Method for producing phosphoric acid

Abstract

Provided is a novel method for producing phosphoric acid. A method for producing phosphoric acid according to one embodiment of the present invention includes the following steps. (a) A step for treating a bone tissue with an acid to obtain an acid extract. (b) A step for adding a base to the obtained acid extract to obtain calcium phosphate thus precipitated. (c) A step for adding an acid to the obtained calcium phosphate precipitate to obtain a calcium phosphate solution. (d) A step for adding, to the obtained calcium phosphate solution, one or more selected from the group consisting of a sulfate, a carbonate and a hydrogencarbonate to remove calcium thus precipitated.

Description

[Technical Field]

[0001] This invention relates to a method for producing phosphoric acid. [Background technology]

[0002] Phosphate is a material widely used in agriculture and industry. While it is generally produced using phosphate rock as a raw material, technologies for recovering phosphoric acid from waste have also been developed. For example, Patent Document 1 discloses a method for recovering phosphoric acid from human waste and septic tank sludge. Patent Document 2 discloses a method for recovering phosphoric acid from steelmaking slag. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2017-196619 [Patent Document 2] Japanese Patent Publication No. 2011-213558 [Overview of the project] [Problems that the invention aims to solve]

[0004] Bone tissue is estimated to contain approximately 33% phosphate based on its weight before drying. Therefore, if phosphate can be recovered from bone tissue, waste bones generated at slaughterhouses and other sources could become a huge source of phosphate. This would be extremely desirable for Japan, which relies entirely on imports for its phosphate rock needs. For this reason, the development of methods for recovering phosphate from bone tissue is urgently needed.

[0005] One aspect of the present invention aims to provide a novel method for producing phosphoric acid. [Means for solving the problem]

[0006] <1> A method for producing phosphoric acid, comprising the following steps. (a) A process of treating bone tissue with acid to obtain an acid extract. (b) Adding a base to the obtained acid extract to obtain precipitated calcium phosphate. (c) Adding acid to the obtained calcium phosphate precipitate to obtain a calcium phosphate solution. (d) Adding one or more salts selected from the group consisting of sulfates, carbonates, and bicarbonates to the obtained calcium phosphate solution to remove the precipitated calcium. <2> The process further includes a step of pre-treating the bone tissue prior to step (a) above, The above pretreatment is one or more selected from the group consisting of heating the bone tissue, heating the bone tissue under pressure, and irradiating the bone tissue with microwaves. <1> The manufacturing method described above. <3> The process further includes removing anions and cations other than phosphate ions and hydrogen ions after step (d) above. <1> or <2> The manufacturing method described above. <4> Steps (b) and (c) above are repeated two or more times. <1> ~ <3> A manufacturing method described in any of the following. <5> In step (a) above, the bone tissue is treated with an acid selected from the group consisting of nitric acid, hydrochloric acid, formic acid, and sulfuric acid. <1> ~ <4> A manufacturing method described in any of the following. <6> In step (d) above, sulfuric acid is not added to the calcium phosphate solution. <1> ~ <5> A manufacturing method described in any of the following. [Effects of the Invention]

[0007] According to one aspect of the present invention, a novel method for producing phosphoric acid is provided. [Brief explanation of the drawing]

[0008] [Figure 1] This is a flowchart illustrating an example of a fertilizer manufacturing method according to one embodiment of the present invention. [Figure 2]This is a flowchart showing another example of the method for producing a fertilizer according to an embodiment of the present invention. [Figure 3] This is a flowchart showing an example of the method for producing phosphoric acid according to an embodiment of the present invention. [Figure 4] This is a diagram showing the results of treating bone tissue obtained by acid-treating pork bones with nitric acid, using a protease. [Figure 5] This is a diagram showing the results of treating bone tissue, obtained by acid-treating pork bones with nitric acid or hydrochloric acid, using actinidain. [Figure 6] This is a diagram showing the results of preparing bone solubilized liquid A without solution exchange using various acids and proteases. [Figure 7] This is a diagram showing the results of an experiment examining the plant growth effect of an acid extract. [Figure 8] This is a diagram showing the results of an experiment examining the plant growth effect of an acid extract, a protease-treated liquid, or bone solubilized liquid A. [Figure 9] This is a diagram showing the results of an experiment comparing the plant growth effect of bone solubilized liquid A and a commercially available culture solution. [Figure 10] This is a diagram showing the results of an experiment comparing the plant growth effect of bone solubilized liquid A and a commercially available culture solution. [Figure 11] This is a diagram showing the results of an experiment comparing changes in the expression level of stress tolerance-related genes caused by bone solubilized liquid A or a commercially available liquid fertilizer. [Figure 12] This is a diagram showing the results of an experiment comparing changes in the expression level of stress tolerance-related genes caused by bone solubilized liquid A or a commercially available liquid fertilizer. [Figure 13] This is a diagram showing the effect of bone tissue pretreatment on the amount of phosphoric acid contained in the acid extract. [Figure 14] This is a diagram showing the results of electrophoretic analysis of protein components contained in the acid extract. [Figure 15] This is a diagram showing the results of preparing a protease-treated liquid using various proteases. [Figure 16]This figure shows the results of analyzing the protein components contained in the protease-treated solution by electrophoresis. [Figure 17] This figure shows the results of preparing bone solubilizing solution A without solution exchange using various acids and proteases. [Figure 18] This figure shows the results of the analysis of differentially expressed genes in the leaves of sprouts grown for 5 days with bone solubilizing solution B or commercially available liquid fertilizer. [Figure 19] This figure shows the results of the analysis of differentially expressed genes in the roots of sprouts cultivated for 5 days with a mixture of bone solubilizing solution B and commercially available liquid fertilizer, or with only commercially available liquid fertilizer. [Figure 20] This figure shows the results of the analysis of differentially expressed genes in the leaves of sprouts grown for 5 days with a mixture of bone solubilizing solution B and commercially available liquid fertilizer, or with only commercially available liquid fertilizer. [Figure 21] This is a dissolution curve showing the results of extracting phosphate from commercially available bone meal. [Figure 22] This diagram shows the results of manufacturing liquid fertilizer using commercially available bone meal as a raw material. Neurase F3G was used as the protease. [Figure 23] This diagram shows the results of manufacturing liquid fertilizer using commercially available bone meal as a raw material. Papain was used as the protease. [Modes for carrying out the invention]

[0009] [1. Method of manufacturing fertilizer] A method for producing fertilizer according to one aspect of the present invention includes one or more of the following steps 1 to 3. In one embodiment, the method for producing fertilizer further includes the following step 4. Steps 2, 3, 4, and 1 will be described in detail below.

[0010] Step 1: A process to produce fertilizer from bone solubilized liquid A obtained by treating bone tissue with a solution containing both acid and protease. Step 2: A process of treating bone tissue with acid and producing fertilizer from the resulting acid extract. Step 3: A process of treating acid-treated bone tissue with protease and producing fertilizer from the resulting protease-treated solution. Step 4: A process to produce fertilizer from the resulting bone-solubilizing solution B by mixing the acid extract and the protease-treated solution.

[0011] [1.1.Process 2] Step 2 is a process of treating bone tissue with acid and producing fertilizer from the resulting acid extract. In Step 2, inorganic components are mainly separated from the bone tissue and eluted into the acid extract. Fertilizer is produced from the resulting acid extract. The acid extract itself may be used as fertilizer, or bone solubilization solution B, which is mixed with protease treatment solution, may be used as fertilizer.

[0012] The bone tissue used in step 2 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 a raw material for fertilizer, and bone tissue from livestock (such as cattle, pigs, sheep, and chickens) is preferably used. The bone tissue may be shredded or crushed before acid treatment. Doing so may shorten the manufacturing time as the bone tissue can be broken down more efficiently.

[0013] The acid used to treat the bone tissue in step 2 is not particularly limited. Examples of acids include hydrochloric acid, nitric acid, formic acid, sulfuric acid, and trichloroacetic acid. Acidic decalcification solutions such as Planck-Luclo solution may also be used. From the viewpoint of availability, one or more acids selected from the group consisting of nitric acid, hydrochloric acid, formic acid, and sulfuric acid are preferred. From the viewpoint of phosphoric acid recovery efficiency, one or more acids selected from the group consisting of hydrochloric acid, sulfuric acid, and nitric acid are preferred, and one or more acids selected from the group consisting of hydrochloric acid and nitric acid are more preferred. From the viewpoint of calcium recovery efficiency, one or more acids selected from the group consisting of hydrochloric acid and formic acid are preferred. Two or more acids may be mixed and used in appropriate ratios.

[0014] In step 2, the bone tissue may be treated with an acid solution obtained by diluting the acid in a solvent. Examples of solvents include water, lower alcohols, glycerol, propane-1,2-diol, and 1,3-propanediol. Two or more solutions may be mixed in appropriate ratios and used.

[0015] The acid concentration can be appropriately determined according to the volume of bone tissue to be treated with acid. If the volume of bone tissue is small, it can be treated with a low concentration of acid. If the volume of bone tissue is large, a higher acid concentration is preferable. When treating with a low concentration of acid, it is preferable to treat the bone tissue in a fine powder form. However, even if the volume of bone tissue is large, inorganic components can be sufficiently extracted by increasing the acid concentration and immersing it for a long time. For example, if the volume is several centimeters... 3 When using the following bone fragments as raw materials, the lower limit of the acid concentration in step 2 may be 0.6 mol / L or higher, 0.7 mol / L or higher, 0.8 mol / L or higher, or 0.9 mol / L or higher. The upper limit of the acid concentration in step 2 may be 2.0 mol / L or lower, 1.5 mol / L or lower, 1.0 mol / L or lower, or 0.9 mol / L or lower. If the acid concentration is within the above range, inorganic components can be extracted efficiently, and since the acid concentration is not too high, the cost of neutralizing the acid extract can be reduced.

[0016] The following are examples of suitable acid concentrations when treating pulverized bone tissue with acid: When the acid is nitric acid, the lower limit of the nitric acid concentration is preferably 0.6 mol / L or higher, and more preferably 0.7 mol / L or higher. The upper limit of the nitric acid concentration is preferably 1.0 mol / L or lower, and more preferably 0.9 mol / L or lower. When the acid is hydrochloric acid, the lower limit of the hydrochloric acid concentration is preferably 0.8 mol / L or higher, and more preferably 0.9 mol / L or higher. The upper limit of the hydrochloric acid concentration is preferably 1.2 mol / L or lower, and more preferably 1.1 mol / L or lower. When the acid is formic acid, the lower limit of the formic acid concentration is preferably 0.8 mol / L or higher, and more preferably 0.9 mol / L or higher. The upper limit of the formic acid concentration is preferably 1.2 mol / L or lower, and more preferably 1.1 mol / L or lower. When the acid is sulfuric acid, the lower limit of the sulfuric acid concentration is preferably 0.8 mol / L or higher, and more preferably 0.9 mol / L or higher. The upper limit of the sulfuric acid concentration is preferably 1.2 mol / L or lower, and more preferably 1.1 mol / L or lower. However, the above concentrations are examples of suitable concentrations when bone tissue is pulverized, and the acid concentration may be increased when bone tissue with a larger volume is used as the raw material.

[0017] For example, a volume of several cubic centimeters 3 When using the following bone fragments as raw materials, the lower limit of the extraction time in step 2 is preferably 6 hours or more, more preferably 8 hours or more, and even more preferably 10 hours or more. The upper limit of the extraction time in step 2 is preferably 48 hours or less, more preferably 24 hours or less, and even more preferably 14 hours or less. If the extraction time is within the above range, inorganic components contained in the bone tissue can be sufficiently extracted.

[0018] For example, a volume of several cubic centimeters 3 When using the following bone crushed materials as raw materials, the acid treatment temperature in step 2 is preferably 5 to 60°C. Performing the acid treatment within this temperature range reduces the generation of unpleasant odors associated with the acid treatment. Therefore, the location requirements for the fertilizer manufacturing plant become more flexible. Furthermore, expensive special equipment is not required to adjust the temperature to below 60°C; the temperature can be kept constant by using a water bath or incubator.

[0019] In step 2, in addition to the acid, a chelating agent capable of capturing calcium ions may be added. Alternatively, in step 2, the bone tissue may be treated with a chelating agent before and after the acid treatment (the solutions may or may not be exchanged between the acid treatment and the chelating agent treatment). Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA, CAS registry number: 60-00-4), glycol etherdiaminetetraacetic acid (EGTA, CAS registry number: 67-42-5), and ethylenediamine-N,N'-disuccinic acid (EDDS, CAS registry number: 20846-91-7). In step 2, the pH of the solution containing the chelating agent is preferably 6.0 to 8.0. For example, if the volume is several cm³ 3 When using the following bone fragments as raw materials, the lower limit of the chelating agent concentration in step 2 may be 0.1 mol / L or higher, 0.2 mol / L or higher, 0.3 mol / L or higher, or 0.4 mol / L or higher. The upper limit of the chelating agent concentration in step 2 may be 0.9 mol / L or lower, 0.8 mol / L or lower, 0.7 mol / L or lower, or 0.6 mol / L or lower.

[0020] In step 2, at least some of the calcium may be removed after the acid treatment. Examples of methods for removing calcium include neutralizing the acid extract (which results in a calcium phosphate precipitate), adding sulfuric acid or sulfates (which results in a calcium sulfate precipitate), adding carbonic acid or carbonates (which results in a calcium carbonate or calcium bicarbonate precipitate), or adding bicarbonates (which results in a calcium carbonate or calcium bicarbonate precipitate).

[0021] Performing the pretreatment steps described later tends to increase the phosphoric acid concentration in the acid extract obtained in step 2. The phosphoric acid concentration in the acid extract can be, for example, 280 mM or higher, 300 mM or higher, or 320 mM or higher.

[0022] [1.2.Process 3] Step 3 is a step in which acid-treated bone tissue is treated with protease, and fertilizer is produced from the resulting protease-treated solution. Fertilizer is produced from the resulting protease-treated solution. The protease-treated solution itself may be used as fertilizer, or bone solubilization solution B, which is mixed with the acid extract, may be used as fertilizer.

[0023] The protease used in step 3 is not particularly limited. Examples of proteases include serine protease, cysteine ​​protease, aspartate protease, glutamate protease, and metalloprotease.

[0024] Specific examples of proteases include trypsin [EC 3.4.21.4], chymotrypsin [EC 3.4.21.1], [EC 3.4.21.2], pepsin [EC 3.4.23.1], ecolysin [EC 3.4.23.19], papain [EC 3.4.22.2], ficin [EC 3.4.22.3], actinidine [EC 3.4.22.14], bromelain [EC 3.4.22.32], cathepsin B [EC 3.4.22.1], cathepsin H [EC 3.4.22.16], cathepsin K [EC 3.4.22.38], cathepsin L [EC 3.4.22.15], and cathepsin S [EC Examples include thermolysin [EC 3.4.24.27] and thermolysin [EC 3.4.24.27].

[0025] Commercially available enzyme preparations may be used as proteases. Examples of such preparations include Neurase F3G (derived from Rhizopus niveus), Orientase AY (derived from Aspergillus niger), Tetrase (derived from Aspergillus niger), Sumizyme AP (derived from Aspergillus niger), Denapsin 2P (derived from Aspergillus genus), Brewers Clarex (derived from Aspergillus niger), Maxipro AFP (derived from Aspergillus niger), Protease S "Amano" G (derived from Bacillus stearothermophilus), Protease N "Amano" G (derived from Bacillus subtilis), Protease NL "Amano" (derived from Bacillus subtilis), Protease A "Amano" G (derived from Aspergillus oryzae), and Umamizyme (Aspergillus Examples include protease P "Amano" 3G (derived from Aspergillus oryzae), protease M "Amano" SD (derived from Aspergillus oryzae), and peptidase R (derived from Rhizopus oryzae). Alternatively, chemically synthesized enzymes, enzymes produced by genetic engineering techniques, or enzymes extracted from living organisms may be used.

[0026] The protease used in step 3 is preferably a protease with an optimal pH of 1.5 to 8.0. Examples of proteases with an optimal pH of 1.5 to 8.0 include: Protease S "Amano" G (optimal pH: 7.0 to 8.5), Protease N "Amano" G (optimal pH: 6.0 to 7.5), Protease NL "Amano" (optimal pH: 6.5 to 7.5), Protease A "Amano" G (optimal pH: 6.0 to 7.5), Umamizyme (optimal pH: 6.0 to 7.5), and Protease M "Amano" G (optimal pH: 3.0 to 6.5). Examples include protease P "Amano" 3G (optimal pH: 7.0~8.0), peptidase R "Amano" (optimal pH: 6.0~8.0), actinidine (optimal pH: 2.5~7.5), papain (optimal pH: 4.0~9.0), pepsin (optimal pH: 1.5~3.0), neurase F3G (optimal pH: 3.0~5.0), trypsin (optimal pH: 7.0~9.0), and chymotrypsin (optimal pH: 7.0~9.0). The protease used is more preferably a protease with an optimal pH of 1.5~5.0. Examples of proteases with an optimal pH of 1.5 to 5.0 include protease M "Amano" (optimal pH: 3.0 to 6.5), actinidine (optimal pH: 2.5 to 7.5), papain (optimal pH: 4.0 to 9.0), pepsin (optimal pH: 1.5 to 3.0), and neurase F3G (optimal pH: 3.0 to 5.0). More preferably, the protease used is one with an optimal pH of 1.5 to 4.0. Examples of proteases with an optimal pH of 1.5 to 4.0 include actinidine (optimal pH: 2.5 to 7.5), pepsin (optimal pH: 1.5 to 3.0), and neurase F3G (optimal pH: 3.0 to 5.0).

[0027] The protease concentration in step 3 can be set as appropriate. The lower limit of the protease concentration may be 2 mg / L or higher, or 10 mg / L or higher. The upper limit of the protease concentration may be 100 mg / L or lower, or 50 mg / L or lower.

[0028] The temperature and pH in step 3 can be set as appropriate. Matching them to the optimal temperature and pH of the protease used is preferable for improving processing efficiency. An example of the reaction system temperature in step 3 is 20 to 60°C. The lower limit of the reaction system temperature in step 2 may be greater than 10°C, 20°C or higher, 25°C or higher, 30°C or higher, 35°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, or 55°C or higher. The upper limit of the reaction system temperature in step 2 may be 60°C or lower, or 55°C or lower.

[0029] In step 3, salt may be added to the reaction system. Depending on the concentration of salt added to the reaction system, the substrate specificity of the protease may change. Therefore, by adding salt to the reaction system, the components contained in the resulting protease-treated solution can be altered.

[0030] Examples of salts to be added to the reaction system in step 3 include chloride salts. Examples of chloride salts include NaCl, KCl, LiCl, and MgCl2. The lower limit of the concentration of the salt added to the reaction system in step 3 may be greater than 0 mmol / L, 20 mmol / L or more, 100 mmol / L or more, 150 mmol / L or more, 200 mmol / L or more, 500 mmol / L or more, 1000 mmol / L or more, 1500 mmol / L or more, or 2000 mmol / L or more. The upper limit of the concentration of the salt added to the reaction system in step 3 may be 4000 mmol / L or less, or 2000 mmol / L or less.

[0031] In one embodiment, the size of the peptide fragment produced in step 3 is 10,000 Da or less, 8,000 Da or less, 6,000 Da or less, or 4,000 Da or less. Peptide fragments cleaved to this size are highly likely to have lost their physiological activity. Furthermore, peptide fragments cleaved to this size are easily absorbed as nutrients by plants and may function as active ingredients in fertilizers. In one embodiment, the peptide fragment is derived from one or more selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan, and SPARC.

[0032] [1.3.Step 4] Step 4 is a process of mixing the acid extract obtained in Step 2 with the protease-treated solution obtained in Step 3 to produce fertilizer from the resulting bone-solubilizing solution B. Fertilizer is produced from the resulting bone-solubilizing solution B. In Step 4, the mixing ratio of the acid extract and the protease-treated solution is not particularly limited. The mixing ratio can be appropriately selected to obtain the bone-solubilizing solution B with a desired composition.

[0033] [1.4.Process 1] Step 1 is a process of treating bone tissue with a solution containing both acid and protease to produce fertilizer from the resulting bone solubilized liquid A. In Step 1, the acid treatment in Step 2 and the protease treatment in Step 3 proceed simultaneously. In Step 1, there is no need to change the solution during the acid treatment and protease treatment.

[0034] In step 1, the order in which the bone tissue is brought into contact with the acid and protease is not particularly limited. For example, the following order is possible. Of these orders, order 1 is preferred. Procedure 1: Immerse bone tissue in an acid-containing solution, and after a predetermined time has elapsed, add protease to the solution. Before adding the protease, the pH of the solution may be adjusted to the optimal pH for the protease. Procedure 2: Immerse bone tissue in a solution containing protease, and after a predetermined time has elapsed, add acid to the solution. Step 3: Prepare a solution containing both acid and protease, and immerse the bone tissue in the solution.

[0035] The preferred conditions for the acid treatment and protease treatment in Step 1 are to be referred to in Sections [1.1] and [1.2].

[0036] [1.5. Other processes] A method for producing fertilizer according to one embodiment of the present invention may further include steps that are normally performed in the production of fertilizer, in addition to steps 1 to 4. Examples of such steps include a pretreatment step, a component addition step, a drying step, a crushing step, a coated granulation step, and a packaging step.

[0037] The pretreatment step is a step that precedes step 1 or step 2. In the pretreatment step, the bone tissue, which is the raw material, is pretreated. By performing the pretreatment step, the amount of phosphate extracted from the bone tissue can be increased (see Example 4).

[0038] In one embodiment, the bone tissue is heated in the pretreatment step. The heating temperature at this time may be 30°C or higher, or 40°C or higher; or 100°C or lower, or 80°C or lower. The bone tissue may also be heated while immersed in acid.

[0039] In one embodiment, the pretreatment step involves heating the bone tissue under pressure. The pressure at this time may be 200 kPa or more or 1 MPa or more; 500 MPa or less or 800 MPa or less. The heating temperature at this time may be 10°C or more or 50°C or more; 120°C or less or 200°C or less.

[0040] In one embodiment, the pretreatment step involves irradiating bone tissue with microwaves. Heating, heating under pressure, and microwave irradiation may be performed in any combination. Of these, microwave irradiation is more preferred as a pretreatment step because it is effective in a short time. The microwave irradiation time may be 5 seconds or more, 10 seconds or more, or 15 seconds or more; or 10 minutes or less, 7 minutes or less, or 5 minutes or less.

[0041] When a pretreatment process is applied, the proteins contained in the bone tissue are denatured. Therefore, the fertilizer obtained after the pretreatment process contains denatured proteins (or fragments thereof). Denatured proteins (or fragments thereof) have lost the physiological activity that the original proteins possessed.

[0042] The component addition step is a process of adding additional fertilizer components. By including a component addition step, it becomes possible to produce fertilizers with a composition suitable for the plants being cultivated. Examples of fertilizer components added in the component addition step include potassium components (potassium oxide, potassium hydroxide, potassium chloride, potassium sulfate, etc.), nitrogen components (urea, ammonium nitrate, etc.), magnesium components (magnesium phosphate, magnesium chloride, magnesium sulfate, etc.), vitamins, manganese, boron, iron, copper, zinc, and molybdenum. In addition, the acid extract, protease treatment solution, bone solubilization solution A, or bone solubilization solution B may be mixed with other fertilizers (inorganic fertilizers, organic fertilizers, etc.).

[0043] The drying process involves removing excess water from the acid extract, protease-treated solution, bone solubilization solution A, or bone solubilization solution B. Through the drying process, a solid or paste-like fertilizer is obtained. The solid fertilizer may be cut or crushed into sizes and shapes that are easy to apply, as needed.

[0044] The coated granulation process is a process of coating and granulating solid fertilizers. For example, coating fertilizers with silicate compounds can adjust the timing of fertilizer release, prevent the immobilization of phosphorus and calcium, prevent fertilizer runoff, and prevent damage to the fertilizer due to impact.

[0045] The packaging process involves packaging the acid extract, protease treatment solution, bone solubilizer A, or bone solubilizer B into containers so that they can be distributed or sold as fertilizer. The packaging process may also include instructions for using these as fertilizer. These instructions may be printed on the container or provided separately as physical or electronic documents. The instructions may include information on the fertilizer formulation, application method, application timing, and target crops.

[0046] [1.6. Exemplary Fertilizer Manufacturing Method] An example of a method for producing fertilizer according to one embodiment of the present invention will be described with reference to an exemplary flowchart.

[0047] Figure 1 is an exemplary flowchart representing a manufacturing method including steps 2, 3, and / or 4. In this flowchart, an acid extract is obtained by going through steps S1, S2, S3, and S4. A protease-treated solution is obtained by going through steps S1, S2, and S5. Furthermore, bone solubilization solution B is obtained by going through step S6 with the acid extract and the protease-treated solution. The acid extract, protease-treated solution, and bone solubilization solution B can all be used as components of fertilizer or mixed fertilizer. The acid extract contains phosphorus, an essential nutrient for plants, so it can also be used as fertilizer on its own.

[0048] Step S1 involves pretreatment of the bone tissue. Step S1 is optional and does not need to be performed. Pretreatment of the bone tissue can increase the amount of phosphoric acid contained in the acid extract. This step is described in Section [1.5].

[0049] In step S2, the bone tissue is treated with acid. The supernatant obtained after step S2 is the acid extract. When fertilizer is produced from the acid extract, it becomes step 2 as described above. The acid treatment of the bone tissue is as explained in section [1.1].

[0050] In step S3, a sulfate, carbonate, or bicarbonate is added. Step S3 is optional and may not be performed. In step S3, calcium ions contained in the acid extract precipitate as calcium sulfate, calcium carbonate, or calcium bicarbonate. Examples of sulfates include sodium sulfate, potassium sulfate, ammonium sulfate, and magnesium sulfate. Examples of carbonates include potassium carbonate and ammonium carbonate. An example of a bicarbonate is potassium bicarbonate. Preferably, the sulfate is one or more selected from the group consisting of potassium sulfate, ammonium sulfate, and magnesium sulfate. Preferably, the carbonate is potassium carbonate. Preferably, the bicarbonate is potassium bicarbonate. When these sulfates, carbonates, or bicarbonates are used, potassium, magnesium, and / or ammonium are added to the acid extract. These components are important nutrients for plants.

[0051] In step S4, the acid extract is neutralized with a base. Step S4 is optional and does not need to be performed. Step S4 returns the acid extract to a near-neutral pH. Examples of bases that can be used include sodium hydroxide and potassium hydroxide. Of these, using potassium hydroxide will result in potassium ions being present in the acid extract. Since potassium ions are an important nutrient for plants, neutralization with potassium hydroxide is preferable.

[0052] In step S5, the bone tissue that has undergone acid treatment is treated with protease. The treatment solution obtained after step S5 is the protease-treated solution. When fertilizer is produced from the protease-treated solution, it becomes step 3 as described above. The acid treatment of the bone tissue with protease is as described in section [1.2].

[0053] In step S6, the acid extract and the protease-treated solution are mixed. This yields bone solubilizing solution B. The fertilizer is then produced from bone solubilizing solution B, which is step 4 described above. The preparation of bone solubilizing solution B is as described in section [1.3].

[0054] Figure 2 is an exemplary flowchart representing a manufacturing method including step 1. In this flowchart, bone solubilizing solution A is obtained by going through steps S1, S7, S3, and S4. Bone solubilizing solution A can be used as a component of fertilizer or mixed fertilizer.

[0055] Processes S1, S3, and S4 have been explained above, so we will omit further explanation.

[0056] In step S7, the bone tissue is treated with both acid and protease. This yields bone solubilization solution A. The production of fertilizer from bone solubilization solution A results in step 1 described above. The preparation of bone solubilization solution A is as described in section [1.4].

[0057] [2. Fertilizer] [2.1. Fertilizers identified by manufacturing method] A fertilizer according to one aspect of the present invention is a fertilizer obtained by a method for producing a fertilizer according to one aspect of the present invention. Therefore, a fertilizer according to one aspect of the present invention contains an acid extract, a protease treatment solution, and bone solubilizing solution A or bone solubilizing solution B. Among these, a fertilizer containing bone solubilizing solution A or bone solubilizing solution B is preferred because it has a higher effect in promoting plant growth.

[0058] The lower limit of the proportion of acid extract, protease-treated solution, bone solubilizer A, or bone solubilizer B in the total weight of the fertilizer may be 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more. The upper limit of the proportion of acid extract, protease-treated solution, bone solubilizer A, or bone solubilizer B in the total weight of the fertilizer is 100% by weight. In one embodiment, the fertilizer consists only of acid extract, protease-treated solution, bone solubilizer A, bone solubilizer B, or any mixture thereof.

[0059] The composition of the fertilizer may be changed as needed. For example, some or all of the phosphorus and calcium may be replaced with calcium dihydrogen phosphate or calcium citrate. With such a composition, the timing of fertilizer release can be adjusted so that phosphorus and calcium are supplied according to the growth stage of the plant.

[0060] The fertilizer may be a solid fertilizer or a liquid fertilizer. Since the acid extract, protease treatment solution, bone solubilization solution A, or bone solubilization solution B are obtained as liquids, they can be easily processed into liquid fertilizer. According to the fertilizer manufacturing method of one embodiment of the present invention, the components of bone tissue (for example, all components of bone tissue) can be solubilized, so it is expected that a liquid fertilizer rich in components useful for plant growth can be obtained. Furthermore, while existing bone-derived solid fertilizers (bone meal) are slow-acting and are often used for soil improvement over several years, liquid fertilizers can be expected to have an immediate effect. In addition, bone meal is not suitable for hydroponics, whereas liquid fertilizers can be suitably used in hydroponics. In addition, liquid fertilizers also have the advantage of being easily applicable to foliar application.

[0061] Fertilizers may contain components other than acid extract, protease treatment solution, bone solubilizer A, or bone solubilizer B. Examples of such components include acidic fertilizers, alkaline fertilizers, and other general fertilizers. Examples of acidic fertilizers include ammonium sulfate, superphosphate, potassium sulfate, aluminum sulfate, peat moss, black soil, ash, and alum. Examples of alkaline fertilizers include wood ash, calcium cyanamide, Chilean nitrate, fish fertilizer leaf mold, dolomitic lime, organic lime, slaked lime, calcium cyanamide, limestone, cement, baking soda, seashells, rice hulls, and charcoal. Examples of other general fertilizers include straw, bark, and molasses. Free amino acids (such as γ-aminobutyric acid), plant growth hormones (such as auxin), and trace elements (such as magnesium, sulfur, iron, manganese, zinc, copper, boron, and molybdenum) may further be included.

[0062] [2.2. Fertilizers identified by their components] A fertilizer according to one embodiment of the present invention contains bone tissue degradation products. Therefore, it often contains type I collagen, osteocalcin, alpha-2-HS-glycoprotein, periostin, biglycan, SPARC, or their degradation peptides, which are the main organic components of bone tissue. These components are rarely found in fertilizers obtained by other manufacturing methods. Therefore, a fertilizer containing type I collagen, osteocalcin, alpha-2-HS-glycoprotein, biglycan, SPARC, their degradation peptides, or a combination thereof is highly likely to be a fertilizer produced by the manufacturing method according to one embodiment of the present invention.

[0063] In one embodiment, the fertilizer contains peptide fragments derived from one or more selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan, and SPARC. A method for producing the fertilizer according to one embodiment of the present invention may include protease treatment of bone tissue (steps S5 and S7). The location of the peptide chain cleaved by the protease is determined specifically by that protease. Therefore, when a particular protein is treated with a particular protease, the peptide fragments produced by the treatment are uniquely determined. Since each fragment has a different molecular weight, the protein before protease treatment can be identified from the peptide fragments contained in the fertilizer, for example, by mass spectrometry. Thus, a person skilled in the art can determine whether the peptide fragments contained in the fertilizer are peptide fragments derived from one or more selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan, and SPARC.

[0064] Table 1 shows examples of peptide fragments that may be detected by LC-MS / MS. These peptide fragments may appear when fertilizer is analyzed without trypsin treatment. Because it is not trypsin-treated, the C-terminus of the peptide fragment is an amino acid that is not Lys or Arg.

[0065] [Table 1]

[0066] In one embodiment, peptide fragments derived from one or more selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan, and SPARC have no physiological activity. The method for producing fertilizer according to one embodiment of the present invention includes several steps in which proteins may lose their physiological activity. One is a pretreatment step. In the pretreatment step, bone tissue is heated, heated under pressure, or irradiated with microwaves, causing the proteins to denature and lose their physiological activity. Another is the protease treatment in steps 1 and 3. Proteins treated with protease become fragments and lose their original physiological activity.

[0067] In one embodiment, the size of peptide fragments derived from one or more substances selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan, and SPARC is 10,000 Da or less, 8,000 Da or less, 6,000 Da or less, or 4,000 Da or less. Peptide fragments cleaved to this size are highly likely to have lost their physiological activity. Furthermore, peptide fragments cleaved to this size are easily absorbed as nutrients by plants and may function as active ingredients in fertilizers.

[0068] In this specification, "the physiological activity of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan, and SPARC" refers to the following. The loss of physiological activity of fragments derived from these means that they do not possess the following activities. • Physiological activity of type I collagen: It self-associates under physiological conditions to form fibers. • Physiological activity of alpha-2-HS-glycoprotein: It binds to calcium ions. • Physiological activity of periostin: It functions as a cell adhesion molecule in osteoblast progenitor cells. • Biological activity of biglycans: They bind to type I collagen. • Bioactivity of SPARC: Promotes type I collagen synthesis in cultured dermal fibroblasts.

[0069] In one embodiment, the fertilizer contains phosphoric acid. In a method for producing fertilizer according to one embodiment of the present invention, the concentration of phosphoric acid in the fertilizer tends to increase when a pretreatment step is performed. The concentration of phosphoric acid in the fertilizer may be, for example, 280 mM or more, 300 mM or more, or 320 mM or more.

[0070] In one embodiment, the fertilizer comprises both a peptide fragment derived from one or more selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan, and SPARC, and phosphate.

[0071] [3. Method for producing phosphoric acid] Another aspect of the present invention relates to a method for producing phosphoric acid using bone tissue as a raw material. The method for producing phosphoric acid includes, for example, a step of purifying calcium phosphate and a step of removing calcium.

[0072] An example of a calcium phosphate production process is the following procedure. 1. Add an alkaline solution to the acid extract obtained in step 2 to neutralize it. This will yield a precipitate. 2. Add an acidic solution (such as hydrochloric acid solution) to the precipitate to resolubilize it. 3. Repeat steps 1 and 2 to purify calcium phosphate.

[0073] In the calcium removal step, calcium is removed by adding a chelating agent to the purified calcium phosphate solution. Alternatively, calcium may be precipitated as calcium sulfate or calcium carbonate by adding one or more selected from the group consisting of sulfates, carbonates, and bicarbonates to the purified calcium phosphate solution. Examples of sulfates, carbonates, and bicarbonates include sodium sulfate, potassium sulfate, ammonium sulfate, magnesium sulfate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammonium carbonate, or any mixture of several of these components. The reagents added to the calcium phosphate solution may be added in powder form or as a solution (such as an aqueous solution). The above method can be applied to acidic solutions at room temperature, making it a convenient way to remove calcium.

[0074] [3.1. Method for producing exemplary phosphoric acid] Two methods can be considered for producing phosphate from bone tissue. One method involves incinerating the bones to produce bone ash and then refining the phosphate from the ash. In the process of refining the phosphate from bone ash, the conventional method of refining phosphate from phosphate rock is employed. However, this method consumes a large amount of energy and produces a large amount of CO2, making it unsuitable for achieving the SDGs.

[0075] Another manufacturing method is the one illustrated in the flowchart of Figure 3. Bone tissue contains inorganic components such as calcium and organic components such as collagen, in addition to phosphate. In the method illustrated in Figure 3, phosphate is dissolved in steps S11 and S12, and components other than phosphate and calcium are removed in steps S13 and S14. Steps S13 and S14 may be repeated, and the number of repetitions can be set as appropriate. In step S15, sulfate, carbonate, or bicarbonate is added to remove calcium. The calcium sulfate, calcium carbonate, or calcium bicarbonate produced in step S15 may be used as agricultural fertilizer or industrial raw material. Each step will be described in detail below.

[0076] In step S11, the bone tissue is pretreated. Step S11 is optional and does not need to be performed. Pretreatment of the bone tissue can increase the amount of phosphoric acid contained in the acid extract. This step is the same as step S1 described above, and the details are as explained in section [1.5].

[0077] In step S12, the bone tissue is treated with acid. This step yields an acid extract. This step is the same as step S2 described above, and the conditions for the acid treatment are as described in section [1.1].

[0078] In step S13, the acid extract is neutralized with a base. As a result of step S13, the phosphate ions in the acid extract precipitate as calcium phosphate, and components derived from bone impurities are included in the supernatant. Therefore, phosphate can be purified by recovering the precipitate. Examples of bases that can be used include sodium hydroxide and potassium hydroxide.

[0079] In step S14, an acid is added to the calcium phosphate precipitate. This causes the calcium phosphate that had precipitated to dissolve again. Examples of acids that can be used include hydrochloric acid, nitric acid, and formic acid.

[0080] Steps S13 and S14 may be repeated. Repeating these steps increases the purity of the phosphoric acid. The number of times steps S13 and S14 are repeated may be, for example, two or more times, three or more times, four or more times, or five or more times. From an economic standpoint, the number of times steps S13 and S14 are repeated may be, for example, 10 times or less. After repeating steps S13 and S14, before proceeding to step S15, a washing treatment to rinse away unwanted components contained in the calcium phosphate precipitate with pure water may be performed as many times as necessary.

[0081] In step S15, a sulfate, carbonate, or bicarbonate is added to the calcium phosphate solution. This causes the calcium ions in the solution to precipitate as calcium sulfate, calcium carbonate, or calcium bicarbonate. The precipitated calcium sulfate, calcium carbonate, or calcium bicarbonate is removed by centrifugation or other means. Examples of sulfates include one or more selected from sodium sulfate, potassium sulfate, ammonium sulfate, and magnesium sulfate. Examples of carbonates include one or more selected from sodium carbonate, potassium carbonate, and ammonium carbonate. Examples of bicarbonates include one or more selected from sodium bicarbonate and potassium bicarbonate. In addition, two or more of sulfates, carbonates, and bicarbonates can be arbitrarily combined. Adding sulfate instead of sulfuric acid in step S15 has the following advantages. • Since no liquid is added, the volume increase of the reaction system can be reduced. • Since the reaction system does not become strongly acidic, only a small amount of base is needed for neutralization. • High safety. • High calcium removal rate.

[0082] The amount of sulfate, carbonate, or bicarbonate added in step S15 can be appropriately determined by those skilled in the art. The amount of sulfate, carbonate, or bicarbonate added may be, for example, an amount such that the concentration of sulfate, carbonate, or bicarbonate in the reaction system is 0.2M or higher, 0.4M or higher, 0.6M or higher, 0.8M or higher, or 1.0M or higher. A larger amount of sulfate, carbonate, or bicarbonate added can reduce the amount of residual calcium. The upper limit of the amount of sulfate, carbonate, or bicarbonate added may be, for example, an amount such that the concentration of sulfate in the reaction system is 3.0M or lower, 2.0M or lower, or 1.0M.

[0083] In step S15, due to solubility product considerations, small amounts of sulfate ions, carbonate ions, or bicarbonate ions remain in the supernatant. When removing these ions, carbonate ions or bicarbonate ions are converted to carbon dioxide by heating, so they can be easily removed. Therefore, it is preferable that the salt added in step S15 be one or more types selected from the group consisting of carbonates and bicarbonates.

[0084] In step S16, the supernatant after calcium removal is purified. The supernatant may contain cations such as sodium ions and anions such as chloride ions that were present in the sulfate. High-purity phosphoric acid can be obtained by adsorbing these ions, for example, onto an ion exchange resin. If carbonate ions or bicarbonate ions are present, they can be removed from the system by converting them to carbon dioxide through heating.

[0085] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Examples]

[0086] [Example 1: Preparation of fertilizer containing bone tissue degradation products] [Example 1-1: Preparation of acid extract with nitric acid and component analysis] Bone tissue was treated with nitric acid according to the following procedure to obtain an acid extract. 1. Pork bones obtained from a slaughterhouse were finely ground using a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.). 2. 3g of pork bone (wet weight) was immersed in 40mL of nitric acid solution at 20°C. The concentrations of the nitric acid solution were 0.3mol / L, 0.5mol / L, 0.75mol / L, or 1.0mol / L. The immersion time was 12 hours, 24 hours, or 48 hours. 3. The supernatant was collected and used as the acid extract.

[0087] [Examples 1-2: Preparation of acid extract using hydrochloric acid and component analysis] Bone tissue was treated with hydrochloric acid according to the following procedure to obtain an acid extract. 1. Pork bones obtained from a slaughterhouse were finely ground using a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.). 2. 2 g of pork bone (wet weight) was immersed in 35 mL of hydrochloric acid solution at 20°C. The hydrochloric acid solution was concentrated to 0.3 mol / L, 0.5 mol / L, or 1.0 mol / L. The immersion time was 6 hours, 12 hours, or 24 hours. 3. The supernatant was collected and used as the acid extract.

[0088] [Examples 1-3: Preparation of acid extract using formic acid and component analysis] Bone tissue was treated with formic acid according to the following procedure to obtain an acid extract. 1. Pork bones obtained from a slaughterhouse were finely ground using a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.). 2. 2 g of pork bone (wet weight) was immersed in 35 mL of formic acid solution at 20°C. The formic acid solution was concentrated to 0.3 mol / L, 0.5 mol / L, or 1.0 mol / L. The immersion time was 6 hours, 12 hours, or 24 hours. 3. The supernatant was collected and used as the acid extract.

[0089] [Examples 1-4: Preparation of acid extract using sulfuric acid and component analysis] Bone tissue was treated with sulfuric acid according to the following procedure to obtain an acid extract. 1. Pork bones obtained from a slaughterhouse were finely ground using a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.). 2. 2 g of pork bone (wet weight) was immersed in 35 mL of sulfuric acid solution at 20°C. The sulfuric acid solution was concentrated to 0.3 mol / L, 0.5 mol / L, or 1.0 mol / L. The immersion time was 6 hours, 12 hours, or 24 hours. 3. The supernatant was collected and used as the acid extract.

[0090] Table 2 shows the results of the analysis of total phosphorus in the obtained acid extracts. Component analysis was outsourced to Kurita Analysis Center Co., Ltd. Table 2 shows the measurement results of total phosphorus (mg) per gram of wet bone weight. The analysis results are summarized for each acid, based on the concentration of nitric acid, hydrochloric acid, formic acid, or sulfuric acid, and the immersion time. It was shown that most of the phosphorus could be recovered in about 24 hours in any of the acidic aqueous solutions. Furthermore, it was shown that sufficient phosphorus could be recovered at an acid concentration of about 1 mol / L. Among the four types of acids examined, hydrochloric acid and sulfuric acid showed the highest phosphorus recovery efficiency. From Table 2, it was found that approximately 100 mg of phosphorus can be recovered from 1 g of bone tissue.

[0091] [Table 2]

[0092] Table 3 shows the results of the analysis of total calcium in the obtained acid extracts. Component analysis was commissioned to Kurita Analysis Center Co., Ltd. Table 3 shows the measurement results of total calcium (mg) per gram of wet bone weight. The analysis results are summarized for each acid, based on the concentration of nitric acid, hydrochloric acid, formic acid, and sulfuric acid, and the immersion time. It was shown that most of the calcium could be recovered in about 24 hours in aqueous solutions of nitric acid, hydrochloric acid, and formic acid. In the aqueous solution of sulfuric acid, the free calcium formed insoluble calcium sulfate. Therefore, although the calcium concentration in the analysis results is low, calcium is actually leaching out from the bone tissue. It was shown that sufficient calcium could be recovered at an acid concentration of about 1 mol / L. Among the four types of acids examined, hydrochloric acid and formic acid showed the highest calcium recovery efficiency. From Table 3, it was found that approximately 200 mg of calcium could be recovered from 1 g of bone tissue.

[0093] [Table 3]

[0094] [Examples 1-5: Preparation of protease-treated solution] The acid-treated bone tissue obtained in Examples 1-2 was immersed in a treatment solution containing the following two types of proteases. The protease treatment conditions were: protease concentration: 2% (w / w), temperature: 50°C, pH: optimal pH. • Protease 1: Neurase F3G (Amano Enzyme Co., Ltd., protease derived from the filamentous fungus Rhizopus niveus) • Protease 2: Pepsin (Sigma Aldrich, aspartate protease)

[0095] The results are shown in Figure 4. In Figure 4, A represents the results of treatment with protease 1, and B represents the results of treatment with protease 2. The treatment times were 3.5 hours, 19 hours, or 24 hours. The results of visual inspection of the transparency of the treatment solution and the remaining bone tissue are as follows. 3.5 hours later: In all systems treated with the protease, the bone tissue had almost completely disappeared. • 24 hours later: In all systems treated with proteases, bone tissue completely disappeared, and the clarity of the protease-treated solution also increased.

[0096] Thus, it was found that a protease-treated solution can be obtained by treating acid-treated bone tissue with a commercially available protease.

[0097] [Examples 1-6: Preparation and component analysis of protease-treated solutions] The protease-treated solution was obtained by following the procedure below. 1. Bone tissue was acid-treated by immersing it in a 1 mol / L nitric acid solution or hydrochloric acid solution for 48 hours. 2. The resulting treatment solution was separated into an acid extract and bone tissue residue, and the acid extract was transferred to a separate container. 3. 0.1 mol / L citrate buffer (pH 3.5) was added to the bone tissue residue. The amount added was 10 mL per gram of the initial bone weight. 4. The samples were pretreated with actinidine (a cysteine ​​protease derived from kiwifruit). Specifically, they were exposed to 20 mmol / L phosphate buffer (pH 6.5) containing 10 mmol / L dithiothreitol and 5 mmol / L ethylenediaminetetraacetic acid at 25°C for 90 minutes. 5. The reaction system obtained in step 3 was treated with protease by adding activated actinidine. The treatment conditions were: temperature: 20°C or 50°C, pH: 3.5 (adjusted with 100 mmol / L phosphate buffer), and time: 3 days.

[0098] The results are shown in Figure 5. In both systems treated with nitric acid or hydrochloric acid, bone tissue was almost completely eliminated at a reaction temperature of 50°C.

[0099] The resulting protease-treated solution was passed through a 100 μm filter to remove fine insoluble matter, and a filtrate was obtained. The results of the component analysis of this filtrate are shown in Table 4 (values ​​are shown converted to 1 g of wet bone weight). The component analysis was outsourced to Kurita Analysis Center Co., Ltd.

[0100] [Table 4]

[0101] [Examples 1-7: Preparation and component analysis of bone solubilizing solution A] Bone solubilizing solution A was prepared in a single step without solution exchange between acid treatment and protease treatment. The specific procedure is as follows. 1. Bone tissue was acid-treated by immersing it in a 1 mol / L aqueous solution of nitric acid, hydrochloric acid, formic acid, or sulfuric acid for 48 hours. 2. Citrate buffer (pH 3.5) was added to the reaction system to achieve a final concentration of 0.1 mol / L. 3. The pH was adjusted to the optimal pH for each protease using 5N NaOH and 5N HCl. Specifically, actinidine was adjusted to pH 3.5, pepsin to pH 3.0, and neurase F3G to pH 3.0. 4. Each protease was added to a concentration of 1% (w / w). The protease reaction conditions were 50°C for 4 days. Actinidine (cysteine ​​protease derived from kiwifruit) was pre-treated with 20 mmol / L phosphate buffer (pH 6.5) containing 10 mmol / L dithiothreitol and 5 mmol / L ethylenediaminetetraacetic acid at 25°C for 90 minutes.

[0102] The results are shown in Figure 6. In all systems, the bone tissue was reduced in size when the reaction temperature was 50°C. Thus, bone solubilization solution A was obtained from bone tissue in a single step without the need for solution exchange between the acid treatment and the protease treatment. In particular, the combination of pepsin with hydrochloric acid or nitric acid dissolved the bone tissue well, and only a small amount of bone tissue remained.

[0103] The obtained bone solubilization solution A was passed through a 100 μm filter to remove fine insoluble matter and obtain a filtrate. The calcium concentration of this filtrate was measured and the results are shown in Table 5 (values ​​are shown converted to per gram of bone weight). Calcium was measured using a compact calcium ion meter LAQUAtwin-Ca-11 (Horiba Advanced Technology Co., Ltd.).

[0104] [Table 5]

[0105] [Examples 1-8: Extraction of Phosphate] Crudely purified phosphate was obtained from bone tissue using the following procedure. 1. Bone tissue was acid-treated by immersing it in a 1 mol / L aqueous solution of nitric acid, hydrochloric acid, formic acid, or sulfuric acid for 48 hours. 2. 0.15 volume of 5N NaOH was added to the reaction system and mixed thoroughly, then allowed to stand at room temperature for 1 hour. A white solution was obtained. 3. The obtained solution was centrifuged at 10,000 g for 10 minutes at room temperature, and the supernatant was removed. The same amount of 1N HCl added in step 1 was added to the white precipitate and mixed thoroughly. A clear solution was obtained. 4. Add Na2SO4, K2SO4, MgSO4, (NH4)2SO4, Na2CO3, or NaHCO3 to the reaction system in an amount equal to the calcium concentration present, mix well, and let stand at room temperature for 1 hour. This yielded a white solution. 5. The obtained solution was centrifuged at 10,000 g for 10 minutes at room temperature, and the supernatant was collected. The resulting clear solution contained phosphoric acid.

[0106] By immersing bone tissue in sulfuric acid, a phosphoric acid-containing solution from which some calcium has been removed can be obtained. However, treatment with sulfuric acid increases the volume, makes the solution highly acidic, and introduces many impurities, making purification difficult. On the other hand, the above process allows for the simple acquisition of a highly pure phosphoric acid-containing solution.

[0107] [Example 2: Growth evaluation and component analysis of plants grown with fertilizer containing bone tissue degradation products] [material and method] 1. Radish (Raphanus sativus L) seeds (Takii Seed Co., Ltd.) were sown on a damp paper towel. The towel was left undisturbed in the dark at 22°C for two days to allow the seeds to germinate. 2. The obtained plants were transplanted into hydroponic urethane cubes (2cm x 2cm x 2cm). 3. Urethane cubes were immersed in acid extract, protease treatment solution, or bone solubilizing solution A and allowed to grow for 5 days. The light conditions during this period were: Photosynthetic photon flux density: approximately 150 μmol / m² 2 The experiment consisted of a 16-hour light period and an 8-hour dark period. A fluorescent lamp designed for cultivating plants (Biorux A, NEC Lighting Corporation) was used as the light source. 4. Growth evaluation and component analysis were performed on the grown plants. Specifically, the following was done: • Fresh weight of above-ground parts: The plant was cut at the boundary between the hypocotyl and roots, and the weight of the hypocotyl side was measured. • Dry weight of above-ground parts: The plant portion whose fresh weight was measured was dried in an 80°C oven for two days. Its weight was then measured. • Above-ground moisture content: Calculated based on the difference between the fresh weight and the dry weight of the above-ground parts. • Leaf area: The cotyledons of the plant were scanned, and the leaf area was measured using ImageJ. • Total polyphenol content: 50 mg of cotyledons were crushed in a 90% methanol solution. The total polyphenol content was measured using the supernatant obtained by centrifugation by the Forin-Ciocalt method.

[0108] [Results and Discussion] (1) Effect of acid extract concentration on growth Plants were grown under conditions where they were given acid extracts diluted 500-fold or 2000-fold. The results are shown in Figure 7. Plants grown with the 500-fold diluted acid extract showed an increase in above-ground fresh weight compared to control plants. From these results, it was found that acid extracts diluted to about 500-fold have a growth-promoting effect on plants. In subsequent experiments, acid extracts diluted 500-fold and protease-treated solutions were used.

[0109] (2) Effects of acid extract, protease-treated solution, or bone solubilizing solution A on growth Plants were grown under the following six conditions, and the fresh weight of the above-ground parts was compared. • Provide only water (control) • Provide an unheated protease treatment solution (protease treatment solution 1). • Provide a protease-treated solution (protease-treated solution 2) that has been heat-treated at 50°C. • Give the acid extract. • Provide bone solubilizing solution A, which is a mixture of protease-treated solution 1 and acid extract. • Provide bone solubilizing solution A, which is a mixture of protease-treated solution 2 and acid extract. The results are shown in Figure 8. Plants treated with protease solution 1 and protease solution 2 showed accelerated growth compared to the control. Similarly, plants treated with acid extract also showed accelerated growth compared to the control (as shown in (1)). Furthermore, plants treated with bone solubilization solution A, a mixture of protease solution and acid extract, showed an even greater growth-promoting effect compared to the control. From these results, it can be seen that both acid extract and protease solution have a growth-promoting effect on plants. Moreover, it was found that the growth-promoting effect of bone solubilization solution A was higher than that of acid extract or protease solution alone.

[0110] (3) Comparison of effects with commercially available culture media The growth-promoting effects of bone solubilizing solution A and a commercially available culture medium were compared. For the commercially available culture medium, a 1 / 6 dilution of formula A from OAT House (OAT Agrio Co., Ltd.) was used. This dilution was adjusted to achieve the same nitrate ion concentration (approximately 600 ppm) as bone solubilizing solution A.

[0111] The results are shown in Figures 8 and 9. Plants given commercially available culture medium showed growth promotion to the same extent as plants given bone solubilizer A. Plants given both bone solubilizer A and culture medium showed an even greater growth promotion effect than plants given each individually. This suggests that bone solubilizer A can further enhance the growth promotion effect of commercially available culture medium.

[0112] The above-ground water content of the plant increased with the application of bone solubilizer A, and further increased with the application of both bone solubilizer A and the culture solution. The total polyphenol content per cotyledon increased with the application of bone solubilizer A, and further increased with the application of both bone solubilizer A and the culture solution. This is thought to be due to the increased cotyledon weight resulting from the promotion of plant growth.

[0113] [Example 3: RNA expression analysis of plants 1] [Total RNA extraction from plant tissue] 1. Seeds of Japanese radish (Raphanus sativus L) (Takii & Co., Ltd.) were sown on wet paper towels. The seeds were allowed to stand for 2 days at 22°C under dark conditions with 100% humidity to germinate. 2. The obtained plant bodies were transplanted onto urethane sponges. The urethane sponges were impregnated with any one of (1) water, (2) bone-solubilized liquid A, (3) a commercially available liquid fertilizer, or (4) a mixed liquid of bone-solubilized liquid A and a commercially available liquid fertilizer. 3. The plant bodies were grown at 22°C for 1 day, 3 days, or 5 days. The light conditions during this period were: light intensity: about 100 μmol / m 2 ·s, light period: 16 hours, dark period: 8 hours. As the light source, a fluorescent lamp for cultivating and growing plants (Biolux A, NEC Lighting, Ltd.) was used. 4. Approximately 0.1 g each of leaf and root tissues were collected from the grown plant bodies. Immediately after collection, the tissues were placed in a mortar, liquid nitrogen was added, and the tissues were ground in a frozen state. 5. Total RNA was extracted from the ground tissues using the RNeasy Plant Mini Kit (Qiagen). 6. The concentration of total RNA in the aqueous solution was measured using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and a fluorophotometer (Qubit-4). In addition, the absorbance at 230 nm, 260 nm and 280 nm (A 230 , A 260 and A 280 ) was measured with a spectrophotometer to examine the purity of the extracted RNA.

[0114] The results are shown in Table 6. At the time point up to 5 days after the start of culture, 13.2 to 88.2 μg of total RNA was extracted from approximately 0.1 g of plant leaf. 8.4 to 43.8 μg of total RNA was extracted from approximately 0.1 g of plant root. A 260 / A 280 was about 2.0, and it was confirmed that there was no problem with the RNA purity.

[0115] [Table 6]

[0116] [Construction of RNA sequencing libraries and RNA sequence analysis using next-generation sequencers] The preparation of a cDNA library from total RNA extracted from sprouts and the gene expression analysis using a next-generation sequencer were outsourced to Azenta Co., Ltd. The analysis procedure is as follows: 1. The quality (degree of RNA degradation) of the extracted total RNA was evaluated using BioAnalyzer (Agilent Technologies). 2. From the extracted total RNA, polyA-mRNA was enriched using beads conjugated with polyT oligoDNA. 3. A cDNA library was prepared using reverse transcriptase. 4. The cDNA was sequenced using a next-generation sequencer (DNBSEQ-G400, MGI tech). This allowed for comprehensive determination of the sequence of the expressed mRNA (RNA-seq analysis). Sprout gene assembly was performed using Stringtie software. For this purpose, the whole genome sequence of sprouts, publicly available in the database of the Kazusa DNA Research Institute (http: / / radish.kazusa.or.jp), was referenced.

[0117] [Analysis of genes with altered expression levels] 1. Genes whose expression levels significantly changed due to differences in cultivation conditions were identified based on FPKM values ​​(fragments per kilobase of exon per million reads mapped), which are obtained by correcting the number of reads for each gene by the transcript length. DESeq2 software was used for identification. 2. Genes (DEGs) with significantly increased or decreased expression levels were comprehensively identified. The analysis results were displayed using a volcano plot. Expression levels were compared in four groups: (1) water only, (2) bone solubilizing solution A, (3) commercially available liquid fertilizer, and (4) a mixture of bone solubilizing solution A and commercially available liquid fertilizer. Genes with significantly increased or decreased expression levels were identified in groups (2) or (4). 3. GO enrichment analysis was performed on the identified differentially expressed genes using GOseq software. This allowed for the analysis of the biological function of the differentially expressed genes.

[0118] [Quantitative RT-PCR] We targeted five genes closely related to stress tolerance and examined their expression using quantitative RT-PCR. The target genes were glutathione S-transferase τ19 (GSTU19), catalase 2 (CAT2), auxin transporter-like protein 2-1 (LAX2-1), glutathione S-transferase 12 (GST12), and calmodulin 5 (CaM5). The specific procedure is as follows. 1. Total RNA was extracted from the leaves and roots of plants grown with (1) water, (2) bone solubilizing solution A, (3) commercially available liquid fertilizer, or (4) a mixture of bone solubilizing solution A and commercially available liquid fertilizer. 2. A reverse transcriptase reaction (RT reaction) was performed using random primers. PrimeScript RT Master Mix (Takara Bio Inc.) was used as the reverse transcriptase. 3. The target gene was subjected to quantitative PCR. For quantitative PCR, a specific primer pair using 2.0 ng of cDNA as a template, TB Green Premix Ex Taq II (Takara Bio Inc.), was used. The instrument used to perform quantitative PCR was the Thermal Cycler Dice Real Time System TP850 (Takara Bio Inc.).

[0119] In quantitative RT-PCR, the expression levels of each target gene were standardized relative to the expression level of the actin gene. The nucleotide sequences of the primer pairs used to amplify the target genes and actin gene are shown in Table 7 (Sequence IDs 1-12, from top to bottom).

[0120] [Table 7]

[0121] [result] The expression level of the gene encoding glutathione S-transferase τ19 (GSTU-19) was not detected before transplantation into the urethane sponge. Similarly, on day 1 after transplantation, it was not detected in the leaves and roots of plants grown in a system containing (1) water, (2) bone solubilizing solution A, and (3) commercially available liquid fertilizer. However, on day 1 after transplantation, the expression level of GSTU-19 was significantly elevated in the leaves and roots of plants grown in a system containing (4) a mixture of bone solubilizing solution A and commercially available liquid fertilizer (Figure 11). Likewise, on day 1 after transplantation, the expression levels of the genes encoding catalase 2 (CAT2), auxin transporter-like protein 2-1 (LAX2-1), glutathione S-transferase 12 (GST12), and calmodulin 5 (CaM5) were significantly elevated in the leaves of plants grown in a system containing (4) a mixture of bone solubilizing solution A and commercially available liquid fertilizer (Figure 12).

[0122] Thus, it was shown that using the fertilizer according to one embodiment of the present invention in combination with existing liquid fertilizers promotes the expression of genes involved in plant stress tolerance. This result suggests that the stress tolerance of plants can be improved by using the fertilizer according to one embodiment of the present invention in combination with existing liquid fertilizers.

[0123] [Example 4: Improvement of phosphoric acid extraction rate by pretreatment] We confirmed that pretreatment of bone tissue increased the amount of phosphate in the acid extract. Specifically, the acid extract was prepared using the following procedure, and the amount of phosphate contained therein was quantified. 1. Pork bones obtained from a slaughterhouse were finely ground using a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.). 2. 5g of crushed pork bone was subjected to one of the following pretreatments (1) to (5). (1) No processing (2) Immerse in 30 mL of nitric acid (1 mol / L) at 50°C for 1 day. (3) Irradiate with 500W microwaves for 30 seconds. (4) Irradiate with 500W microwaves for 60 seconds. (5) Irradiate with 500W microwaves for 120 seconds. 3. 30 mL of nitric acid (1 mol / L) was added to the pork bones from (1) and (3) to (5). The pork bones from (1) to (5) were immersed in 30 mL of nitric acid and left to soak at 20°C for 48 hours while shaking (100 rpm). In this way, the bone tissue was demineralized. 4. The supernatant was collected and used as the acid extract. 5. Phosphate concentration was measured using the Malachite Green Phosphate Assay Kit (BioAssay Systems). The measurement method followed the product manual. The supernatant was diluted with distilled water for measurement.

[0124] [result] The results are shown in Figure 13. From bone tissue (2) pretreated by heating at 50°C, 1.18 times more phosphate was extracted than from bone tissue (1) that was not pretreated. Furthermore, from bone tissues (3) to (5) pretreated by microwave irradiation, up to 1.26 times more phosphate was extracted than from bone tissue (1) that was not pretreated.

[0125] Thus, it was found that the amount of phosphoric acid extracted into the acid extract increases by appropriate pretreatment. In particular, microwave irradiation is a preferred embodiment because it can be completed in a short time, does not require heating, can be applied to large bone tissues, and can further improve the phosphoric acid extraction rate.

[0126] [Example 5: Analysis of protein components contained in the acid extract] The protein components contained in the acid extract were analyzed using the following procedure. 1. Pork bones obtained from a slaughterhouse were finely ground using a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.). 2. 2 g of pork bone (wet weight) was immersed in 30 mL of hydrochloric acid (1 mol / L) at 20°C for 48 hours while shaking (100 rpm). The bone tissue was demineralized in this manner. 4. The supernatant was collected and used as the acid extract. 5. The obtained acid extracts were divided into three solutions: (1) undiluted acid extract, (2) 1 / 2 diluted acid extract, and (3) 1 / 4 diluted acid extract. 6. An appropriate amount of 1 mol / L NaOH solution was added to neutralize the pH. 5. Equal amounts of dithiothreitol were added as a reducing agent, and the mixture was heated at 95°C for 10 minutes. 6. Electrophoresis was performed using a 5% polyacrylamide gel (for high molecular weight components) and a 12.5% ​​polyacrylamide gel (for low molecular weight components) by a conventional method.

[0127] [result] The results are shown in Figure 14. As a control sample, type I collagen, a protein abundant in bone, and a molecular weight marker were subjected to electrophoresis on the same gel. As can be seen from the figure, no proteins with a molecular weight of 10,000 Da or more, which are stained by a highly sensitive silver staining kit, were detected in the acid extract. In other words, it was found that the acid extract contained almost no protein components. This is thought to be because the peptide bonds of the proteins were cleaved by treatment with hydrochloric acid, a strong acid, resulting in the breakdown of low molecular weight peptides and amino acids.

[0128] [Example 6: Preparation of protease-treated solution and analysis of protein components] [Example 6-1: Preparation of protease-treated solution] The protease-treated solution was obtained by following the procedure below. 1. Bone tissue was immersed in a 1 mol / L hydrochloric acid solution for 48 hours to treat it with acid. 2. The resulting treatment solution was separated into an acid extract and bone tissue residue, and the acid extract was transferred to a separate container. 3. 0.1 mol / L citrate buffer (pH 3.5) was added to the bone tissue residue. The amount added was 10 mL per gram of the initial bone weight. 4. The obtained bone tissue residue was immersed in a treatment solution containing the following three types of proteases. The protease treatment conditions were: protease concentration: 2% (w / w), temperature: 50°C, pH: optimal pH. • ProLeather FG-F (Amano Enzyme Co., Ltd., derived from Bacillus sp.) • Protease P "Amano" 3G (Amano Enzyme Co., Ltd., derived from Aspergillus melleus) • Protease M "Amano" SD (Amano Enzyme Co., Ltd., derived from Aspergillus oryzae)

[0129] [result] The appearance of the treated solution is shown in Figure 15. For reference, the results of Examples 1-5 (Neurase F3G and Pepsin) and 1-6 (Actinidine) are also shown. As can be seen from the figure, it was found that the bone decomposition residue was also decomposed by the proteases examined in this example, and a protease-treated solution was obtained. A precipitate was observed in the protease-treated solution obtained in this example, which is thought to be the precipitation of calcium salts due to the near-neutral pH of the solution. Taking into account the results of Example 6-2, which will be described later, it can be said that the proteins contained in the bone decomposition residue were sufficiently decomposed by the protease treatment.

[0130] [Example 6-2: Analysis of protein components contained in protease-treated solution] A protease-treated solution was prepared using the following proteases in the same manner as in Examples 1-5, 1-6, and 6-1. • Pepsin (Sigma Aldrich, aspartate protease) • Peptidase R (Amano Enzyme Co., Ltd., derived from the genus Rhizopus) • Protease P "Amano" 3G (Amano Enzyme Co., Ltd., derived from Aspergillus melleus) • ProLeather FG-F (Amano Enzyme Co., Ltd., derived from Bacillus sp.) • Neurase F3G (Amano Enzyme Co., Ltd., derived from Rhizopus niveus) • Protease M "Amano" SD (Amano Enzyme Co., Ltd., derived from Aspergillus oryzae) • Actinidine (derived from kiwifruit, cysteine ​​protease)

[0131] The resulting protease-treated solution was diluted 10-fold (lane A) or 5-fold (lane B) and subjected to electrophoresis using a 16% polyacrylamide gel. The electrophoresed proteins were stained with silver. As a control sample, a solution containing only the enzyme (lane C) was also subjected to electrophoresis.

[0132] The results are shown in Figure 16. As shown in the figure, each protease-treated solution contained protein components with molecular weights different from the enzyme (lane C). In other words, regardless of which protease was used for treatment, the proteins contained in the bone tissue residue were broken down. These broken-down peptides are then utilized by the plant as a nutrient source.

[0133] [Example 7: Recovery of phosphate from bone tissue] Phosphoric acid was recovered as a precipitate from the acid extract using the following procedure. The recovery rate of phosphoric acid was also calculated. 1. Pork bones obtained from a slaughterhouse were finely ground using a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.). 2. The crushed pork bones were immersed in a 1N nitric acid solution or a 1N hydrochloric acid solution for 48 hours. 3. The supernatant was collected to obtain the acid extract. 4. To 0.5 mL of the acid extract, 30 μL, 50 μL, 70 μL, 100 μL, or 120 μL of 5N sodium hydroxide aqueous solution were added and allowed to stand at room temperature for 1 hour. This precipitated phosphoric acid. 5. The reaction mixture was separated into supernatant and precipitate by centrifugation. 6. 0.5 mL of 1N hydrochloric acid solution was added to the precipitate to redissolve the phosphoric acid. 7. The phosphate content was measured in the redissolved precipitate obtained in step 5 and in the supernatant obtained in step 4. A Malachite Green Phosphate Assay Kit (BioAssay Systems) was used for the measurements. The phosphate recovery rate was calculated based on the phosphate content in the acid extract obtained in step 3.

[0134] [Table 8]

[0135] [result] The results are shown in Table 8. In the acid extract using nitric acid, more than 70% of the phosphoric acid could be recovered as a precipitate when the amount of NaOH aqueous solution added was 100 μL or more. On the other hand, the amount of phosphoric acid in the supernatant decreased to about 10% when the amount of NaOH aqueous solution added was 120 μL. In the acid extract using hydrochloric acid, more than 70% of the phosphoric acid could be recovered as a precipitate when the amount of NaOH aqueous solution added was 70 μL or more. On the other hand, the amount of phosphoric acid in the supernatant decreased to about 10% when the amount of NaOH aqueous solution added was 100 μL. Thus, by adding an appropriate amount of NaOH aqueous solution to the acid extract, phosphoric acid could be recovered as a precipitate and crudely purified.

[0136] Incidentally, the phosphoric acid content in the acid extract using nitric acid was 24.6 mg. This represents 34.7% of the wet weight of the pork bone used in step 2 (71 mg). The phosphoric acid content in the acid extract using hydrochloric acid was 15.1 mg. This represents 20.2% of the wet weight of the pork bone used in step 2 (74.5 mg). Since the weight of phosphoric acid in bone tissue before drying is said to be 25-35%, it means that up to 99% (=34.7 / 35 × 100) of phosphoric acid can be extracted by preparing the acid extract.

[0137] [Example 8: Purification of phosphoric acid by addition of sulfate] [Example 8-1: Investigation of phosphate recovery rate in the supernatant] Calcium was removed as calcium sulfate precipitate from the redissolved phosphate precipitate obtained in Example 7 by following the procedure described below. 1. A redissolution solution of the phosphate precipitate was prepared in the same manner as in Example 7. The acid used in step 2 of Example 7 was a 1N aqueous nitric acid solution or a 1N aqueous hydrochloric acid solution. The amount of 5N aqueous sodium hydroxide solution added in step 4 of Example 7 was 100 μL. 2. Sulfuric acid or sulfate (sodium sulfate, potassium sulfate, ammonium sulfate, or magnesium sulfate) was added. The amount of sulfuric acid or sulfate added was such that the final concentration was 0.4 M, 0.6 M, 0.8 M, or 1.0 M. 3. The resulting calcium sulfate precipitate was removed by centrifugation. 4. The weight of phosphate contained in the supernatant was measured. A Malachite Green Phosphate Assay Kit (BioAssay Systems) was used for the measurement. The phosphate recovery rate was calculated based on the phosphate content in the precipitate (the precipitate obtained in step 5 of Example 7) formed by adding aqueous sodium hydroxide solution to the acid extract before adding sulfuric acid or sulfate.

[0138] [Table 9]

[0139] [result] The results are shown in Table 9. It was found that, by appropriately setting the sulfate concentration, a phosphoric acid recovery rate comparable to that of sulfuric acid can be achieved with sulfate. Furthermore, at a low concentration of 0.4 M, the phosphoric acid concentration obtained by adding sulfate tended to be slightly higher than that obtained by adding sulfuric acid. Importantly, adding sulfate not only reduces the volume of the system compared to adding sulfuric acid, but also yields a phosphoric acid concentration approximately 1.5 times higher. In this respect, the method according to one embodiment of the present invention is a useful method for purifying phosphoric acid. Moreover, the fact that a smaller amount of sulfuric acid or sulfate is preferable when commercializing the technology further demonstrates the effectiveness of the calcium removal effect of sulfate.

[0140] Using sulfates instead of sulfuric acid for calcium precipitation offers the following advantages: • The phosphate concentration is about 1.5 times higher. • Since no liquid is added, the volume increase of the reaction system can be reduced. • Since the reaction system does not become strongly acidic, only a small amount of base is needed for neutralization. • High safety.

[0141] [Example 8-2: Examination of calcium removal ability from the supernatant] The calcium content of the supernatant obtained in step 4 of Example 8-1 was measured. LAQUAtwin-Ca-11 was used for the measurement. In addition, the calcium residue rate was calculated based on the calcium content of the precipitate (the precipitate obtained in step 5 of Example 7) formed by adding an aqueous sodium hydroxide solution to the acid extract before adding sulfuric acid or sulfate.

[0142] [Table 10]

[0143] [result] The results are shown in Table 10. When compared at the same concentration, sulfates generally resulted in lower calcium residue than sulfuric acid. For example, in an experimental system where 0.4 M sodium hydroxide was added after acid treatment with hydrochloric acid, the amount of calcium in the solution was 1,210 μg for H2SO4, compared to 155-270 μg for sulfates. In other words, the calcium removal ability under these conditions was up to 7.8 times higher for sulfates. Importantly, when comparing the solution with added sulfates to the solution with added sulfuric acid, the volume of the former solution was about half that of the latter, yet the calcium concentration in the former solution was reduced to about half that of the latter. In other words, adding sulfates allows for the efficient removal of calcium that is unnecessary for the purification of phosphoric acid.

[0144] When comparing the acids used for acid treatment, treatment with hydrochloric acid resulted in a lower calcium residue rate than treatment with nitric acid. The system that removed the most calcium was the one in which 1M potassium sulfate was added to the acid extract obtained by acid treatment with hydrochloric acid.

[0145] Table 11 shows the relative ratio (%) of calcium amount (mg) / phosphate amount (mg) calculated based on the results in Tables 9 and 10. Although phosphate can also be recovered by adding sulfuric acid, the amount of residual calcium was shown to be higher than in the experimental system with added sulfate at all concentrations. On the other hand, when phosphate was recovered by adding sulfate, the amount of residual calcium was significantly lower than in the experimental system with added sulfuric acid, and the amount of recovered phosphate was also shown to be relatively higher. Thus, by adding an appropriate sulfate, the amount of residual calcium can be reduced to 0.2% by weight without reducing the amount of recovered phosphate. Therefore, it can be said that it is preferable to use sulfate rather than sulfuric acid for the purification of phosphate extracted from bone tissue. [Table 11]

[0146] [Example 8-3: Investigation of phosphate leaching rate into precipitate] The phosphoric acid content in the calcium sulfate precipitate obtained in step 3 of Example 8-1 was measured according to the following procedure. 1. EDTA (pH 7.4) and 5N sodium hydroxide were added to the calcium sulfate precipitate and redissolved. 2. The phosphoric acid content in the obtained solution was measured. A Malachite Green Phosphate Assay Kit (BioAssay Systems) was used for the measurement. In addition, the phosphoric acid leaching rate was calculated based on the phosphoric acid content in the precipitate (the precipitate obtained in step 5 of Example 7) formed by adding an aqueous sodium hydroxide solution to the acid extract before adding sulfuric acid or sulfate.

[0147] [Table 12]

[0148] [result] The results are shown in Table 12. As can be seen from the table, by appropriately adjusting the sulfate concentration, the phosphate content in the calcium sulfate precipitate could be reduced to a level similar to that of sulfuric acid. A sulfate concentration of 0.4 M was sufficient to sufficiently reduce the amount of phosphate that leaked out.

[0149] [Example 8-4: Investigation of total phosphate recovery rate] The sum of the phosphate recovery rate in the supernatant examined in Example 8-1 and the phosphate leaching rate to the precipitate examined in Example 8-3 was investigated. The phosphate contained in the precipitate is likely to be recovered by washing the precipitate. For example, the phosphate contained in the precipitate (calcium sulfate) can be recovered by washing it with pure water. Therefore, the higher the total phosphate content, the greater the potential amount of phosphate that can be recovered.

[0150] [Table 13]

[0151] [result] The results are shown in Table 13. It was found that, with appropriate concentration adjustment, a phosphoric acid recovery rate comparable to that of sulfuric acid could be achieved with sulfates. The total recovery rate was higher with the acid extract using hydrochloric acid, but the absolute phosphoric acid content was similar between the acid extracts using hydrochloric acid and those using nitric acid.

[0152] [Example 9: RNA expression analysis of plants 2] [CDNA library construction and gene expression analysis using next-generation sequencing] The procedure was carried out in the same manner as in Example 3.

[0153] [Analysis of genes with altered expression levels] The procedure was carried out in the same manner as in Example 3. The expression levels were compared in two sets: bone solubilizer B vs. commercially available liquid fertilizer, and a mixture of bone solubilizer B and commercially available liquid fertilizer vs. commercially available liquid fertilizer alone.

[0154] [result] Figure 18 shows the results of gene expression analysis in sprout leaves cultivated for 5 days with bone solubilizing solution B or commercially available liquid fertilizer. 1,568 genes showed a doubling or greater increase in FPKM value. 1,654 genes showed a decrease of half or less in FPKM value. Therefore, the expression levels of a total of 3,222 genes were altered. GO enrichment analysis revealed that among the genes with altered expression levels were 65 genes related to growth control, 44 genes related to photosynthesis, and 26 genes related to light harvesting in photosystem I of photosynthesis.

[0155] Figure 19 shows the results of gene expression analysis in sprout roots cultivated for 5 days with a mixture of bone solubilizing solution B and commercially available liquid fertilizer, or with only commercially available liquid fertilizer. 766 genes showed a doubling or greater increase in FPKM value. 1,235 genes showed a decrease of half or less in FPKM value. Therefore, the expression levels of a total of 2,001 genes were altered. GO enrichment analysis revealed that the genes with altered expression levels included 17 genes related to nitrate assimilation, 16 genes related to root hair elongation, and 12 genes related to nitrate uptake.

[0156] Figure 20 shows the results of gene expression analysis in sprout leaves cultivated for 5 days with a mixture of bone solubilizing solution B and commercially available liquid fertilizer, or with only commercially available liquid fertilizer. 582 genes showed a doubling or greater increase in FPKM value. 662 genes showed a decrease of half or less in FPKM value. Therefore, the expression levels of a total of 1,244 genes were altered. GO enrichment analysis revealed that the genes with altered expression levels included 32 genes related to the response to abscisic acid (a plant hormone), 13 genes related to root hair elongation, and 6 genes related to germination promotion.

[0157] The results shown in Figures 18-20 indicate that bone solubilizer B alters gene expression in sprouts. In particular, the expression of gene groups related to tissue growth in the leaves or roots, photosynthesis in the leaves, and nutrient absorption in the roots were significantly altered.

[0158] [Example 10: Investigation of calcium ion removal efficiency by adding sulfate] Calcium ions were removed as calcium carbonate precipitate by adding sulfate to the redissolution solution of the phosphate precipitate. The calcium ion content in the supernatant after precipitate removal was measured to examine the calcium removal efficiency. The specific procedure is as follows: 1. A redissolution solution of the phosphate precipitate was prepared in the same manner as in Example 7. The acid used in step 2 of Example 7 was a 1N hydrochloric acid aqueous solution. The amount of 5N sodium hydroxide aqueous solution added in step 4 of Example 7 was 100 μL. 2. Liquid sulfuric acid (H2SO4) or solid sulfates (Na2SO4, K2SO4, Mg2SO4, and (NH4)2SO4) were added. The amount of sulfuric acid or sulfate added was such that the final concentration was 0.4 M. 3. The resulting calcium sulfate precipitate was removed by centrifugation. 4. The weight (mg) of calcium ions contained in the supernatant was measured. Calcium ion measurement was performed under precise measurement conditions using a high-performance ion chromatography IC-8100EX (Tosoh Corporation) connected to a TSKgel SuperIC-Cation HSII (4.6 mm ID × 10 cm). A mixture of 3.0 mmol / L metasulfonic acid and 2.7 mmol / L 18-crown-6 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. The electroelectricity (μS) was measured, and the calcium ion concentration was determined by calculating a regression equation from the area of ​​the standard substance. From the calcium ion concentration, the calcium ion content per sample (30 μL) was determined.

[0159] [Table 14]

[0160] [result] The results are shown in Table 14. The system with added liquid sulfuric acid not only increased the overall volume of the system, but also had the lowest calcium ion removal rate at 73%. The system with added solid sulfate showed less increase in overall volume and a higher calcium ion removal rate of 91-94%. Furthermore, since it does not use sulfuric acid, a strong acid, the method of adding sulfate is also safer. In addition, by appropriately increasing or decreasing the amount of sulfate added, it was possible to retain the desired concentration of calcium.

[0161] When producing phosphorus or phosphorus-containing fertilizers from bone tissue, calcium ions present in the bone tissue contribute to reduced phosphorus recovery. This is because, in the neutral pH range, calcium ions combine with phosphate ions to form calcium phosphate precipitate. Therefore, it is desirable to remove calcium ions by precipitation, and it has been shown that this can be successfully achieved by adding sulfate.

[0162] The potassium, magnesium, or ammonium ions contained in sulfates are essential components for plant growth. Therefore, using potassium sulfate, magnesium sulfate, or ammonium sulfate as the sulfate allows the fertilizer to contain these essential components for plant growth. Alternatively, changing the base added in step 4 of Example 7 from sodium hydroxide to potassium hydroxide also allows the fertilizer to contain these essential components for plant growth. By adopting such a manufacturing method, a fertilizer with increased value can be produced.

[0163] [Example 11: Purification of phosphoric acid by adding carbonate] Calcium was removed as a calcium carbonate precipitate from the redissolved phosphate precipitate obtained in Example 7 by following the procedure described below. 1. A redissolution solution of the phosphate precipitate was prepared in the same manner as in Example 7. The acid used in step 1 of Example 7 was a 1N aqueous nitric acid solution or a 1N aqueous hydrochloric acid solution. The amount of 5N aqueous sodium hydroxide solution added in step 4 of Example 7 was 100 μL. 2. A carbonate (solid sodium bicarbonate or sodium carbonate) was added. The amount of sodium bicarbonate added was such that the final concentration was 0.4 M. The amount of sodium carbonate added was such that the final concentration was 0.6 M. 3. The resulting calcium carbonate precipitate was removed by centrifugation. 4. The concentration (ppm) of phosphate ions in the supernatant was measured. The phosphate ion concentration was measured using a high-performance ion chromatograph 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 electroelectricity (μS) was measured, and the phosphate ion concentration was determined by calculating a regression equation from the area of ​​the standard material. From the phosphate ion concentration, the phosphate ion content per sample (30 μL) was determined.

[0164] [Table 15]

[0165] [result] The results are shown in Table 15. Upon addition of carbonate, calcium ions precipitated as calcium carbonate, while phosphate ions remained in the supernatant. For example, in a system where sodium bicarbonate was added to an acid extract with hydrochloric acid, the supernatant contained more than 9,000 ppm of phosphate. The carbonate ions remaining in the supernatant can be easily removed by heating, as they are converted to carbon dioxide.

[0166] [Example 12: Removal of cations contained in sulfate] [Example 12-1: Removal of sodium or potassium ions] The following procedure was used to remove the cations (sodium or potassium ions) contained in the sulfate added to remove calcium ions. Specifically, sodium or potassium ions were adsorbed using a strong cation exchange gel. 1. TSKgel SP-TOYOPEARL 650M gel (Tosoh Corporation) was decanted with pure water and packed into a microspin column (GE Healthcare). 2. An appropriate amount of the supernatant, after removing calcium sulfate by adding sulfate, was added to the upper layer of the gel. 3. The supernatant was passed through the gel by centrifugation using a benchtop centrifuge. The fraction that passed through the gel was collected. 3. The sodium ion or potassium ion content (mg) in the pass-through fraction was measured. LAQUAtwin-Na-11 (Horiba Advanced Technology Co., Ltd.) was used for sodium ion measurement. LAQUAtwin-K-11 (Horiba Advanced Technology Co., Ltd.) was used for potassium ion measurement.

[0167] [Table 16]

[0168] [result] The results are shown in Table 16. The supernatant after precipitating calcium sulfate by adding sodium sulfate or potassium sulfate contains sodium or potassium ions. These ions could be removed by passing the solution through a strong cation exchange gel. Specifically, 83% of sodium ions were removed and 86% of potassium ions were removed. By repeatedly passing the solution through the strong cation exchange gel, even more cations could be removed.

[0169] Thus, it has been shown that in the method of adding sulfates, the cations added as sulfates can be removed by a strong cation exchange gel. However, when producing fertilizer from bone tissue, potassium ions, magnesium ions, and ammonium ions do not need to be removed because they are important nutrients for plant growth.

[0170] [Example 12-2: Removal of magnesium ions or ammonium ions] The following procedure was used to remove cations (magnesium ions or ammonium ions) contained in the sulfate added to remove calcium ions. Specifically, magnesium ions or ammonium ions were adsorbed using a strong cation exchange gel. 1. TSKgel SP-TOYOPEARL 650M gel (Tosoh Corporation) was decanted with pure water and packed into a microspin column (GE Healthcare). 2. An appropriate amount of the supernatant, after removing calcium sulfate by adding sulfate, was added to the upper layer of the gel. 3. The supernatant was passed through the gel by centrifugation using a benchtop centrifuge. The fraction that passed through the gel was collected. 3. The content (μg) of magnesium ions, ammonium ions, or calcium ions in the pass-through fraction was measured. Ion concentrations were measured using a high-performance ion chromatograph 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 metasulfonic acid and 2.7 mmol / L 18-crown-6 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 ion concentration was determined by measuring the electroelectricity (μS) and deriving a regression equation from the area of ​​the standard material. From the ion concentrations, the ion content per sample (30 μL) was determined.

[0171] [Table 17]

[0172] [result] The results are shown in Table 17. It was shown that magnesium ions, ammonium ions, and calcium ions could also be removed from the supernatant by contact with the strong cation exchange gel. By repeatedly passing the solution through the strong cation exchange gel, even more cations, including calcium ions, can be removed. This suggests that the purity of phosphate can be increased.

[0173] [Example 13: Purification of phosphate from commercially available bone meal] The following procedure was used to solubilize commercially available steamed bone meal (Omiya Green Service Co., Ltd.) and recover phosphoric acid. 1. To 1 g of steamed bone meal, 10 mL of sulfuric acid (1N), hydrochloric acid (1N) or nitric acid (1N) was added, and the mixture was shaken at 25°C for 19 hours. 2. The obtained acid extract was separated into a supernatant and a precipitate using a centrifuge. 3. To 0.5 mL of the supernatant, 0.1 mL of an aqueous NaOH solution (5N) was added, and the mixture was shaken for 1 hour. 4. The reaction solution was separated into a supernatant and a precipitate by centrifugation. 5. An appropriate amount of hydrochloric acid (1N) was added to the precipitate to dissolve the precipitate. 6. Steps 3 to 5 were repeated multiple times. Thereafter, the precipitate was dissolved in 0.5 mL of hydrochloric acid (0.4N). Thereby, a precipitate-dissolved solution with concentrated phosphoric acid was obtained. 7. The anion content in the obtained precipitate-dissolved solution was measured. The measurement was carried out under precise measurement conditions using high-performance ion chromatography IC-8100EX (Tosoh Corporation) connected with TSKgel SuperIC-Anion HS (4.6 mm I.D. × 10 cm). 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 electric conductivity (μS) was measured, and the anion concentration was determined by obtaining a regression equation from the peak area of the standard substance. From the anion concentration, the anion content per one sample (30 μL) was calculated.

[0174] [Table 18]

[0175] [Results] The results are shown in Table 18 and Figure 21. Even using commercially available steamed bone meal (bone tissue heat-treated under high pressure) as a raw material, phosphoric acid could be recovered efficiently and relatively safely by the method for producing phosphoric acid according to one embodiment of the present invention. According to Table 18, the phosphoric acid recovery efficiency is higher when using an acid extract with hydrochloric acid or nitric acid than when using an acid extract with sulfuric acid. The phosphoric acid recovery efficiency of the system using hydrochloric acid or nitric acid reached 3.2 to 3.4 times that of the system using sulfuric acid.

[0176] This is also shown in Figure 21. According to the ion chromatography elution curves shown in the same figure, in the systems using hydrochloric acid or nitric acid, there were almost no anions other than chloride ions, except for phosphate ions (hydrochloric acid was used to dissolve the precipitate in step 5). On the other hand, in the system using sulfuric acid, sulfate ions were detected in addition to phosphate ions.

[0177] The results of this embodiment demonstrate that phosphate can be extracted from commercially available bone meal (bone tissue heated under pressure). This proves that the method for producing phosphate according to one embodiment of the present invention is a sustainable technology with lower energy costs and less environmental impact than methods for recovering phosphate from phosphate rock ore or dried sewage sludge.

[0178] [Example 14: Production of protease-treated solution from commercially available bone meal] A protease-treated solution was prepared by solubilizing commercially available steamed bone meal (Omiya Green Service Co., Ltd.) using the following procedure. 1. Add 10 mL of sulfuric acid (1N), hydrochloric acid (1N), or nitric acid (1N) to 1 g of steamed bone meal and shake at 25°C for 24 hours. 2. The obtained acid extract was separated into supernatant (acid extract) and precipitate (bone residue) using a centrifuge and collected. 3. To the precipitate, 10 mL of 0.1 mol / L citrate buffer (pH 3) or 0.1 mol / L Tris-HCl buffer (pH 8) was added. 4. A protease-treated solution was prepared from the precipitate according to S5 in Figure 1. Neurase F3G (Amano Enzyme Co., Ltd.) or papain W-40 (Amano Enzyme Co., Ltd.) was used as the protease, and the treatment was carried out in the same procedure as in Examples 1-5. Specifically, it was as follows: • Neurase F3G system: Protease concentration = 1% of initial bone weight (w / w), buffer = citrate buffer (pH 3), temperature = 37°C, immersion time = 3 days Papain W-40 series: Protease concentration = 1% of initial bone weight (w / w), buffer = Tris-HCl buffer (pH 8), temperature = 37°C, immersion time = 3 days 5. The reaction mixture was centrifuged to separate the precipitate from the supernatant.

[0179] [result] The results are shown in Figures 22 and 23. Commercially available steamed bone meal is a mixture of pork bones and chicken bones that have been heat-treated under high pressure. Using this bone meal as a raw material, a protease-treated solution could be prepared by the fertilizer manufacturing method according to one embodiment of the present invention. The protease-treated solution can be used as a fertilizer itself, or it can be used as a raw material for bone solubilization solution B. Liquid fertilizer is expected to have an immediate plant growth effect that cannot be expected with solid bone meal. Furthermore, liquid fertilizer can be applied to hydroponics and foliar spraying.

[0180] Regarding protease treatment using New Larse F3G, bone residue treated with hydrochloric acid was almost completely dissolved by protease treatment. Bone residue treated with nitric acid was also completely dissolved by protease treatment. Bone residue treated with sulfuric acid still showed a white precipitate after protease treatment. This precipitate appeared to be calcium sulfate, suggesting that at least some of the protein components were decomposed.

[0181] Regarding protease treatment using papain W-40, bone residue treated with hydrochloric acid or nitric acid was almost completely dissolved by protease treatment. Bone residue treated with sulfuric acid still showed a white precipitate after protease treatment. This precipitate appeared to be calcium sulfate, suggesting that at least some of the protein components were decomposed.

[0182] From the above results, it was found that when obtaining a protease-treated solution from an acid extract using sulfuric acid as a raw material, it may be necessary to remove precipitate by centrifugation or other means. When obtaining a protease-treated solution from an acid extract using hydrochloric acid or nitric acid as a raw material, precipitate removal is not necessary, and in this respect, it is preferable to use hydrochloric acid or nitric acid in the acid treatment process. Furthermore, the supernatant after centrifugation of the solution after protease treatment was clear when treated with sulfuric acid, and cloudy when treated with nitric acid or hydrochloric acid. From these results, it was found that acid treatment with hydrochloric acid or nitric acid is more preferable for more complete decomposition of bone residue. However, it should be noted that a protease-treated solution can also be prepared using sulfuric acid.

[0183] [Example 15: Measurement of peptide concentration in acid extract using commercially available bone meal as a raw material] An acid extract was prepared from commercially available steamed bone meal (Omiya Green Service Co., Ltd.) using the following procedure. The peptide concentration in the acid extract was measured. 1. Add 10 mL of sulfuric acid (1N), hydrochloric acid (1N), or nitric acid (1N) to 1 g of steamed bone meal and shake at 25°C for 24 hours. 2. The obtained acid extract was separated into supernatant and precipitate using a centrifuge. The supernatant was collected as the acid extract. 3. A 0.4 mol / L potassium hydroxide solution was added to 0.5 mL of the supernatant to precipitate the discrete calcium. 4. The peptide concentration in the supernatant obtained by removing the precipitate from the reaction mixture obtained in step 3 was measured. A protein assay BCA kit (Catalog No. 297-73101, Fujifilm Wako) was used for the measurement. The measurement method followed the product manual. Absorbance at 540 nm was measured. Higher absorbance indicates a higher peptide concentration. 5. The precipitate was recovered from the reaction solution obtained in step 3. Only the systems treated with hydrochloric acid or nitric acid in step 1 were recovered. 6. The precipitate was completely dissolved in 1N hydrochloric acid. Then, 0.4 mol / L potassium hydroxide was added again to precipitate calcium phosphate. This dissolution-precipitation process was repeated three times. 7. In Step 6, after the first and third repetitions were completed, the peptide concentration in the supernatant obtained by removing the precipitate from the reaction solution was measured. For the measurement, a Protein Assay BCA Kit (product number 297-73101, Fujifilm Wako) was used. The measurement method followed the product manual. The absorbance at 540 nm was measured. The higher the absorbance, the higher the peptide concentration.

[0184]

Table 19

[0185] [Results] The results are shown in Table 19. Peptides were detected in the acid extract prepared using commercially available steamed bone meal as the raw material. The peptide concentration increased in the order of the acid extract treated with hydrochloric acid, the acid extract treated with sulfuric acid, and the acid extract treated with nitric acid. This result indicated that the acid extract treated particularly with hydrochloric acid contained a large amount of peptides. Peptides are organic components contained in bone tissue. Therefore, it was suggested that the acid extract contains components that bring about plant growth effects.

[0186] In Example 5, it was shown that the acid extract contained almost no protein components stained with a silver staining kit (see also Figure 14). Regarding this reason, it was presumed in Example 5 that the peptide bonds of proteins were cleaved to form low-molecular-weight peptides and amino acids. This Example supports this presumption.

[0187] Peptides become impurities in the production of phosphoric acid. It was found that peptides as impurities can be almost removed by repeating the calcium phosphate dissolution-precipitation process (Step S13 and Step S14 in Figure 3). As shown in Table 19, by repeating the dissolution-precipitation process 3 times, the peptide concentration was reduced to 1 / 100. This result suggested that peptides can be successfully removed particularly from the acid extract treated with hydrochloric acid or nitric acid to increase the phosphoric acid concentration.

Industrial Applicability

[0188] This invention can be used for the production of phosphoric acid, among other applications.

Claims

1. A method for producing phosphoric acid, comprising the following steps. (a) A process of treating bone tissue with acid to obtain an acid extract. (b) A step in which a base is added to the obtained acid extract to obtain precipitated calcium phosphate. (c) A step in which an acid is added to the obtained calcium phosphate precipitate to obtain a calcium phosphate solution. (d) Adding one or more salts selected from the group consisting of sulfates, carbonates, and bicarbonates to the obtained calcium phosphate solution to remove the precipitated calcium.

2. The process further includes a step of pre-treating the bone tissue prior to step (a) above, The manufacturing method according to claim 1, wherein the above pretreatment is one or more selected from the group consisting of heating the bone tissue, heating the bone tissue under pressure, and irradiating the bone tissue with microwaves.

3. The manufacturing method according to claim 1, further comprising a step of removing anions and cations other than phosphate ions and hydrogen ions after step (d) above.

4. The manufacturing method according to claim 1, wherein step (b) and step (c) above are repeated two or more times.

5. The manufacturing method according to claim 1, wherein in step (a) above, the bone tissue is acid-treated with one or more selected from the group consisting of nitric acid, hydrochloric acid, formic acid, and sulfuric acid.

6. The manufacturing method according to claim 1, wherein in step (d) above, sulfuric acid is not added to the calcium phosphate solution.

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