibacillus sp., And the like, with more preferred examples being Paenibacillus massiliensis, Paenibacillus popilliae, Paenibacillus popilliae, Paenibacillus sp. , Paenibacillus sp., More preferred examples include Paenibacillus polymyxa ATCC39564, Paenibacillus sp. KMC150, Paenibacillus glucanolyticus KMC129.

The spores of the Paenibacillus bacterium may be in the state of floating cells or may be present in a biofilm. In either case, aspartic acid exerts an excellent germination inhibitory effect on the spores of Paenibacillus bacteria.

The aspartic acid used in the present invention may be either L-aspartic acid or D-aspartic acid. In addition, the aspartic acid may be in the form of a free form or a salt. Examples of the salt include acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts and the like. Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, nitrate and phosphate, and acetate, maleate, fumarate, citrate, malate, lactate and α-ketoglutal. Examples thereof include organic acid salts such as acid salts, gluconates and caprylates. Examples of the metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, zinc salt and the like. Examples of the ammonium salt include salts such as ammonium and tetramethylammonium. Examples of the organic amine addition salt include salts such as morpholine and piperidine. Examples of the amino acid addition salt include salts with arginine, lysine and the like. The aspartic acid used in the present invention may be at least one selected from the group consisting of L-aspartic acid, D-aspartic acid, and salts thereof.

Aspartic acid or a salt thereof used in the present invention can be produced by a conventional method. For example, a free form of aspartic acid or a salt thereof can be obtained by a method of isolating and purifying from animals and plants containing them, chemical synthesis, fermentative production and the like. Alternatively, a commercial product may be purchased.

In the present invention, the target to which the aspartic acid is applied includes the spores of the Paenibacillus bacterium described above, or a substance containing or may contain the spores of the Paenibacillus bacterium. Preferably, the aspartic acid in the present invention contains or may contain Paenibacillus bacterial spores, a substance that wants to suppress the germination of Paenibacillus bacterial spores, a substance that wants to suppress the growth of Paenibacillus bacteria, and Paenibacillus. It can be applied to substances that want to prevent bacterial contamination by bacteria. Examples of such applicable substances include foods, their packaging or storage containers, fertilizers or microbial preparations containing Paenibacillus bacteria, liquids or biofilms containing or may contain Paenibacillus bacteria, and the like. Be done.

As used herein, "food" includes solid, semi-solid or liquid foods, beverages, feeds, pet foods and the like, and their raw materials. Examples of the foods include breads, noodles, rice, sweets such as cookies, tablets, jellies, dairy products, soups, frozen foods, instant foods, processed starch products, processed fish meat products, and other processed foods. , Seasonings, nutritional supplements, and beverages such as tea beverages, coffee beverages, dairy beverages, fruit beverages, near water, carbonated beverages, jelly-like beverages, and the like.

As used herein, "fertilizer" includes forms known in the art, such as solid, liquid or powder fertilizers. Preferably, the fertilizer is an organic biofertilizer. In addition to the above-mentioned Paenivacillus bacteria, the fertilizers include organic fertilizer materials, micronutrient fertilizer materials, pesticides, herbicides, plant growth improvers, fungicides, shell-killing agents, algae-killing agents, bacterial inoculum materials, and fungal inoculum. It may further include materials, or combinations thereof, and the like. The fertilizer may be applied to a plant growth medium (eg, soil, solution, etc.) or directly to the plant or seed.

In the present specification, the "microbial preparation" includes a microbial preparation generally used in the field of horticulture or agriculture for the purpose of promoting plant growth, controlling biological effects, and the like. The microbial preparation has the same form as that of a normal horticultural or agricultural microbial preparation (for example, in the form of powder, wettable powder, granule, emulsion, liquid, suspension, flowable agent, coating agent, etc.). good. The microbial preparation may contain the culture solution, high-concentration product or dried product of the Paenibacillus bacterium alone, or in addition to the Paenibacillus bacterium, to plants such as other microorganisms, solid carriers and auxiliary agents. It may further contain other optional components to which the application of. The microbial preparation may be applied to a plant growth medium (for example, soil, solution, etc.), may be applied to fertilizer or water given to a plant, or may be applied directly to a plant or seed.

The Paenibacillus bacterium used for the purpose of promoting plant growth or controlling the organism in the fertilizer and microbial preparation is preferably Paenibacillus polymixa, Paenibacillus tibensis, Paenibacillus glucanoricus, Paenibacillus maciliensis, Paenibacillus. Popiliae, Paenibacillus albay, etc. can be mentioned.

When suppressing the sprouting of bacterial follicles of the genus Paenibacillus contained in the above-mentioned applicable substance, aspartic acid is added to the substance, a liquid containing aspartic acid is applied or sprayed on the surface of the substance, or aspartic acid. The substance may be immersed in a liquid containing. When suppressing the germination of Paenibacillus bacterial spores present in foods, fertilizers or microbial preparations, it is preferable to add aspartic acid to the foods, fertilizers or microbial preparations. When suppressing the germination of bacterial spores of the genus Paenibacillus present in a food package or storage container, it is preferable to apply or spray a liquid containing aspartic acid on the surface of the package or storage container to attach the spores. When suppressing the germination of bacterial spores of the genus Paenibacillus present in the biofilm, the biofilm is coated with or sprayed with a liquid containing aspartic acid, or the biofilm is immersed in the liquid containing aspartic acid. It is preferable to allow aspartic acid to permeate the inside of the biofilm.

In the present invention, the applicable concentration of aspartic acid may be an effective amount capable of suppressing the germination of Paenibacillus bacterial spores in the application target, and is not particularly limited, but is preferably 2 mM or more, more preferably 3 mM or more, and further. It is preferably 5 mM or more, and on the other hand, from the viewpoint of economic efficiency, it is preferably 300 mM or less, more preferably 30 mM or less (aspartic acid free form conversion, the same applies hereinafter in the present specification). Examples of the applicable concentration range of aspartic acid in the present invention include 2 to 300 mM, 2 to 30 mM, 3 to 300 mM, 3 to 30 mM, 5 to 300 mM and 5 to 30 mM.

Therefore, according to a preferred embodiment of the present invention, for example, the concentration of aspartic acid in the substance to be applied such as food, fertilizer, microbial preparation, etc. is 0.027 to 4.0% by mass, 0. 027 to 0.40% by mass, 0.040 to 4.0% by mass, 0.040 to 0.40% by mass, 0.067 to 4.0% by mass, or 0.067 to 0.40% by mass. Aspartic acid may be added as described above. Alternatively, when the applicable substance is a food package or storage container, 0.027 to 4.0% by mass, 0.027 to 0.40% by mass, 0.040 to 4.0% by mass, 0.040. A liquid containing ~ 0.40% by mass, 0.067 to 4.0% by mass, or 0.067 to 0.40% by mass of aspartic acid is applied or sprayed on the substance, or the substance is applied to the liquid. The liquid may be attached to the surface of the substance by immersing it. Alternatively, when the substance to be applied is a biofilm, a liquid containing aspartic acid is applied or sprayed on the substance, or the substance is immersed in the liquid, and the concentration of aspartic acid in the substance is 0. 027 to 4.0% by mass, 0.027 to 0.40% by mass, 0.040 to 4.0% by mass, 0.040 to 0.40% by mass, 0.067 to 4.0% by mass, or 0 It may be set to 0.067 to 0.40% by mass.

In a preferred embodiment, the concentration of aspartic acid in the agent for suppressing the germination of Paenibacillus spores, the agent for suppressing the growth of Paenibacillus bacteria, and the agent for preventing microbial contamination by Paenibacillus bacteria is preferably 2 to 300 mM, 2 to 2. It is 30 mM, 3 to 300 mM, 3 to 30 mM, 5 to 300 mM or 5 to 30 mM.

In the present invention, the aspartic acid may be used in combination with another substance having an action of suppressing germination of Paenibacillus spores. Examples of such other substances include indole and its analogs. Furthermore, the aspartic acid of the present invention may be used in combination with other substances as long as the action of suppressing the germination of Paenibacillus spores is not impaired. For example, it can be used in combination with an existing bactericidal agent, preservative, or the like.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited thereto.

(Method)
(1) Preparation of spore culture solution The bacteria of the genus Paenibacillus were Paenibacillus polymyxa ATCC39564, which was distributed from the American Type Culture Collection (ATCC), and Paenibacillus sp. , And any of Paenibacillus glucanolyticus KMC129, which was separated from the raw material by a conventional method and identified by the sequence of 16S ribosomal RNA, was used. The above-mentioned Paenibacillus bacteria were cultured overnight in TSB medium (BACTO Tryptic Soy broth: Becton, Dickinson and Company, catalog # 21182). Then, the culture medium of Paenibacillus polymyxa ATCC39564 and Paenibacillus sp. KMC150 was mixed with 2 × SGG (Schaeffer's glucose supplemented with 1% [wt / vol] glycerol) medium (composition is Journal of) so that _{OD 600 = 0.01.} (According to bacteriology, 2007, 189 (13): 4920-4931) Suspended in 100 mL. JASCO's V-530 was used for the measurement of OD _600. In addition, the culture medium of Paenibacillus glucanolyticus KMC129 was prepared according to DS (Difco suspension) medium (composition is Molecular biological methods for Bacillus, CR Harwood, and SM Cutting (eds.), Wiley, 1990 so that _{OD 600 = 0.01.} ) Suspended in 100 mL. A suspension of Paenibacillus polymyxa ATCC39564 was allowed to stand in a 500 mL beaker at 30 ° C. for 200 hours to obtain a culture solution containing a biofilm containing spores. In addition, the suspension of each strain was cultured in a 500 mL Erlenmeyer flask with blades at 30 ° C. and 150 rpm with shaking (60 hours for Paenibacillus polymyxa ATCC39564, 50 hours for Paenibacillus glucanolyticus KMC129, 96 hours for Paenibacillus sp. KMC150). A culture medium containing suspended cells containing Paenibacillus bacterial blasts was obtained.

(2) Preparation of purified spore solution The culture solution of (1) was observed with a phase-contrast microscope to confirm the formation of spores. Spores were purified from the culture medium of each bacterium. Paenibacillus polymyxa spores are collected by centrifuging the culture solution at 4 ° C., 9000 G for 15 minutes, then suspending the collected bacteria in sterile water and centrifuging at 4 ° C., 9000 G, 15 minutes to spores. Purified by precipitation. The spores of Paenibacillus sp. Or Paenibacillus glucanolyticus were collected by centrifuging the culture solution at 4 ° C, 3000 G for 15 minutes, and then the collected bacteria were suspended in sterile water and centrifuged at 4 ° C, 7000 G, 15 minutes. Purification was performed by separating and precipitating spores. The procedure for purifying spores was repeated until 90% or more of the cells became spores after removing cell debris and vegetative cells when the purified product was observed with a phase contrast microscope. The obtained pellets of spores were suspended in cold sterilized water, and this was used as a spore purification solution. The purified solution was stored at 4 ° C. until use.

(3) Culture and spore observation in the presence of aspartic acid L-aspartic acid (L-Asp) or D in the spore purification solution of Paenibacillus polymyxa derived from the biofilm (BF) or suspension culture obtained in (2). -Nutrient Broth containing aspartic acid (D-Asp) was added (final concentration of L-Asp was 0, 1, 2, 3, 4, 5 or 30 mM, final concentration of D-Asp was 0 or 30 mM) and tested. The cells were cultured in a tube at 30 ° C. The spore purification solution of Paenibacillus sp. Or Paenibacillus glucanolyticus obtained in (2) was heated at 80 ° C. for 15 minutes and then aGFK solution (100 mM L) containing 0 or 30 mM L-Asp. -Asparagine, 100 mM D-glucose, 10 mM D-fructose, 100 mM KCl, 10 mM Tris-HCl; pH 8.4), or 1/2 AGFK solution containing 0 or 30 mM D-Asp and in vitro. It was cultured at 30 ° C. Germinated spores are observed as black spores by phase contrast microscopy. Therefore, the spore culture medium was observed using a phase-contrast microscope 30 minutes after culturing and after 16, 24 and 28 hours. In addition, when spores germinate, they absorb water and the turbidity decreases. _{Therefore, the turbidity OD 660} of the spore culture solution was measured after 0 to 120 minutes of culturing, and the turbidity change rate based on the initial turbidity (0 minutes of culturing) was calculated and used as an index of germination efficiency. A miniphoto 518R manufactured by TAITEC was used for the measurement of OD _660.

(Example 1) Suppression of germination of Paenibacillus polymyxa spores by L-Asp Phase-contrast microscopic observation of BF-derived Paenibacillus polymyxa ATCC39564 spores cultured in the presence (30 mM) and non-existence (0 mM) of L-Asp for 30 minutes and 28 hours. Are shown in FIGS. 1 and 2, respectively. In both 30 minutes and 28 hours after culturing, the proportion of Dark spore (germinated spores) in the total spores was lower in the presence of L-Asp than in the absence.

Table 1 shows the results of turbidity measurement of the BF-derived Paenibacillus polymyxa ATCC39564 spore culture medium cultured in the presence or absence of L-Asp. It was shown that the decrease in turbidity due to culture was suppressed in the presence of L-Asp of 2 mM or more, and the germination of spores was inhibited.

Phase-contrast microscopic images of suspension-cultured Paenibacillus polymyxa ATCC39564 spores cultured in the presence (3-30 mM) or in the absence (0 mM) of L-Asp for 30 minutes and 28 hours are shown in FIGS. 3 and 4, respectively. In both 30 minutes and 28 hours after culturing, the proportion of Dark spores in the total spores decreased in the presence of L-Asp as compared with the absence. Furthermore, after 30 minutes of culturing, the proportion of Dark spore in whole spores decreased in a concentration-dependent manner of L-Asp.

(Example 2) Suppression of germination of Paenibacillus polymyxa spores by D-Asp Phase-contrast microscopic observation of Paenibacillus polymyxa ATCC39564 spores cultured in the presence (30 mM) and non-existence (0 mM) of D-Asp (culture 30 minutes) Later) is shown in FIG. Spore germination was suppressed in the presence of D-Asp.

(Example 3) Suppression of germination of Paenibacillus sp. Spores by L-Asp Phase-contrast microscopic observation image (culture) of Paenibacillus sp. KMC150 spores derived from floating cells cultured in the presence (30 mM) and in the absence (0 mM) of L-Asp. After 24 hours) is shown in FIG. Spore germination was suppressed in the presence of L-Asp.

(Example 4) Suppression of germination of Paenibacillus sp. Spores by D-Asp Phase-difference microscopic observation image (culture) of Paenibacillus sp. KMC150 spores derived from floating cells cultured in the presence (30 mM) and in the absence (0 mM) of D-Asp. 16 hours later) is shown in FIG. Spore germination was suppressed in the presence of D-Asp.

(Example 5) Suppression of germination of Paenibacillus glucanolyticus spores by L-Asp Phase-contrast microscopic observation of Paenibacillus glucanolyticus KMC129 spores cultured in the presence (30 mM) and non-existence (0 mM) of L-Asp (24 hours of culture) Later) is shown in FIG. Spore germination was suppressed in the presence of L-Asp.

(Example 6) Suppression of germination of Paenibacillus glucanolyticus spores by D-Asp Phase-contrast microscopic observation of Paenibacillus glucanolyticus KMC129 spores cultured in the presence (30 mM) and non-existence (0 mM) of D-Asp (culture 16 hours) Later) is shown in FIG. Spore germination was suppressed in the presence of D-Asp.

Claims (10)

A germination inhibitor for Paenibacillus bacterial spores containing aspartic acid as an active ingredient. A growth inhibitor for Paenibacillus bacteria containing aspartic acid as an active ingredient. An inhibitor of microbial contamination by Paenibacillus bacteria containing aspartic acid as an active ingredient. An agent for improving the stability of fertilizers or microbial preparations containing Paenibacillus bacteria containing aspartic acid as an active ingredient. The agent according to any one of claims 1 to 4, wherein the Paenibacillus bacterium is Paenibacillus polymixa, Paenibacillus sp., Or Paenibacillus glucanoriticus. A method for suppressing germination of Paenibacillus bacterial spores, which comprises applying aspartic acid to Paenibacillus bacterial spores. A method for suppressing the growth of Paenibacillus bacteria, which comprises applying aspartic acid to Paenibacillus bacterium spores. A method for preventing contamination of a substance by Paenibacillus bacteria, which comprises applying aspartic acid to a substance containing or potentially containing Paenibacillus spores. A method for improving the stability of a fertilizer or microbial preparation containing Paenibacillus bacterial spores, which comprises applying aspartic acid to a fertilizer or microbial preparation containing Paenibacillus bacterial spores. The method according to any one of claims 6 to 9, wherein the Paenibacillus bacterium is Paenibacillus polymixa, Paenibacillus sp., Or Paenibacillus glucanoriticus.

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