Biostimulant and method for producing same

Abstract

Provided is a method for producing a biostimulant using a bone tissue as a raw material. A first production method according to the present disclosure comprises: step S11, in which a bone tissue is brought into contact, in a solution, with a substance containing an anion capable of forming a poorly soluble calcium salt; step S12, in which the poorly soluble calcium salt formed in the step S11 is removed; step S13, in which the solution obtained in the step S12 is brought into contact with a substance containing a cation capable of forming a poorly soluble phosphoric acid salt; and step S14, in which the poorly soluble phosphoric acid salt formed in the step S13 is removed. A second production method according to the present disclosure comprises: step S15, in which a bone tissue is brought into contact with a strong acid; step S16, in which the solution obtained in the step S15 is brought into contact with a base; and step S17, in which the poorly soluble phosphoric acid salt formed in the step S16 is removed.

Description

Biostimulant and method for producing the same

[0001] The present invention relates to a biostimulant and a method for producing the same.

[0002] Conventionally, fertilizers using bone tissue as a raw material and methods for producing the same are known (see, for example, Patent Document 1).

[0003] International Publication No. 2023 / 167166

[0004] Patent Document 1 discloses a fertilizer using bone tissue as a raw material, but does not disclose processing the fertilizer into further other products.

[0005] An aspect of the present invention aims to provide a biostimulant using bone tissue as a raw material and a method for producing the same.

[0006] The present invention includes the following aspects: <1> A method for producing a biostimulant comprising the following steps: Step S11: A step of contacting bone tissue with a substance containing an anion that can form a sparingly soluble calcium salt in a solution; Step S12: A step of removing the sparingly soluble calcium salt formed in step S11; Step S13: A step of contacting the solution obtained in step S12 with a substance containing a cation that can form a sparingly soluble phosphate; Step S14: A step of removing the sparingly soluble phosphate formed in step S13. <2> A method for producing a biostimulant comprising the following steps: Step S15: A step of contacting bone tissue with a strong acid; Step S16: A step of contacting the solution obtained in step S15 with a base; Step S17: A step of removing the sparingly soluble phosphate formed in step S16. <3> The manufacturing method according to <1>, wherein the substance containing anion capable of forming the above-mentioned sparingly soluble calcium salt is sulfuric acid, sulfate, tartaric acid, tartrate, citric acid, citrate, maleic acid, maleate, malic acid, malate, or any combination thereof. <4> The manufacturing method according to <1> or <3>, wherein the substance containing a cation capable of forming the above-mentioned sparingly soluble phosphate is a calcium salt, magnesium salt, ammonium salt, aluminum salt, or any combination thereof. <5> The manufacturing method according to <2>, wherein the above-mentioned strong acid is hydrochloric acid, nitric acid, sulfuric acid, or any combination thereof. <6> The manufacturing method according to <2> or <5>, wherein the above-mentioned base is sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium hydroxide, or any combination thereof. <7> The manufacturing method according to any one of <1> to <6>, wherein in the above-mentioned biostimulant, the calcium concentration is 400 mmol / L or less, and the phosphoric acid concentration is 400 mmol / L or less. <7a> A biostimulant obtained by the manufacturing method described in any of <1> to <7>.<8> A biostimulant obtained by removing at least a portion of calcium and phosphate from bone tissue, wherein the calcium concentration is 400 mmol / L or less and the phosphate concentration is 400 mmol / L or less. <9> The biostimulant described in <8> for use in one or more of the following uses: (i) to elongate plant roots; (ii) to increase the root mass of plants; (iii) to increase the weight of plants; (iv) to promote plant growth; (v) to improve the taste of plants.

[0007] The present invention also includes the following embodiments: <1A> A method for producing a biostimulant, comprising the step of removing calcium and phosphate from bone tissue in a solution. <2A> A method for producing the biostimulant according to <1A>, comprising the steps of removing calcium and phosphate from the bone tissue as follows: Step S11: A step of contacting bone tissue with a substance containing an anion that can form a sparingly soluble calcium salt in a solution; Step S12: A step of removing the sparingly soluble calcium salt formed in step S11; Step S13: A step of contacting the solution obtained in step S12 with a substance containing a cation that can form a sparingly soluble phosphate; Step S14: A step of removing the sparingly soluble phosphate formed in step S13. <3A> A method for producing the biostimulant described in <1A>, comprising the following steps to remove calcium and phosphate from the bone tissue: Step S15: A step of contacting the bone tissue with a strong acid; Step S16: A step of contacting the solution obtained in step S15 with a base; Step S17: A step of removing the sparingly soluble phosphate formed in step S16. <4A> The method for producing the biostimulant described in <2A>, wherein the substance containing anion capable of forming the sparingly soluble calcium salt is sulfuric acid, sulfate, tartaric acid, tartrate, citric acid, citrate, maleic acid, maleate, malic acid, malate, or any combination thereof. <5A> The method for producing the biostimulant described in <2A> or <4A>, wherein the substance containing a cation capable of forming the sparingly soluble phosphate is calcium salt, magnesium salt, ammonium salt, aluminum salt, or any combination thereof. <6A> The method for producing the biostimulant described in <3A>, wherein the strong acid is hydrochloric acid, nitric acid, sulfuric acid, or any combination thereof. <7A> The manufacturing method according to <3A> or <6A>, wherein the base is sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium hydroxide, or any combination thereof. <8A> The manufacturing method according to any one of <1A> to <7A>, wherein in the biostimulant, the calcium concentration is 400 mmol / L or less, and the phosphoric acid concentration is 400 mmol / L or less.<9A> A biostimulant obtained by a manufacturing method described in any of <1A> to <8A>. <10A> A biostimulant obtained by removing at least a portion of calcium and phosphate from bone tissue, wherein the calcium concentration is 400 mmol / L or less and the phosphate concentration is 400 mmol / L or less. <11A> The biostimulant described in <10A> for use in one or more of the following uses: (i) to elongate plant roots; (ii) to increase the root mass of plants; (iii) to increase the weight of plants; (iv) to promote plant growth; (v) to improve the taste of plants.

[0008] According to one aspect of the present invention, a biostimulant using bone tissue as a raw material and a method for producing the same are provided.

[0009] This is a flowchart showing an example of a method for producing a biostimulant according to one aspect of the present invention. This is a flowchart showing an example of a method for producing a biostimulant according to another aspect of the present invention. This is a diagram showing the results of Example 2-1. It shows the appearance of frilly lettuce after the seedling period. This is a diagram showing the results of Example 2-1. It shows the appearance of the roots of frilly lettuce after the seedling period. This is a diagram showing the results of Example 2-2. It shows the weight of frilly lettuce after the seedling period. This is a diagram showing the results of Example 2-3. This is a graph showing the rooting state of spray chrysanthemum cuttings.

[0010] One embodiment of the present invention is described below. However, the present invention is not limited to the configurations described below. The present invention can be modified in various ways within the scope of the claims. The technical scope of the present invention also extends to embodiments or examples obtained by appropriately combining the multiple technical means disclosed herein. In this case, the multiple technical means may be disclosed across multiple embodiments or examples.

[0011] Unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B".

[0012] In this specification, "biostimulant" refers to a substance that acts on the physiology of a plant to produce a favorable effect on that plant. In one embodiment, the biostimulant does not contain, or does not intentionally contain, chemical components essential for the plant to maintain its life activities, i.e., fertilizer components (nitrogen, phosphorus, potassium, etc.). In this respect, biostimulants differ from fertilizers. However, it is possible that a biostimulant may be included as one of the components in a fertilizer that is a blend of various components. Furthermore, it cannot be denied that the biostimulants described herein may fall under the definitions of fertilizers, pesticides, or soil conditioners according to various regulatory laws.

[0013] In this specification, substances containing anions that can form sparingly soluble calcium salts are sometimes abbreviated as "calcium-removing substances." In this specification, substances containing cations that can form sparingly soluble phosphates are sometimes abbreviated as "phosphate-removing substances."

[0014] In this specification, the term "ion" may be omitted from the names of substances. For example, "phosphate ion" may be expressed as "phosphate," and "calcium ion" as "calcium." Whether these substances exist in ionic form will be easily understood by those skilled in the art. For example, substances contained in aqueous solutions may exist in ionic form. In one embodiment, phosphate is a phosphate ion. In one embodiment, calcium is a calcium ion.

[0015] In one embodiment, "slowly soluble calcium salt" refers to a calcium salt whose solubility in 100 mL of water at 25°C is 1.0 g or less, 0.5 g or less, 0.4 g or less, or 0.3 g or less. In another embodiment, "slowly soluble phosphate" refers to a phosphate salt whose solubility in 100 mL of water at 25°C is 1.0 g or less, 0.5 g or less, 0.4 g or less, or 0.3 g or less.

[0016] [1. Method for Producing Biostimulants (Part 1)] The method for producing biostimulants according to one aspect of the present invention will be described below with reference to the illustrative Figure 1. In this method, at least a portion of calcium is first removed from bone tissue (steps S11 and S12), and then at least a portion of phosphate is removed (steps S13 and S14). Bone tissue mainly consists of calcium phosphate, but also contains peptides, amino acids, vitamins, fatty acids, sugars, trace metals, etc., and it is thought that these components function as biostimulants.

[0017] [1.1. Step S11] In step S11, bone tissue is brought into contact with a substance containing anions that can form sparingly soluble calcium salts (calcium removal substance) in a solution. This converts the calcium contained in the bone tissue into sparingly soluble calcium salts. Only one type of calcium removal substance may be used, or two or more types may be used in combination.

[0018] Anions that can form sparingly soluble calcium salts include sulfate ions, tartrate ions, citrate ions, maleate ions, and malate ions. Therefore, examples of calcium removal substances in step S11 include acids or salts containing these anions. Examples of acids include sulfuric acid, tartaric acid, citric acid, maleic acid, and malic acid. Examples of salts include sulfates, tartrates, citrates, maleates, and malates. These salts may be sodium salts, potassium salts, magnesium salts, ferric salts, ammonium salts, etc. Tartaric acid, tartrates, malic acid, and malates may be D-isomers, L-isomers, or mixtures thereof.

[0019] In one embodiment, the calcium-removing substance is one or more selected from the group consisting of sulfuric acid, sulfates, tartaric acid, tartrate, citric acid, citrate, maleic acid, maleate, malic acid, and malate. Examples of sulfates include sodium sulfate, sodium bisulfate, potassium sulfate, potassium bisulfate, sodium potassium sulfate, ammonium sulfate, ammonium bisulfate, and magnesium sulfate. Examples of tartrates include sodium tartrate, sodium bisulfate, potassium tartrate, potassium bisulfate, sodium potassium tartrate, ammonium tartrate, and magnesium tartrate. Examples of citrates include trilithium citrate, dilithium hydrogen citrate, lithium dihydrogen citrate, trisodium citrate, disodium hydrogen citrate, dipotassium citrate, dipotassium hydrogen citrate, dipotassium citrate, triammonium citrate, diammonium hydrogen citrate, ammonium dihydrogen citrate, and ammonium iron citrate. An example of a maleate is disodium maleate. Examples of malate salts include sodium malate, disodium malate, potassium malate, dipotassium malate, and ammonium hydrogen malate. In one embodiment, the calcium removal substance is one or more selected from the group consisting of tartaric acid, citric acid, maleic acid, potassium bisulfate, and malic acid.

[0020] The bone tissue used as raw material in step S11 may be derived from any organism. Examples of organisms include mammals, birds, amphibians, and fish. In order to mass-produce fertilizer, it is preferable to be able to obtain a large quantity of bone tissue to be used as raw material for fertilizer, and bone tissue from livestock (cattle, pigs, sheep, chickens, etc.) or fish is suitably used. Bone tissue that has been heated (e.g., by steaming) or frozen may be used as raw material. Alternatively, bone tissue that has been shredded or crushed may be used as raw material. Calcium and phosphate can be removed efficiently from such bone tissue, which may shorten the manufacturing time.

[0021] Examples of solvents for the solution used to bring bone tissue into contact with the calcium-removing substance include water, lower alcohols, glycerol, propane-1,2-diol, and 1,3-propanediol. A mixed solvent, obtained by mixing two or more solvents in appropriate proportions, may also be used.

[0022] In step S11, the concentration of the calcium removal substance may be appropriately selected by a person skilled in the art. The lower limit of the concentration of the calcium removal substance may be 0.01 mol / L or more, 0.05 mol / L or more, 0.1 mol / L or more, 0.2 mol / L or more, 0.3 mol / L or more, 0.4 mol / L or more, 0.5 mol / L or more, 0.6 mol / L or more, 0.7 mol / L or more, 0.8 mol / L or more, 0.9 mol / L or more, 1.0 mol / L or more, 1.5 mol / L or more, or 2.0 mol / L or more. The upper limit of the concentration of the calcium-removing substance may be 5.0 mol / L or less, 4.5 mol / L or less, 4.0 mol / L or less, 3.5 mol / L or less, 3.0 mol / L or less, 2.5 mol / L or less, 2.0 mol / L or less, 1.5 mol / L or less, 1.0 mol / L or less, 0.9 mol / L or less, 0.8 mol / L or less, 0.7 mol / L or less, 0.6 mol / L or less, 0.5 mol / L or less, 0.4 mol / L or less, 0.3 mol / L or less, 0.2 mol / L or less, or 0.1 mol / L or less.

[0023] In step S11, the contact time may be appropriately selected by a person skilled in the art. The lower limit of the contact time may be 1 minute or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, or 48 hours or more. The upper limit of the contact time may be 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2.5 days or less, 2 days or less, 1.5 days or less, 1 day or less, 18 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less.

[0024] [1.2. Step S12] In step S12, the sparingly soluble calcium salt formed in step S11 is removed. In one embodiment, the sparingly soluble calcium salt forms a precipitate. The sparingly soluble calcium salt can be removed by centrifugation, filtration, or pressing.

[0025] By going through step S12, at least a portion of the calcium contained in the bone tissue is removed. A higher calcium removal rate is preferable, and may be 70% by weight or more, 75% by weight or more, 80% by weight or more, 85% by weight or more, 90% by weight or more, or 95% by weight or more. Here, the calcium removal rate is calculated based on the amount of calcium that dissolves into the supernatant when the same amount of bone tissue is immersed in 30 mL of hydrochloric acid (0.8 N) at room temperature (20°C) for 48 hours. Therefore, the calcium removal rate in step S12 is determined by the following formula: Calcium removal rate (by weight) = {(Calcium content in the supernatant of the hydrochloric acid treatment - Calcium content in the solution obtained after step S12) ÷ Calcium content in the supernatant of the hydrochloric acid treatment} × 100

[0026] [1.3. Step S13] In step S13, the solution obtained in step S12 is brought into contact with a substance containing a cation capable of forming a sparingly soluble phosphate (phosphate removal substance). This converts the phosphoric acid contained in the solution into a sparingly soluble phosphate. Only one type of phosphate removal substance may be used, or two or more types may be used in combination. In one embodiment, the phosphoric acid before contact with the phosphate removal substance is dissolved in the solution as phosphate ions.

[0027] Examples of cations that can form sparingly soluble phosphates include calcium ions, magnesium ions, ammonium ions, and aluminum ions. Therefore, examples of phosphate removal substances in step S13 include salts or hydroxides containing these cations. Examples of salts include calcium salts, magnesium salts, ammonium salts, and aluminum salts. Specific examples of calcium salts include calcium carbonate and calcium bicarbonate. Specific examples of magnesium salts include magnesium chloride and magnesium carbonate. An example of an ammonium salt is ammonium chloride. Examples of hydroxides include calcium hydroxide, magnesium hydroxide, aluminum hydroxide, and ammonium hydroxide.

[0028] In step S13, the concentration of the phosphate removal substance may be appropriately selected by a person skilled in the art. The lower limit of the concentration of the phosphate removal substance may be 0.01 mol / L or more, 0.05 mol / L or more, 0.1 mol / L or more, 0.2 mol / L or more, 0.3 mol / L or more, 0.4 mol / L or more, 0.5 mol / L or more, 0.6 mol / L or more, 0.7 mol / L or more, 0.8 mol / L or more, 0.9 mol / L or more, 1.0 mol / L or more, 1.5 mol / L or more, or 2.0 mol / L or more. The upper limit of the concentration of the phosphate removal substance may be 5.0 mol / L or less, 4.5 mol / L or less, 4.0 mol / L or less, 3.5 mol / L or less, 3.0 mol / L or less, 2.5 mol / L or less, 2.0 mol / L or less, 1.5 mol / L or less, 1.0 mol / L or less, 0.9 mol / L or less, 0.8 mol / L or less, 0.7 mol / L or less, 0.6 mol / L or less, 0.5 mol / L or less, 0.4 mol / L or less, 0.3 mol / L or less, 0.2 mol / L or less, or 0.1 mol / L or less.

[0029] In step S13, the contact time may be appropriately selected by a person skilled in the art. The lower limit of the contact time may be 1 minute or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, or 48 hours or more. The upper limit of the contact time may be 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2.5 days or less, 2 days or less, 1.5 days or less, 1 day or less, 18 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less.

[0030] [1.4. Step S14] In step S14, the sparingly soluble phosphate formed in step S13 is removed. In one embodiment, the sparingly soluble phosphate forms a precipitate. The sparingly soluble phosphate can be removed by centrifugation, filtration, or pressing.

[0031] By going through step S14, at least a portion of the phosphoric acid contained in the solution is removed. A higher phosphoric acid removal rate is preferable, and may be 70% by weight or more, 75% by weight or more, 80% by weight or more, 85% by weight or more, 90% by weight or more, or 95% by weight or more. Here, the phosphoric acid removal rate is calculated based on the amount of phosphoric acid that dissolves into the supernatant when the same amount of bone tissue is immersed in 30 mL of hydrochloric acid (0.8 N) at room temperature (20°C) for 48 hours. Therefore, the phosphoric acid removal rate in step S14 is determined by the following formula: Phosphoric acid removal rate (by weight) = {(Phosphoric acid content in the supernatant of the hydrochloric acid treatment - Phosphoric acid content in the solution obtained after step S14) ÷ Phosphoric acid content in the supernatant of the hydrochloric acid treatment} × 100

[0032] As a result, after step S14, a substance is obtained from which at least some of the calcium and phosphate have been removed from the bone tissue. This substance has low levels of nitrogen, phosphate, and potassium, the three major nutrients for plants, and therefore has little function as a fertilizer. Instead, this substance can act on the physiology of plants and can exert various effects (such as promoting root elongation and increasing root mass). In other words, a biostimulant can be produced by going through steps S11 to S14.

[0033] In one embodiment, the biostimulant obtained by this manufacturing method has a low content of calcium and phosphate. The calcium concentration or phosphate concentration in the biostimulant can be independently 400 mmol / L or less, 300 mmol / L or less, 200 mmol / L or less, 100 mmol / L or less, 90 mmol / L or less, 80 mmol / L or less, 70 mmol / L or less, 60 mmol / L or less, 50 mmol / L or less, 40 mmol / L or less, 30 mmol / L or less, 20 mmol / L or less, or 10 mmol / L or less. In one embodiment, the calcium concentration is the concentration of calcium ions. In one embodiment, the phosphate concentration is the concentration of phosphate ions.

[0034] [1.5. Other Processes] In addition to steps S11 to S14, the method for producing a biostimulant according to one embodiment of the present invention may further include steps that are normally performed in the production of a biostimulant. Examples of such steps include a pretreatment step, a filtration step, a component addition step, a concentration step, a dilution step, a cooling step, a freezing step, a heating step, a drying step, a granulation step, a grinding step, a coating granulation step, and a packaging step.

[0035] The pretreatment step is a step of pretreatment of the bone tissue before subjecting it to step S11. Examples of pretreatment include heating the bone tissue, heating the bone tissue under pressure, and irradiating the bone tissue with microwaves. Multiple pretreatments may be combined.

[0036] The filtration process is a process that removes components whose particle size falls within a predetermined range. By using a filter with an appropriate pore size in the filtration process, at least some of insoluble matter, suspended solids, bacteria, fungi, and other elements can be removed.

[0037] The component addition process is a process of adding additional components. By including a component addition process, it becomes possible to produce biostimulants with a suitable composition depending on the plant being cultivated and the desired function. Examples of components added in the component addition process include potassium components (potassium oxide, potassium hydroxide, potassium chloride, potassium sulfate, etc.), nitrogen components (urea, ammonium nitrate, etc.), phosphorus components (phosphate, ammonium phosphate, etc.), magnesium components (magnesium phosphate, magnesium chloride, magnesium sulfate, etc.), vitamins, manganese, boron, iron, copper, zinc, and molybdenum. The product obtained through the component addition process may be a fertilizer, pesticide, or soil conditioner.

[0038] The concentration step is a step to increase the concentration of the biostimulant. The dilution step is a step to dilute the concentration of the biostimulant. By appropriately incorporating a concentration step or a dilution step, the concentration of the biostimulant can be brought within a desired range.

[0039] The cooling process is a process of lowering the temperature of the biostimulant. The freezing process is a process of freezing the biostimulant. By going through these processes, it is possible to remove components that become insoluble at low temperatures, for example. As a result, the effect of the biostimulant can be enhanced or its concentration can be adjusted.

[0040] The heating process involves raising the temperature of the biostimulant. This heating process can kill microorganisms and fungi, for example. As a result, the effectiveness and safety of the biostimulant can be enhanced.

[0041] The drying process removes excess moisture from the biostimulant. Through this drying process, a solid or paste-like biostimulant is obtained.

[0042] The granulation step is a step of granulating a biostimulant. A solid or paste-like biostimulant can be produced through the granulation step.

[0043] The pulverization step is a step of cutting and pulverizing a solid biostimulant into a size and shape that are easy to use.

[0044] The coating granulation step is a step of coating and granulating a solid biostimulant. For example, coating a biostimulant with a silicic acid compound or the like can prevent the biostimulant from being washed away, and prevent damage to the biostimulant caused by impact.

[0045] The packaging step is a step of packaging the biostimulant in a container so that it can be distributed or sold. In the packaging step, the biostimulant may be combined with an instruction manual therefor. This instruction manual may be printed on the container, or may be prepared separately from the biostimulant packaged as a physical or electronic document. The instruction manual may describe the formulation, method of use, timing of use, target crops, and the like of the biostimulant.

[0046] [2. Method for producing biostimulant (part 2)] Next, a method for producing a biostimulant according to another aspect of the present invention will be described with reference to FIG. 2. In this production method, calcium phosphate in bone tissue is dissolved with a strong acid (step S15), then a base is added to form calcium phosphate and precipitate it (step S16). Further, at least part of the calcium phosphate precipitate is removed (step S17).

[0047] [2.1. Step S15] In step S15, bone tissue is brought into contact with a strong acid. Thereby, phosphoric acid and calcium contained in the bone tissue are dissolved into phosphate ions and calcium ions. In one embodiment, the strong acid is a strong acid that dissolves bone tissue. In one embodiment, bone tissue and the strong acid are brought into contact in a solution. Only one type of strong acid may be used, or two or more types may be used in combination.

[0048] Examples of strong acids include hydrochloric acid, nitric acid, and sulfuric acid.

[0049] In step S15, the concentration of the strong acid may be appropriately selected by a person skilled in the art. The lower limit of the concentration of the strong acid may be 0.01 mol / L or more, 0.05 mol / L or more, 0.1 mol / L or more, 0.2 mol / L or more, 0.3 mol / L or more, 0.4 mol / L or more, 0.5 mol / L or more, 0.6 mol / L or more, 0.7 mol / L or more, 0.8 mol / L or more, 0.9 mol / L or more, 1.0 mol / L or more, 1.5 mol / L or more, or 2.0 mol / L or more. The upper limit of the concentration of a strong acid may be 5.0 mol / L or less, 4.5 mol / L or less, 4.0 mol / L or less, 3.5 mol / L or less, 3.0 mol / L or less, 2.5 mol / L or less, 2.0 mol / L or less, 1.5 mol / L or less, 1.0 mol / L or less, 0.9 mol / L or less, 0.8 mol / L or less, 0.7 mol / L or less, 0.6 mol / L or less, 0.5 mol / L or less, 0.4 mol / L or less, 0.3 mol / L or less, 0.2 mol / L or less, or 0.1 mol / L or less.

[0050] In step S15, the contact time may be appropriately selected by a person skilled in the art. The lower limit of the contact time may be 1 minute or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, or 48 hours or more. The upper limit of the contact time may be 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2.5 days or less, 2 days or less, 1.5 days or less, 1 day or less, 18 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less.

[0051] [2.2. Step S16] In step S16, a base is added to the solution. Only one type of base may be used, or two or more types may be used in combination. This causes the phosphate ions and calcium ions dissolved in step S15 to combine and form calcium phosphate. In one embodiment, the calcium phosphate forms a precipitate. On the other hand, the biostimulant components derived from bone tissue that were eluted in step S15 remain in the solution.

[0052] By going through step S16, at least a portion of the calcium contained in the solution can be precipitated and removed. A higher calcium removal rate is preferable. In one embodiment, the calcium removal rate may be 70% by weight or more, 75% by weight or more, 80% by weight or more, 85% by weight or more, 90% by weight or more, or 95% by weight or more. Here, the calcium removal rate is calculated based on the amount of calcium that dissolves into the supernatant when the same amount of bone tissue is immersed in 30 mL of hydrochloric acid (0.8 N) at room temperature (20°C) for 48 hours. Therefore, the calcium removal rate in step S16 can be determined by the following formula: Calcium removal rate (by weight) = {(Calcium content in the supernatant of the hydrochloric acid treatment - Calcium content in the solution obtained after step S16) ÷ Calcium content in the supernatant of the hydrochloric acid treatment} × 100

[0053] Similarly, by going through step S16, at least a portion of the phosphoric acid contained in the solution precipitates and can be removed. A higher phosphoric acid removal rate is preferable. In one embodiment, the phosphoric acid removal rate may be 70% by weight or more, 75% by weight or more, 80% by weight or more, 85% by weight or more, 90% by weight or more, or 95% by weight or more. Here, the phosphoric acid removal rate is calculated based on the amount of phosphoric acid that dissolves into the supernatant when the same amount of bone tissue is immersed in 30 mL of hydrochloric acid (0.8 N) at room temperature (20°C) for 48 hours. Therefore, the phosphoric acid removal rate in step S16 can be determined by the following formula: Phosphoric acid removal rate (by weight) = {(Phosphoric acid content in the supernatant of the hydrochloric acid treatment - Phosphoric acid content in the solution obtained after step S16) ÷ Phosphoric acid content in the supernatant of the hydrochloric acid treatment} × 100

[0054] Examples of bases used in step S16 include sodium hydroxide, potassium hydroxide, ammonium hydroxide, and magnesium hydroxide.

[0055] [2.3. Step S17] In step S17, the sparingly soluble calcium phosphate generated in step S16 is removed. Calcium phosphate can be removed by centrifugation, filtration, or pressing.

[0056] As a result, after step S17, a substance is obtained in which at least some of the calcium and phosphate have been removed from the bone tissue. This substance has a low content of phosphate, one of the three major nutrients for plants. Instead, this substance can act on the physiology of plants and can have various effects (such as promoting root elongation and increasing root mass). In other words, a biostimulant can be produced by going through steps S15 to S17. The nitrogen and potassium content in the biostimulant is not particularly limited. For example, if nitric acid is used as the strong acid in step S15, a biostimulant with a high nitrogen content may be obtained. For example, if potassium hydroxide is used as the base in step S16, a biostimulant with a high potassium content may be obtained.

[0057] In one embodiment, the biostimulant obtained by this manufacturing method has a low content of calcium and phosphate. The calcium concentration or phosphate concentration in the biostimulant can be independently 400 mmol / L or less, 300 mmol / L or less, 200 mmol / L or less, 100 mmol / L or less, 90 mmol / L or less, 80 mmol / L or less, 70 mmol / L or less, 60 mmol / L or less, 50 mmol / L or less, 40 mmol / L or less, 30 mmol / L or less, 20 mmol / L or less, or 10 mmol / L or less. In one embodiment, the calcium concentration is the concentration of calcium ions. In one embodiment, the phosphate concentration is the concentration of phosphate ions.

[0058] [2.4. Other Steps] A method for producing a biostimulant according to another embodiment of the present invention may further include steps that are commonly performed in the production of a biostimulant, in addition to steps S15 to S17. Examples of such steps are those described in Section [1.5], and therefore, a further description is omitted in this section.

[0059] [3. Biostimulants] One embodiment of the present invention is a biostimulant. The biostimulant is obtained by removing at least a portion of calcium and at least a portion of phosphate from bone tissue. Therefore, the concentrations of calcium and phosphate in the biostimulant are low. The calcium concentration or the phosphate concentration can be independently 400 mmol / L or less, 300 mmol / L or less, 200 mmol / L or less, 100 mmol / L or less, 90 mmol / L or less, 80 mmol / L or less, 70 mmol / L or less, 60 mmol / L or less, 50 mmol / L or less, 40 mmol / L or less, 30 mmol / L or less, 20 mmol / L or less, or 10 mmol / L or less. In one embodiment, the calcium concentration is the concentration of calcium ions. In one embodiment, the phosphate concentration is the concentration of phosphate ions.

[0060] In one embodiment, the concentrations of phosphate and calcium in the biostimulant are not limited. The composition of the biostimulant may be determined solely by components other than phosphate or calcium (for example, by the components described in the examples).

[0061] Another aspect of the present invention is a biostimulant obtained by the manufacturing method described in Section [1].

[0062] In one embodiment, the biostimulant contains a small amount of plant nutrients. The nitrogen concentration or potassium concentration in the biostimulant can be independently 200 mmol / L or less, 100 mmol / L or less, 90 mmol / L or less, 80 mmol / L or less, 70 mmol / L or less, 60 mmol / L or less, 50 mmol / L or less, 40 mmol / L or less, 30 mmol / L or less, 20 mmol / L or less, or 10 mmol / L or less. In one embodiment, the nitrogen concentration is the total concentration of nitrate ions and ammonium ions. In one embodiment, the potassium concentration is the concentration of potassium ions.

[0063] In one embodiment, the biostimulant contains a component derived from bone tissue. The lower limit of the concentration of the bone tissue-derived component in the biostimulant may be 1 nmol / L or more, 10 nmol / L or more, 100 nmol / L or more, 1 μmol / L or more, 10 μmol / L or more, 100 μmol / L or more, or 1 mmol / L or more, independently for each component. The upper limit of the concentration of the bone tissue-derived component in the biostimulant may be 100 mmol / L or less, 10 mmol / L or less, 1 mmol / L or less, 100 μmol / L or less, 10 μmol / L or less, or 1 μmol / L or less, independently for each component.

[0064] Examples of components derived from bone tissue include hydroxyproline or peptides containing it. The number of amino acids in a peptide containing hydroxyproline may be two, three, four, five, six, seven, eight, nine, ten, or more. Examples of such peptides include Ala-Hyp, Gly-Hyp, Pro-Hyp, and Gly-Pro-Hyp. Other examples of components derived from bone tissue include γ-carboxyglutamic acid and phosphate ethanolamine. Conversely, biostimulants containing these components are presumed to be made from bone tissue and may employ a manufacturing method according to one aspect of the present invention.

[0065] In one embodiment, the biostimulant does not fall under the category of fertilizer as defined in the Fertilizer Control Act. In one embodiment, the biostimulant does not fall under the category of pesticide as defined in the Agricultural Chemicals Control Act.

[0066] Biostimulants are substances, microorganisms, or mixtures thereof that support the natural functions of plants and the surrounding soil. When biostimulants are applied to plants or soil, they have a beneficial effect on the plants, regardless of the nutrients they contain. Examples include improved absorption of nutrients from the soil, improved efficiency of nutrient uptake and utilization, and improved resistance to abiotic stresses such as drought, high temperatures, and salinity. As a result, biostimulants improve the quality or yield of plants such as crops.

[0067] Known biostimulants that have been reported include amino acids, humic acids (humic acid, fulvic acid, etc.), seaweed extracts, microorganisms (Trichoderma, mycorrhizal fungi, yeast, Bacillus subtilis, rhizobia, etc.), minerals and vitamins, food residues, and chitin and chitosan. In addition to the biostimulant according to one aspect of the present invention, these known biostimulants may be used in combination. The biostimulant according to one aspect of the present invention and the known biostimulants may be used individually or in combination of two or more types.

[0068] The effects of biostimulants on plants are not particularly limited. Examples of such effects include: • Promotion or inhibition of physiological activities (photosynthesis, transpiration, root and stem elongation, flowering, fruiting, etc.) • Increase or enlargement of the plant or parts thereof (leaves, stems, roots, fruits, seeds, etc.) • Improved tolerance to various stresses (high temperature, low temperature, ultraviolet radiation, high salt concentration, drought, etc.) • Improved taste of edible parts (sweetness, umami, crunchiness, texture, etc.) • Activation and proliferation of beneficial soil microorganisms for plants (rhizobia, etc.) • Inactivation and reduction of harmful soil microorganisms for plants

[0069] In one embodiment, the biostimulant is intended for use in one or more of the following applications. In one embodiment, the biostimulant has one or more of the following functions: - Promotes the elongation of plant roots - Increases the root mass of plants - Increases the weight of plants (such as the weight of edible parts) - Promotes plant growth - Improves the taste of the edible parts of plants - Improves heat tolerance - Promotes symbiosis with symbiotic microorganisms (rhizobia).

[0070] The biostimulant may be a solid or a liquid. Since the substance obtained in step S14 or step S17 is a liquid, it can be easily processed into a liquid biostimulant. The liquid biostimulant can be easily applied to soil application, rice cultivation, hydroponics (hydroponics, solid substrate cultivation, or spray cultivation), foliar application, etc.

[0071] Biostimulants may or may not contain other components. Examples of other components include straw, bark, molasses, free amino acids (such as gamma-aminobutyric acid), fatty acids, sugars, plant growth hormones (such as auxin), and trace elements (such as magnesium, sulfur, iron, manganese, zinc, copper, boron, and molybdenum). Biostimulants may also be one component of the fertilizer.

[0072] [Example 1] [Example 1-1] A biostimulant was produced by removing calcium and phosphate from bone tissue using the method for producing a biostimulant (Part 1). The specific procedure is as follows: 1. 5 g of bone meal was immersed in 50 mL of tartaric acid aqueous solution at 20°C. The concentration of the tartaric acid aqueous solution was 0.6 mol / L, and the immersion time was 48 hours. Bovine bone meal X was used as the bone meal. 2. The supernatant was collected, and the precipitated calcium tartrate was removed. 3. By adding calcium hydroxide to the supernatant, the free phosphate was precipitated as calcium phosphate. The concentration of calcium hydroxide added was varied in the molar ratio with phosphate as shown in Table 1. 4. By removing the precipitate, a biostimulant was obtained from which calcium and phosphate had been partially removed from the bone meal. The concentrations of phosphate and calcium in the biostimulant were determined. The phosphate concentration and calcium ion concentration were quantified by ion chromatography.

[0073] The concentration of phosphoric acid was measured using a high-performance ion chromatography system IC-8100EX (Tosoh Corporation) connected to a TSKgel SuperIC-Anion HS (4.6 mm ID × 10 cm), under precise measurement conditions. A mixture of 7.5 mmol / L sodium bicarbonate and 0.8 mmol / L sodium carbonate was used as the eluent. The measurement temperature was 40°C, the flow rate was 1.5 mL / min, and the injection volume was 30 μL. The phosphoric acid concentration was determined by measuring the electrical conductivity (μS) and deriving a regression equation from the area of ​​the standard material. The phosphoric acid content (mg) was calculated based on the phosphoric acid concentration and is shown as the amount per gram of bone meal raw material.

[0074] Calcium concentration was measured using a high-performance ion chromatography system IC-8100EX (Tosoh Corporation) connected to a TSKgel SuperIC-Cation HSII (4.6 mm ID × 10 cm), under precise measurement conditions. A mixture of 3.0 mmol / L methanesulfonic acid and 2.7 mmol / L 18-crown-6-ether was used as the eluent. The measurement temperature was 40°C, the flow rate was 1.0 mL / min, and the injection volume was 30 μL. Calcium concentration was determined by measuring electrical conductivity (μS) and deriving a regression equation from the area of ​​the standard material. Calcium content (mg) was calculated based on the calcium concentration and expressed as the amount per gram of bone meal.

[0075] A baseline calcium concentration was determined for calculating the calcium removal rate. Bovine bone meal X was immersed in 30 mL of hydrochloric acid (0.8 N) at 20°C for 48 hours. The amount of calcium dissolved in the supernatant, converted to a value per gram of raw material bovine bone meal X, was 171 mg. Using this value as the baseline, the calcium removal rate can be calculated using the following formula: Calcium removal rate (weight %) = (171 - Calcium content of the obtained biostimulant (mg)) ÷ 171 × 100

[0076] The standard phosphate concentration used to calculate the phosphate removal rate was determined. Bovine bone meal X was immersed in 30 mL of hydrochloric acid (0.8 N) at 20°C for 48 hours. The amount of phosphate dissolved in the supernatant, converted to a value per gram of raw material bovine bone meal X, was 194 mg. Using this value as the standard, the phosphate removal rate can be calculated using the following formula: Phosphate removal rate (weight %) = (194 - Phosphate content of the obtained biostimulant (mg)) ÷ 194 × 100

[0077] (Results) The results are shown in Table 1.

[0078] As can be seen from Table 1, the concentrations of calcium and phosphate decreased in a manner dependent on the concentration of added calcium hydroxide. This result indicates that, for example, by adding calcium hydroxide in an amount of 1 to 2 times the number of moles of phosphate, most of the calcium and phosphate can be removed from a solution derived from bone tissue. The residue from which calcium and phosphate have been removed contains a large amount of organic components found in bone and can therefore be used as a biostimulant.

[0079] [Example 1-2] A biostimulant was produced by removing calcium and phosphate from bone tissue using the method for producing a biostimulant (Part 1). The specific procedure is as follows: 1. 3 g of bovine bone meal X was immersed in 30 mL of malic acid aqueous solution at 20°C. The concentration of the malic acid aqueous solution was 1 mol / L, and the immersion time was 48 hours. 2. The supernatant was collected, and the precipitated calcium malate was removed. 3. By adding an equimolar amount of calcium hydroxide to the phosphate contained in the supernatant, the free phosphate was precipitated as calcium phosphate. 4. By removing the precipitate, a biostimulant was obtained from which calcium and phosphate had been partially removed from the bone meal. The content of phosphate and calcium in the biostimulant was determined. The method for measuring the content was the same as in Example 1-1.

[0080] (Results) The results are shown in Table 2.

[0081] As can be seen from Table 2, sequential treatment of bovine bone meal X with malic acid and calcium hydroxide significantly reduced both the phosphate and calcium concentrations. The decrease in calcium and phosphate concentrations depended on the concentration of added calcium hydroxide. This result indicates that calcium and phosphate can be removed from a solution derived from bone tissue by adding, for example, an amount of calcium hydroxide equivalent to the number of moles of phosphate. The residue from which calcium and phosphate have been removed contains a large amount of organic components found in bone and can therefore be used as a biostimulant.

[0082] [Examples 1-3] Biostimulants were produced by removing calcium and phosphate from bone tissue using the method for producing biostimulants (Part 2). The specific procedure is as follows: 1. 0.1 g of raw pork bone was immersed in either 1 mL each of 1 mol / L hydrochloric acid or nitric acid at 20°C for 48 hours. 2. The supernatant was collected, and 1 mol / L sodium hydroxide was added to each to precipitate the free phosphate as calcium phosphate. The amount of sodium hydroxide added was changed as shown in Table 3. 3. By removing the precipitate, a biostimulant was obtained from raw pork bone from which calcium and phosphate had been partially removed. The concentration of phosphate in the biostimulant was determined. The phosphate concentration was quantified by ion chromatography.

[0083] (Results) The results are shown in Table 3.

[0084] By immersing raw pork bones in hydrochloric acid or nitric acid, phosphate and calcium could be solubilized. Adding sodium hydroxide to this solution reduced the phosphate to below the detection limit. This solution contained virtually no phosphate, making it suitable for use as a biostimulant.

[0085] [Examples 1-4] Biostimulants were produced by the method for producing biostimulants (Part 1) by changing the type of bone meal or the calcium removal substance. The method for quantifying phosphate was the same as the method for producing biostimulants in Example 1-1. 1. 1 mL of calcium removal substance was added to 1 g of bone meal and immersed at 20°C for 48 hours. Chicken / pork bone meal, beef bone meal, or fish bone meal was used as the bone meal. Tartaric acid, potassium bisulfate, malic acid, or maleic acid was used as the calcium removal substance. The concentration of the calcium removal substance was 1.0 mol / L in all cases. The combinations are as shown in Table 4. 2. The supernatant was collected and the precipitated sparingly soluble calcium salts were removed. 3. By adding an equimolar amount of calcium hydroxide (solid) to the phosphate contained in the supernatant, the free phosphate was precipitated as calcium phosphate. 4. By removing the precipitate, a biostimulant was obtained from which calcium and phosphate had been partially removed from the bone meal. The concentration of phosphate in the biostimulant was determined. The phosphate concentration was quantified by high-performance ion chromatography. The weight of phosphate (mg) was determined by multiplying the solution volume (mL) by the concentration (mg / mL).

[0086] (Results) The results are shown in Table 4.

[0087] As can be seen from Table 4, the concentration of phosphoric acid in the solution decreased significantly after going through steps 3 and 4. This trend remained consistent even when the type of bone meal or the type of calcium removal agent was changed.

[0088] [Example 2] Frilly lettuce was cultivated using fertilizer containing a biostimulant derived from bone tissue. In addition, the effect of the bone tissue-derived biostimulant on the growth of frilly lettuce was investigated by comparing it with a negative control.

[0089] [Example 2-1] Frilly lettuce was grown hydroponically in a cultivation environment similar to that of a plant factory, using a biostimulant derived from bone tissue during the seedling stage.

[0090] (Production of Bone Tissue-Derived Biostimulants) Two types of biostimulants were produced by changing the type of bone tissue as described below. The specific procedure was the same as in Example 1-1. • Biostimulant A: Calcium and phosphate were removed from bovine bone meal Y using tartaric acid and calcium hydroxide. • Biostimulant B: Calcium and phosphate were removed from fish bone meal X using tartaric acid and calcium hydroxide.

[0091] (Preparation of cultivation solution) The solution was prepared according to the following procedure: 1. Five types of commercially available hydroponic fertilizers (OAT House No. 2, OAT House No. 3, OAT House No. 5, OAT House No. 6, and OAT House No. 9 (all from OAT Agrio Co., Ltd.)) were prepared. 2. The five types of commercially available hydroponic fertilizers were diluted with tap water. 3. Following Formula B described in the instructions provided by the manufacturer, 10 mL each of the five types of hydroponic fertilizers was mixed with 940 mL of tap water. This mixture was used as the negative control. 4. 10 mL of biostimulant A was added to the negative control. This mixture was used as cultivation solution A. 5. 10 mL of biostimulant B was added to the negative control. This mixture was used as cultivation solution B.

[0092] Table 5 shows the composition of the prepared nutrient solutions. As shown in the table, the concentrations of major fertilizer components were similar across all nutrient solutions. Therefore, by comparing the results of cultivation using nutrient solution A or nutrient solution B with the results of cultivation using a negative control, the growth effect of biostimulant A or biostimulant B on plants can be estimated.

[0093] (Hydroponic cultivation) Using the prepared nutrient solution, frilly lettuce was cultivated from seed to seedling under the following cultivation conditions. ◆Germination period: 2 days, Planting density: approximately 1666 plants / m ２ ・Light irradiation time: 0 hours / day ・Temperature: 23 to 25℃ ・Humidity: 65 to 80% RH ・Carbon dioxide concentration: 1500 ppm ◆Seedling raising period: 12 days ・Planting density: Approximately 1666 plants / m ２• Light exposure time: 24 hours / day • Temperature: 23-25°C • Humidity: 65-80% RH • Carbon dioxide concentration: 1500 ppm

[0094] (Results) The results are shown in Figures 3 and 4. As shown in Figure 3, seedlings grown densely were obtained when cultivated using a biostimulant derived from bone tissue. As shown in Figure 4, frilly lettuce cultivated using biostimulant A or biostimulant B showed promoted root elongation, with many roots extending beyond the sponge. On the other hand, frilly lettuce cultivated using a negative control did not show as many roots extending beyond the sponge. Thus, even when the type of bone tissue used as raw material was different, the growth of frilly lettuce (especially root elongation) was promoted by the biostimulant.

[0095] [Example 2-2] The weight of seedlings at the end of the seedling period was compared between nutrient solution B and the negative control in Example 2-1.

[0096] (Results) The results are shown in Figure 5. As shown in the figure, seedlings grown using nutrient solution B were significantly heavier than seedlings grown using the negative control. Thus, the growth of frilly lettuce was promoted by a biostimulant derived from bone tissue.

[0097] [Example 2-3] The rooting-promoting effect of the biostimulant produced in Example 2-1 was investigated. Spray chrysanthemums were used as the experimental plant. 1. Side shoots were collected from the spray chrysanthemums. 2. The cut ends of six side shoots per test plot were immersed in the solution. The types of solutions used for immersion are shown in Table 6. Oxyberon (Bayer CropScience Co., Ltd.) was used as a positive control. Oxyberon is known to have a rooting-promoting effect. 3. 2-3 cm from the cut end of the immersed cuttings were inserted into the growing medium. Perlite was used as the growing medium. 4. The transplanted cuttings were watered with the solution. The types of solutions used for watering are shown in Table 6. OAT House No. 9 (OAT Agrio Co., Ltd.) was used as a positive control. 5. Fourteen days after step 3, the number of rooted roots was counted and the weight of the roots was measured.

[0098] (Results) The results are shown in Figure 6. As can be seen from the figure, cuttings treated with biostimulant A for immersion or watering of the cut end had significantly higher root count and root weight than cuttings in test plot F, which was the negative control. Notably, even in test plot A, which was not treated with oxyberon, an increase in root count was observed, almost the same as in test plot C, and the root weight was significantly higher than in test plot F. These results suggest that biostimulants derived from bone tissue promoted plant rooting.

[0099] [Example 2-4] The rooting-promoting effect of the biostimulant produced in Example 2-1 was investigated. Seiorbia (Inochio Seikouen Co., Ltd.) was used as the experimental plant. 1. New branches of Seiorbia were collected. 2. The cut ends of six branches per test plot were immersed in a 100-fold dilution of Oxyberon (Bayer CropScience Co., Ltd.). 3. 2-3 cm from the cut end of the immersed branches were inserted into the growing medium. The growing medium used was a mixture of perlite and peat moss in a 5:1 volume ratio. 4. The transplanted cuttings were watered with the solution. The watered solution was either a 1,000-fold dilution of biostimulant A or deionized water. 5. Fourteen days after step 3, the number of rooted roots was counted and the weight of the roots was measured.

[0100] (Results) The results are shown in Table 7. As can be seen from Table 7, cuttings treated with biostimulant A showed a 44% increase in the number of roots and a 67% increase in root weight compared to cuttings treated with deionized water. Thus, the rooting-promoting effect of biostimulant A was confirmed in different types of plants.

[0101] [Example 3] Metabolites contained in the biostimulant were analyzed.

[0102] [Example 3-1] Three types of biostimulants were prepared in the same manner as in Example 1-1. • Biostimulant X: A biostimulant obtained by removing most of the calcium and phosphate from bovine bone meal Y using tartaric acid and calcium hydroxide. • Biostimulant Y: A biostimulant obtained by removing most of the calcium and phosphate from chicken / pork bone meal X using tartaric acid and calcium hydroxide. • Biostimulant Z: A biostimulant obtained by removing most of the calcium and phosphate from fish bone meal X using tartaric acid and calcium hydroxide.

[0103] The metabolites contained in the obtained biostimulants were examined by metabolome analysis. The metabolome analysis was outsourced to Human Metabolome Technology, Inc. This allowed us to identify metabolites that were significantly more prominent in each of the three types of biostimulants compared to the solution from which only calcium had been removed (the solution obtained via step S12).

[0104] (Results) Table 8 summarizes the increase in metabolites before and after phosphate removal. Table 8A shows the metabolites detected with biostimulant X, Table 8B with biostimulant Y, and Table 8C with biostimulant Z. These results indicate that removing phosphate relatively increases the content of many metabolites. Surprisingly, all biostimulants contained multiple metabolites that enhance plant activity. Furthermore, some metabolites contained in the biostimulants were common to all types of bone meal, while others differed.

[0105] Table 8 lists the plant activities reported for each metabolite. However, it has been suggested that some biostimulants according to one embodiment of the present invention may have unknown functions not included in these plant activities. Examples of such functions include promoting root growth, improving stress tolerance, and increasing fruit sweetness.

[0106] A biostimulant according to one aspect of the present invention may contain one or any combination of two or more metabolites listed in Table 8. The lower limit of the concentration of these metabolites may be 1 / 1000, 1 / 100, 1 / 10, 1 / 5, 1 / 2, or 1 of the concentrations listed in the table. The upper limit of the concentration of these metabolites may be 1000, 100, 10, 5, 2, or 1 of the concentrations listed in the table. For example, when the concentration of metabolite X is 10 μmol / L in Table 8, the lower limit of the concentration of metabolite X contained in the biostimulant may be 0.01 μmol / L or higher, 0.1 μmol / L or higher, 1 μmol / L or higher, 2 μmol / L or higher, 5 μmol / L or higher, or 10 μmol / L or higher, and the upper limit may be 10,000 μmol / L or lower, 1,000 μmol / L or lower, 100 μmol / L or lower, 50 μmol / L or lower, 20 μmol / L or lower, or 10 μmol / L or lower.

[0107]

[0108] As shown in Table 8, heptanoic acid and butyric acid were components commonly found in multiple biostimulants.

[0109] [Example 3-2] The following five types of biostimulants were prepared in the same manner as in Example 1-1. • Biostimulant (1): A biostimulant obtained by removing most of the calcium and phosphate from bovine bone meal Y using tartaric acid and calcium hydroxide. • Biostimulant (2): A biostimulant obtained by removing most of the calcium and phosphate from bovine bone meal Y using potassium bisulfate and calcium hydroxide. • Biostimulant (3): A biostimulant obtained by removing most of the calcium and phosphate from fish bone meal X using tartaric acid and calcium hydroxide. • Biostimulant (4): A biostimulant obtained by removing most of the calcium and phosphate from chicken / pork bone meal X using tartaric acid and calcium hydroxide. • Biostimulant (5): A biostimulant obtained by removing most of the calcium and phosphate from bovine bone meal Y using sulfuric acid and calcium hydroxide.

[0110] The components contained in the obtained biostimulants (1) to (5) were examined by metabolome analysis. The metabolome analysis was outsourced to Human Metabolome Technology, Inc. Based on the analysis results, components detected in both typical biostimulants (4) and (5) were extracted.

[0111] (Results) Metabolome analysis revealed a wide variety of organic compounds, including amino acids, dipeptides, tripeptides, nucleotides, phosphate compounds, sugars, and other low molecular weight molecules, from typical biostimulants (4) and (5). Although not shown in Table 9, biostimulants (1) to (3) showed similar results, albeit with differences in concentration. Many of these metabolites were involved in root elongation, fruit growth, above-ground growth, flowering promotion, increased stress tolerance (high temperature, low temperature, high salt concentration, ultraviolet radiation, drought, etc.), and immune system enhancement. In other words, bone-derived metabolites were suggested to enhance the biological activity of plants.

[0112] A biostimulant according to one aspect of the present invention may contain one or any combination of two or more metabolites listed in Table 9. The lower limit of the concentration of these metabolites may be 1 / 1000, 1 / 100, 1 / 10, 1 / 5, 1 / 2, or 1 of the concentrations listed in the table. The upper limit of the concentration of these metabolites may be 1000, 100, 10, 5, 2, or 1 of the concentrations listed in the table. For example, when the concentration of metabolite X is 10 μmol / L in Table 9, the lower limit of the concentration of metabolite X contained in the biostimulant may be 0.01 μmol / L or higher, 0.1 μmol / L or higher, 1 μmol / L or higher, 2 μmol / L or higher, 5 μmol / L or higher, or 10 μmol / L or higher, and the upper limit may be 10,000 μmol / L or lower, 1,000 μmol / L or lower, 100 μmol / L or lower, 50 μmol / L or lower, 20 μmol / L or lower, or 10 μmol / L or lower.

[0113]

[0114] [Example 3-3] Table 10 shows representative estimated components identified by comparing the m / z and MT of the detection peak of biostimulant (2) in the metabolome analysis performed in Example 3-2 with the HMT database. Some of these components were not detected by any of the biostimulants (1) to (5). A biostimulant according to one aspect of the present invention may contain one or any combination of two or more components from those listed in Table 10.

[0115]

[0116] [Example 3-4] Of the metabolites detected in the biostimulants (4) and (5) analyzed in Example 3-2, those metabolites that are abundant in bone tissue were extracted. The relative concentrations of the extracted metabolites are shown in Table 11.

[0117] Ninety percent of the proteins in bone tissue are type I collagen, which is rich in glycine (Gly), proline (Pro), and hydroxyproline (Hyp). Typical biostimulants (4) and (5) produced from bone tissue contained large amounts of metabolites containing hydroxyproline (Ala-Hyp, Gly-Hyp, Pro-Hyp, Gly-Pro-Hyp). In addition, metabolites derived from proteins and cellular components that are relatively abundant in bone tissue (γ-carboxyglutamic acid, phosphate ethanolamine) were also detected. These components were also present in biostimulants (1) to (3). It is presumed that biostimulants containing these metabolites are derived from bone tissue.

[0118] This invention can be used for the production of biostimulants from bone tissue, among other applications.

Claims

1. A method for producing a biostimulant comprising the following steps: Step S11: A step of contacting bone tissue with a substance containing anion capable of forming a sparingly soluble calcium salt in a solution; Step S12: A step of removing the sparingly soluble calcium salt formed in Step S11; Step S13: A step of contacting the solution obtained in Step S12 with a substance containing a cation capable of forming a sparingly soluble phosphate; Step S14: A step of removing the sparingly soluble phosphate formed in Step S13.

2. A method for producing a biostimulant comprising the following steps: Step S15: A step of bringing bone tissue into contact with a strong acid; Step S16: A step of contacting the solution obtained in step S15 with a base; Step S17: A step to remove the poorly soluble phosphate formed in step S16.

3. The manufacturing method according to claim 1, wherein the substance containing anion capable of forming the above-mentioned sparingly soluble calcium salt is sulfuric acid, sulfate, tartaric acid, tartrate, citric acid, citrate, maleic acid, maleate, malic acid, malate, or any combination thereof.

4. The manufacturing method according to claim 1, wherein the substance containing a cation capable of forming the above-mentioned poorly soluble phosphate is a calcium salt, a magnesium salt, an ammonium salt, an aluminum salt, or any combination thereof.

5. The manufacturing method according to claim 2, wherein the strong acid is hydrochloric acid, nitric acid, sulfuric acid, or any combination thereof.

6. The manufacturing method according to claim 2, wherein the base is sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium hydroxide, or any combination thereof.

7. The method for producing the biostimulant according to claim 1 or 2, wherein the calcium concentration is 400 mmol / L or less and the phosphate concentration is 400 mmol / L or less.

8. A biostimulant obtained by removing at least a portion of calcium and phosphate from bone tissue, wherein the calcium concentration is 400 mmol / L or less, and the phosphate concentration is 400 mmol / L or less.

9. The biostimulant according to claim 8 for use in one or more of the following uses: (i) to elongate plant roots; (ii) to increase the root mass of plants; (iii) to increase the weight of plants; (iv) to promote plant growth; (v) to improve the taste of plants.

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