WO2023167167A1 - リン酸の製造方法 - Google Patents

リン酸の製造方法 Download PDF

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Publication number
WO2023167167A1
WO2023167167A1 PCT/JP2023/007246 JP2023007246W WO2023167167A1 WO 2023167167 A1 WO2023167167 A1 WO 2023167167A1 JP 2023007246 W JP2023007246 W JP 2023007246W WO 2023167167 A1 WO2023167167 A1 WO 2023167167A1
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Prior art keywords
acid
bone
protease
solution
calcium
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PCT/JP2023/007246
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English (en)
French (fr)
Japanese (ja)
Inventor
康一 森本
勝 坂本
善智 田口
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Kindai University
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Kindai University
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Priority to JP2024504689A priority Critical patent/JPWO2023167167A1/ja
Priority to US18/842,505 priority patent/US20250162870A1/en
Priority to AU2023226896A priority patent/AU2023226896A1/en
Priority to CN202380024553.8A priority patent/CN118922379A/zh
Priority to EP23763424.1A priority patent/EP4488225A4/en
Publication of WO2023167167A1 publication Critical patent/WO2023167167A1/ja
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F1/00—Fertilisers made from animal corpses, or parts thereof
    • C05F1/005—Fertilisers made from animal corpses, or parts thereof from meat-wastes or from other wastes of animal origin, e.g. skins, hair, hoofs, feathers, blood
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/16—Oxyacids of phosphorus; Salts thereof
    • C01B25/18—Phosphoric acid
    • C01B25/22—Preparation by reacting phosphate-containing material with an acid, e.g. wet process
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/16—Oxyacids of phosphorus; Salts thereof
    • C01B25/18—Phosphoric acid
    • C01B25/22—Preparation by reacting phosphate-containing material with an acid, e.g. wet process
    • C01B25/2208—Preparation by reacting phosphate-containing material with an acid, e.g. wet process with an acid or a mixture of acids other than sulfuric acid
    • C01B25/2216—Preparation by reacting phosphate-containing material with an acid, e.g. wet process with an acid or a mixture of acids other than sulfuric acid with nitric acid or nitrous vapours in aqueous medium
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/16—Oxyacids of phosphorus; Salts thereof
    • C01B25/18—Phosphoric acid
    • C01B25/234—Purification; Stabilisation; Concentration
    • C01B25/237—Selective elimination of impurities
    • C01B25/238—Cationic impurities, e.g. arsenic compounds
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05B—PHOSPHATIC FERTILISERS
    • C05B11/00—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes
    • C05B11/04—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes using mineral acid
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05B—PHOSPHATIC FERTILISERS
    • C05B11/00—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes
    • C05B11/04—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes using mineral acid
    • C05B11/06—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes using mineral acid using nitric acid (nitrophosphates)
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05B—PHOSPHATIC FERTILISERS
    • C05B11/00—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes
    • C05B11/04—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes using mineral acid
    • C05B11/08—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes using mineral acid using sulfuric acid
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05B—PHOSPHATIC FERTILISERS
    • C05B11/00—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes
    • C05B11/04—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes using mineral acid
    • C05B11/12—Fertilisers produced by wet-treating or leaching raw materials either with acids in such amounts and concentrations as to yield solutions followed by neutralisation, or with alkaline lyes using mineral acid using aqueous hydrochloric acid
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05B—PHOSPHATIC FERTILISERS
    • C05B17/00—Other phosphatic fertilisers, e.g. soft rock phosphates, bone meal
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G5/00—Fertilisers characterised by their form
    • C05G5/20—Liquid fertilisers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)

Definitions

  • the present invention relates to a method for producing phosphoric acid.
  • Phosphoric acid is a material widely used in agriculture and industry. Phosphoric acid is generally produced using phosphate ore as a raw material, but techniques for recovering phosphoric acid from waste have also been developed.
  • Patent Literature 1 discloses a method for recovering phosphoric acid from night soil and septic tank sludge.
  • Patent Literature 2 discloses a method for recovering phosphoric acid from steelmaking slag.
  • Bone tissue is said to contain about 33% phosphoric acid based on the weight before drying. Therefore, if phosphoric acid can be recovered from bone tissue, discarded bones generated at slaughterhouses and the like can become a huge source of phosphoric acid. This is highly desirable for Japan, which depends on imports for all its phosphate rock demand. Therefore, the development of a method for recovering phosphoric acid from bone tissue is desired.
  • An object of one aspect of the present invention is to provide a novel method for producing phosphoric acid.
  • a method for producing phosphoric acid comprising the steps of: (a) a step of acid-treating a bone tissue to obtain an acid extract (b) a step of adding a base to the obtained acid extract to obtain a precipitated calcium phosphate (c) adding an acid to the obtained calcium phosphate precipitate (d) adding one or more selected from the group consisting of sulfates, carbonates and hydrogencarbonates to the resulting calcium phosphate solution to remove precipitated calcium ⁇ 2> Further comprising a step of pretreating the bone tissue prior to step (a); The manufacturing method according to ⁇ 1>, wherein the 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 production method according to ⁇ 1> or ⁇ 2>, further comprising a step of removing anions and cations other than phosphate ions and hydrogen ions after step (d).
  • ⁇ 4> The production method according to any one of ⁇ 1> to ⁇ 3>, wherein the step (b) and the step (c) are repeated twice or more.
  • ⁇ 5> The production method according to any one of ⁇ 1> to ⁇ 4>, wherein in the step (a), 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 production method according to any one of ⁇ 1> to ⁇ 5>, wherein in the step (d), sulfuric acid is not added to the calcium phosphate solution.
  • a novel method for producing phosphoric acid is provided.
  • FIG. 2 is a diagram showing the results of protease treatment of bone tissue obtained by acid-treating pig bones with nitric acid.
  • FIG. 3 is a diagram showing the results of treating bone tissue obtained by acid-treating pig bones with nitric acid or hydrochloric acid with actinidyne.
  • FIG. 10 is a diagram showing the results of preparing bone lysate A with solution exchange using various acids and proteases.
  • FIG. 4 is a diagram showing the results of an experiment in which the effect of acid extracts on plant growth was investigated.
  • FIG. 3 is a diagram showing the results of an experiment in which an acid extract, a protease-treated solution, or a bone solubilizing solution A was used to examine the effect of plant growth.
  • FIG. 10 is a diagram showing the results of an experiment comparing the plant growth effects of bone lysate A and a commercially available culture medium.
  • FIG. 10 is a diagram showing the results of an experiment comparing the plant growth effects of bone lysate A and a commercially available culture medium.
  • FIG. 10 is a diagram showing the results of an experiment comparing changes in expression levels of stress tolerance-related genes with bone lysate A and commercially available liquid fertilizers.
  • FIG. 10 is a diagram showing the results of an experiment comparing changes in expression levels of stress tolerance-related genes with bone lysate A and commercially available liquid fertilizers.
  • FIG. 4 is a diagram showing the effect of pretreatment of bone tissue on the amount of phosphoric acid contained in the acid extract. It is a figure showing the result of having analyzed the protein component contained in the acid extract by electrophoresis.
  • FIG. 4 is a diagram showing the results of preparing protease-treated solutions using various proteases.
  • FIG. 4 is a diagram showing the results of electrophoresis analysis of protein components contained in a protease-treated solution.
  • FIG. 10 is a diagram showing the results of preparing bone lysate A with solution exchange using various acids and proteases.
  • FIG. 10 is a diagram showing analysis results of expression-variable genes in leaves of sprouts cultivated for 5 days with bone lysate B or a commercially available liquid fertilizer.
  • FIG. 10 is a diagram showing analysis results of expression-variable genes in roots of sprouts cultivated for 5 days with a mixture of bone solubilizing solution B and a commercially available liquid fertilizer or only with a commercially available liquid fertilizer.
  • FIG. 10 is a diagram showing analysis results of expression-variable genes in leaves of sprouts cultivated for 5 days with a mixture of bone solubilizing solution B and a commercially available liquid fertilizer or only with a commercially available liquid fertilizer. It is an elution curve showing the result of extracting phosphoric acid using commercially available bone meal as a raw material.
  • a method for producing a 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 step 4 below. Hereinafter, the steps 2, 3, 4 and 1 will be described in detail in this order.
  • Step 1 Process of treating bone tissue with a solution containing both an acid and a protease to produce a fertilizer from the obtained bone lysate A.
  • Step 2 Acid treatment of bone tissue to obtain an acid extract.
  • Step 3 Process of producing fertilizer from protease-treated solution obtained by treating acid-treated bone tissue with protease
  • Step 4 Mixing acid extract solution and protease-treated solution to obtain Process of manufacturing fertilizer from bone lysate B
  • Step 2 is a step of acid-treating the bone tissue and producing a fertilizer from the resulting acid extract.
  • the inorganic components are separated mainly from the bone tissue and eluted into the acid extract.
  • a fertilizer is produced from the acid extract obtained.
  • the acid extract itself may be used as the fertilizer, or the bone solubilizing solution B mixed with the protease-treated solution may be used as the fertilizer.
  • the bone tissue to be subjected to 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 obtain a large amount of bone tissue as a raw material of fertilizer, and for example, bone tissue of domestic animals (cattle, pig, sheep, chicken, etc.) is preferably used.
  • the bone tissue may be pre-shredded and pulverized prior to the acid treatment. In this way, the bone tissue can be decomposed more efficiently, so the manufacturing time can be shortened.
  • the acid used to treat the bone tissue in step 2 is not particularly limited.
  • acids include hydrochloric acid, nitric acid, formic acid, sulfuric acid, trichloroacetic acid.
  • An acidic demineralization solution such as Planck-Lucro solution may also be used.
  • one or more selected from the group consisting of nitric acid, hydrochloric acid, formic acid and sulfuric acid are preferred.
  • one or more selected from the group consisting of hydrochloric acid, sulfuric acid and nitric acid are preferable, and one or more selected from the group consisting of hydrochloric acid and nitric acid are more preferable.
  • calcium recovery efficiency one or more selected from the group consisting of hydrochloric acid and formic acid is preferable.
  • Two or more kinds of acids may be mixed in an appropriate ratio and used.
  • the bone tissue may be acid-treated with a solution of acid diluted in a solvent.
  • solvents include water, lower alcohols, glycerol, propane-1,2-diol, 1,3-propanediol. Two or more types of solutions may be mixed at an appropriate ratio and used.
  • the acid concentration can be appropriately determined according to the volume of bone tissue to be acid-treated. If the volume of bone tissue is small, it can be treated with low concentrations of acid. When the volume of bone tissue is large, a higher concentration of acid is preferred. When acid treatment is performed with a low-concentration acid, it is preferable to treat pulverized bone tissue. However, even when the volume of the bone tissue is large, if the acid concentration is increased and the bone tissue is immersed for a long period of time, the inorganic components can be sufficiently extracted.
  • the lower limit of the acid concentration in step 2 is 0.6 mol/L or more, 0.7 mol/L or more, 0.8 mol/L or more, or 0 .9 mol/L or more.
  • the upper limit of the acid concentration in step 2 can be 2.0 mol/L or less, 1.5 mol/L or less, 1.0 mol/L or less, or 0.9 mol/L or less. If the concentration of the acid is within the above range, the inorganic components can be efficiently extracted, and since the acid concentration is not too high, the cost of neutralizing the acid extract can be reduced.
  • Suitable acid concentrations for acid treatment of pulverized bone tissue are as follows.
  • the lower limit of the nitric acid concentration is preferably 0.6 mol/L or more, more preferably 0.7 mol/L or more.
  • the upper limit of the concentration of nitric acid is preferably 1.0 mol/L or less, more preferably 0.9 mol/L or less.
  • the lower limit of the concentration of hydrochloric acid is preferably 0.8 mol/L or more, more preferably 0.9 mol/L or more.
  • the upper limit of the concentration of hydrochloric acid is preferably 1.2 mol/L or less, more preferably 1.1 mol/L or less.
  • the lower limit of the concentration of formic acid is preferably 0.8 mol/L or more, more preferably 0.9 mol/L or more.
  • the upper limit of the concentration of formic acid is preferably 1.2 mol/L or less, more preferably 1.1 mol/L or less.
  • the lower limit of the sulfuric acid concentration is preferably 0.8 mol/L or more, more preferably 0.9 mol/L or more.
  • the upper limit of the sulfuric acid concentration is preferably 1.2 mol/L or less, more preferably 1.1 mol/L or less.
  • the above concentration is an example of a suitable concentration when bone tissue is pulverized, and when bone tissue having a larger volume is used as a raw material, the acid concentration may be increased.
  • the lower limit of the extraction time in step 2 is preferably 6 hours or longer, more preferably 8 hours or longer, and even more preferably 10 hours or longer.
  • 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, the inorganic components contained in the bone tissue can be sufficiently extracted.
  • the acid treatment temperature in step 2 is preferably 5 to 60°C. If the acid treatment is carried out within the above temperature range, it is possible to reduce the occurrence of unpleasant odors associated with the acid treatment. As a result, the location requirements for fertilizer manufacturing plants become more lenient. Moreover, in order to adjust the temperature to 60° C. or less, no expensive special equipment is required, and the temperature can be kept constant by using a water bath or an incubator.
  • a chelating agent capable of capturing calcium ions may be added in addition to the acid.
  • 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).
  • chelating agents include ethylenediaminetetraacetic acid (EDTA, CAS Registry Number: 60-00-4), glycol etherdiaminetetraacetic acid (EGTA, CAS Registry Number: 67-42-5), ethylenediamine-N,N'- and disuccinic acid (EDDS, CAS registry number: 20846-91-7).
  • the pH of the solution containing the chelating agent is preferably 6.0-8.0.
  • the lower limit of the concentration of the chelating agent in step 2 is 0.1 mol/L or more, 0.2 mol/L or more, 0.3 mol/L or more, or It can be 0.4 mol/L or more.
  • the upper limit of the concentration of the chelating agent in step 2 can be 0.9 mol/L or less, 0.8 mol/L or less, 0.7 mol/L or less, or 0.6 mol/L or less.
  • step 2 at least part of calcium may be removed after the acid treatment.
  • methods for removing calcium include neutralizing the acid extract (causing calcium phosphate precipitation), adding sulfuric acid or sulfate (causing calcium sulfate to precipitate), adding carbonic acid or carbonate (calcium carbonate Alternatively, calcium hydrogencarbonate is precipitated), and a method of adding a hydrogencarbonate (calcium carbonate or calcium hydrogencarbonate is precipitated).
  • the concentration of phosphoric acid in the acid extract obtained in step 2 tends to increase when the pretreatment step described later is performed.
  • the concentration of phosphoric acid contained in the acid extract can be, for example, 280 mM or higher, 300 mM or higher, or 320 mM or higher.
  • Step 3 is a step of protease-treating the acid-treated bone tissue and producing a fertilizer from the resulting protease-treated solution.
  • a fertilizer is produced from the resulting protease-treated solution.
  • the protease-treated solution itself may be used as the fertilizer, or the bone solubilizing solution B mixed with the acid extract may be used as the fertilizer.
  • protease used in step 3 is not particularly limited.
  • proteases include serine proteases, cysteine proteases, aspartic proteases, glutamic proteases, metalloproteases.
  • 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], actinidyne [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], cathepsin S [EC 3.4.22.27], thermolysin [EC 3.4.24.27].
  • a commercially available enzyme preparation may be used as the protease.
  • examples of such formulations include Neurase F3G (from Rhizopus niveus), Orientase AY (from Aspergillus niger), Tetrase (from Aspergillus niger), Sumizyme AP (from Aspergillus niger), Denapsin 2P (from Aspergillus genus), Brewer's 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” (Bacillus subtilis) (derived from Aspergillus oryzae), Protease A "Amano” G (derived from Aspergillus oryzae), Umamizyme (derived from Aspergillus oryzae), Prot
  • the protease used in step 3 is preferably a protease with an optimum pH of 1.5-8.0.
  • proteases having an optimum pH of 1.5 to 8.0 include Protease S "Amano" G (optimum pH: 7.0 to 8.5), Protease N “Amano” G (optimum pH: 6.0 to 7.5), protease NL “Amano” (optimum pH: 6.5 to 7.5), protease A “Amano” G (optimum pH: 6.0 to 7.5), Umamizyme ( Optimum pH: 6.0-7.5), Protease M “Amano” G (optimum pH: 3.0-6.5), Protease P "Amano” 3G (optimum pH: 7.0-8.
  • protease R "Amano" optimum pH: 6.0-8.0
  • actinidyne optimum pH: 2.5-7.5
  • papain optimum pH: 4.0-9.0
  • pepsin optimum pH: 1.5-3.0
  • Neurase F3G optimum pH: 3.0-5.0
  • trypsin optimum pH: 7.0-9.0
  • chymotrypsin optimum pH: 7.0 to 9.0
  • the protease to be used is more preferably a protease having an optimum pH of 1.5-5.0.
  • proteases having an optimum pH of 1.5-5.0 include Protease M "Amano" (optimum pH: 3.0-6.5), actinidin (optimum pH: 2.5-7.
  • protease to be used is more preferably a protease having an optimum pH of 1.5 to 4.0.
  • proteases with an optimum pH of 1.5-4.0 include actinidyne (pH optimum: 2.5-7.5), pepsin (pH optimum: 1.5-3.0), Lase F3G (optimal pH: 3.0-5.0).
  • the concentration of protease in step 3 can be set as appropriate.
  • the lower protease concentration limit can be 2 mg/L or higher or 10 mg/L or higher.
  • the upper limit of protease concentration can be 100 mg/L or less or 50 mg/L or less.
  • the temperature and pH in step 3 can be set as appropriate. Matching the optimum temperature and optimum pH of the protease to be used is preferable for improving the treatment efficiency.
  • Examples of the temperature of the reaction system in step 3 include 20 to 60°C.
  • the lower limit of the temperature of the reaction system in step 2 may be higher than 10°C, higher than 20°C, higher than 25°C, higher than 30°C, higher than 35°C, higher than 40°C, higher than 45°C, higher than 50°C, or higher than 55°C.
  • the upper limit of the temperature of the reaction system in step 2 may be 60° C. or lower or 55° C. or lower.
  • salt may be added to the reaction system.
  • the substrate specificity of the protease may change depending on the salt concentration added to the reaction system. Therefore, by adding a salt to the reaction system, the components contained in the resulting protease-treated solution can be changed.
  • Examples of salts added to the reaction system in step 3 include chloride salts.
  • Examples of chloride salts include NaCl, KCl, LiCl, MgCl2 .
  • the lower limit of the concentration of the salt added to the reaction system in step 3 is more 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. /L or more, 2000mmol/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.
  • the size of the peptide fragment generated 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. It is highly probable that a peptide fragment that has been cleaved to such a size has lost its physiological activity. In addition, peptide fragments that have been cleaved to such a size are easily absorbed by plants as nutrients, and may function as active ingredients of fertilizers.
  • 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.
  • Step 4 is a step of mixing the acid extract solution obtained in step 2 and the protease-treated solution obtained in step 3, and producing a fertilizer from the bone solubilized solution B obtained. Fertilizer is produced from the obtained bone lysate B.
  • the mixing ratio of the acid extract and the protease-treated solution is not particularly limited. In order to obtain a desired composition of the bone solubilized solution B, the mixing ratio can be appropriately selected.
  • Step 1 is a step of treating bone tissue with a solution containing both an acid and a protease, and manufacturing a fertilizer from the obtained solubilized bone solution A.
  • the acid treatment in step 2 and the protease treatment in step 3 proceed simultaneously.
  • step 1 the order of contacting bone tissue with acid and protease is not particularly limited.
  • order 1 Bone tissue is immersed in a solution containing acid, and after a predetermined time has passed, protease is added to the solution. Prior to adding the protease, the pH of the solution may be adjusted to the optimum pH for the protease.
  • Order 2 Bone tissue is immersed in a solution containing protease, and after a predetermined time has passed, acid is added to the solution.
  • Order 3 A solution containing both acid and protease is prepared, and bone tissue is immersed in the solution.
  • the method for producing a fertilizer according to one embodiment of the present invention may further include steps that are commonly performed in the production of fertilizers. Examples of such steps include pretreatment steps, ingredient addition steps, drying steps, grinding steps, coated granulation steps, and packing steps.
  • the pretreatment process is the process that precedes process 1 or process 2.
  • the raw material bone tissue is pretreated.
  • the amount of phosphoric acid extracted from the bone tissue can be increased (see Example 4).
  • the pretreatment step includes heating bone tissue.
  • the heating temperature at this time may be 30° C. or higher or 40° C. or higher; 100° C. or lower or 80° C. or lower.
  • Bone tissue may be heated while immersed in acid.
  • the pretreatment step heats the bone tissue under pressure.
  • the pressure at this time can 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 higher or 50° C. or higher; 120° C. or lower or 200° C. or lower.
  • the bone tissue is irradiated with microwaves in the pretreatment step.
  • microwaves Any combination of heating, heating under pressure and microwave irradiation may be applied.
  • the microwave irradiation is more preferable as the pretreatment step because the effect can be obtained in a short period of time.
  • the microwave irradiation time can be 5 seconds or more, 10 seconds or more, or 15 seconds or more; 10 minutes or less, 7 minutes or less, or 5 minutes or less.
  • the fertilizer obtained by applying the pretreatment step contains denatured proteins (or fragments thereof).
  • a denatured protein (or a fragment thereof) has lost the physiological activity of the native protein.
  • the ingredient addition process is the process of adding additional fertilizer ingredients.
  • fertilizer components added in the component addition process include potassium components (potassium oxide, potassium hydroxide, potassium chloride, potassium sulfate, etc.), nitrogen components (urea, ammonium nitrate, etc.), magnesium components (magnesium phosphate, magnesium chloride , magnesium sulfate, etc.), vitamins, manganese, boron, iron, copper, zinc, and molybdenum.
  • the acid extract, protease-treated solution, bone solubilizing solution A or bone solubilizing solution B may be mixed with other fertilizers (inorganic fertilizers, organic fertilizers, etc.).
  • the drying step is a step of removing excess water from the acid extract, protease-treated solution, bone solubilized solution A or bone solubilized solution B. Through the drying process, a solid or paste fertilizer is obtained. Solid fertilizers may be cut and pulverized into sizes and shapes that facilitate fertilization, if necessary.
  • the coating granulation process is a process of coating and granulating solid fertilizer.
  • fertilizer is coated with a silicic acid compound or the like, it is possible to adjust the effective timing of fertilizer, prevent immobilization of phosphorus and calcium, prevent fertilizer from running out, and prevent fertilizer from being damaged by impact.
  • the packing process is the process of packing the acid extract, protease-treated liquid, bone solubilized solution A or bone solubilized solution B into a container so that it can be distributed or sold as fertilizer.
  • the acid extract, the protease-treated solution, the bone solubilizing solution A or the bone solubilizing solution B, and instructions for using them as a fertilizer may be combined.
  • the instructions may be printed on the container or may be provided separately from the packaged fertilizer as a physical or electronic document.
  • the manual may describe the formulation of the fertilizer, the method of fertilization, the timing of fertilization, the target crops, and the like.
  • FIG. 1 is an exemplary flow chart representing a manufacturing method including steps 2, 3 and/or 4.
  • an acid extract is obtained through steps S1, S2, S3 and S4.
  • a protease-treated solution is obtained through steps S1, S2 and S5.
  • a bone lysate B is obtained by subjecting the acid extract and the protease-treated solution to step S6. All of the acid extract, protease-treated solution and bone solubilizing solution B can be used as components of fertilizers or mixed fertilizers. Since the acid extract contains phosphoric acid, which is an essential nutrient for plants, it can be used as a fertilizer by itself.
  • step S1 bone tissue is pretreated.
  • Step S1 is an optional step and may not be performed. By pretreating the bone tissue, the amount of phosphoric acid contained in the acid extract can be increased. This step is as described in section [1.5].
  • step S2 the bone tissue is acid treated.
  • the supernatant obtained through step S2 is the acid extract.
  • Production of fertilizer from the acid extract results in step 2 above.
  • the acid treatment of bone tissue is as described in section [1.1].
  • step S3 sulfate, carbonate or hydrogen carbonate is added.
  • Step S3 is an optional step and may not be performed.
  • calcium ions contained in the acid extract are precipitated as calcium sulfate, calcium carbonate, or calcium hydrogen carbonate.
  • sulfates include sodium sulfate, potassium sulfate, ammonium sulfate, magnesium sulfate.
  • carbonates include potassium carbonate and ammonium carbonate.
  • Examples of bicarbonates include potassium bicarbonate.
  • the sulfate is one or more selected from the group consisting of potassium sulfate, ammonium sulfate and magnesium sulfate.
  • the carbonate is potassium carbonate.
  • the bicarbonate is potassium bicarbonate. The use of these sulfates, carbonates or bicarbonates results in potassium, magnesium and/or ammonium being included in the acid extract. These components are important nutrients for plants.
  • step S4 the acid extract is neutralized with a base.
  • Step S4 is an optional step and may not be performed.
  • the liquidity of the acid extract is returned from acidity to near neutrality.
  • usable bases include sodium hydroxide and potassium hydroxide. Of these, when potassium hydroxide is used, potassium ions are included in the acid extract. Since potassium ions are an important nutrient for plants, neutralization with potassium hydroxide is preferred.
  • step S5 the acid-treated bone tissue is treated with protease.
  • the treated liquid obtained through step S5 is the protease-treated liquid.
  • the step 3 described above is performed.
  • the protease-acid treatment of bone tissue is as described in Section [1.2].
  • step S6 the acid extract and the protease-treated liquid are mixed.
  • a solubilized bone solution B is thus obtained.
  • the step 4 described above is carried out. Preparation of bone lysate B is as described in section [1.3].
  • FIG. 2 is an exemplary flow chart representing a manufacturing method including step 1.
  • a bone lysate A is obtained through steps S1, S7, S3 and S4.
  • Bone lysate A can be used as a component of fertilizer or mixed fertilizer.
  • step S7 the bone tissue is treated with both acid and protease. Thereby, a bone lysate A is obtained.
  • the fertilizer is produced from the solubilized bone solution A, the step 1 described above is carried out.
  • the preparation of bone lysate A is as described in section [1.4].
  • 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, the fertilizer according to one aspect of the present invention contains acid extract, protease-treated solution, bone solubilizing solution A or bone solubilizing solution B. Among these, the fertilizer containing the solubilized bone A or the solubilized bone B is preferable because it has a higher effect of promoting plant growth.
  • the lower limit of the ratio of acid extract, protease-treated solution, bone solubilizing solution A or bone solubilizing solution B to the total weight of the fertilizer is 0.01 wt% or more, 0.05 wt% or more, 0.1 wt% 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 It can be weight percent or higher, 80 weight percent or higher, or 90 weight percent or higher.
  • the upper limit of the ratio of the acid extract, protease-treated solution, bone solubilizing solution A or bone solubilizing solution B to the total weight of the fertilizer is 100% by weight.
  • the fertilizer consists solely of acid extract, protease treatment, bone lysate A, bone lysate B, or any mixture thereof.
  • the composition of the fertilizer may be changed as necessary.
  • some or all of phosphorus and calcium may be changed to calcium dihydrogen phosphate or calcium citrate.
  • the timing of fertilization can be adjusted so that phosphorus and calcium are supplied in accordance with the growth stage of the plant body.
  • Fertilizer can be either solid fertilizer or liquid fertilizer. Since the acid extract, the protease-treated solution, the bone solubilized solution A, or the bone solubilized solution B are obtained as liquids, they can be easily processed into liquid fertilizers. According to the method for producing a fertilizer according to one embodiment of the present invention, the components of bone tissue (for example, all components of bone tissue) can be solubilized, so a liquid fertilizer rich in components useful for plant growth can be obtained. expected to be In addition, while existing bone-derived solid fertilizers (bone meal) are slow-acting and often used for soil improvement over several years, liquid fertilizers are expected to be immediate-acting. Furthermore, while bone meal is not suitable for hydroponics, liquid fertilizers are suitable for hydroponics. In addition, liquid fertilizers have the advantage of being easily applied for foliar applications.
  • the fertilizer may contain components other than the acid extract, the protease-treated solution, the bone solubilizing solution A, or the bone solubilizing solution B.
  • ingredients include acid fertilizers, alkaline fertilizers, and other common fertilizers.
  • acid fertilizers include ammonium sulfate, lime superphosphate, potassium sulfate, aluminum sulfate, peat moss, black earth, ash, and alum.
  • alkaline fertilizers include grass and wood ash, lime nitrogen, chile saltpeter, fish compost, magnesia lime, organic lime, slaked lime, lime nitrogen, limestone, cement, sodium bicarbonate, shells, rice husks and charcoal.
  • Other common fertilizers include straw, bark and molasses.
  • Free amino acids ⁇ -aminobutyric acid, etc.
  • plant growth hormones auxin, etc.
  • trace elements magnesium, sulfur, iron, manganese, zinc, copper, boron, molybdenum, etc.
  • a fertilizer according to an aspect of the present invention contains a decomposition product of bone tissue. Therefore, it often contains type I collagen, osteocalcin, alpha-2-HS-glycoprotein, periostin, biglycan, SPARC or its degradation peptides, which are the main organic components of bone tissue. These ingredients are less likely to be contained in fertilizers obtained by other manufacturing methods. Therefore, a fertilizer containing type I collagen, osteocalcin, alpha-2-HS-glycoprotein, biglycan, SPARC, degradation peptides thereof, or a combination thereof is produced by the production method according to one embodiment of the present invention. There is a high probability that it is a fertilizer that has been used.
  • 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 a fertilizer according to an 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 uniquely to the protease. Therefore, treatment of a particular protein with a particular protease will uniquely define the peptide fragments produced by the treatment. Since each fragment has a different molecular weight, for example, by mass spectrometry, the protein before protease treatment can be identified from peptide fragments contained in fertilizer.
  • the peptide fragments contained in the fertilizer are peptides derived from one or more selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan and SPARC. Whether or not it is a fragment can be determined.
  • Table 1 shows examples of peptide fragments that can be detected by LC-MS/MS.
  • This peptide fragment is the peptide fragment that can appear when manure is analyzed without trypsinization. Since it has not been trypsinized, the C-terminus of the peptide fragment is an amino acid that is not Lys or Arg.
  • peptide fragments derived from one or more selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan, and SPARC do not have bioactivity.
  • a method for producing a fertilizer according to an embodiment of the present invention includes a plurality of steps in which proteins can lose their physiological activity.
  • One is a pretreatment step.
  • the pretreatment step the bone tissue is heated, the bone tissue is heated under pressure, or the bone tissue is irradiated with microwaves, so that proteins are denatured and lose their physiological activity.
  • the other is the protease treatment in steps 1 and 3. Proteins treated with proteases become fragments and lose their original physiological activity.
  • the size of the peptide fragment derived from one or more selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan and SPARC is 10,000 Da or less. 000 Da or less, 6,000 Da or less, or 4,000 Da or less. It is highly probable that a peptide fragment that has been cleaved to such a size has lost its physiological activity. In addition, peptide fragments that have been cleaved to such a size are easily absorbed by plants as nutrients, and may function as active ingredients of fertilizers.
  • physiological activities of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan and SPARC are as follows. The fact that fragments derived from them lack physiological activity means that they do not have the following activities.
  • - Physiological activity of type I collagen It self-assembles to form fibrils under physiological conditions.
  • - Biological activity of alpha-2-HS-glycoprotein binds calcium ions.
  • periostin It functions as a cell adhesion molecule for osteoprogenitor cells.
  • biglycan binds to type I collagen.
  • SPARC Promotes type I collagen synthesis in cultured dermal fibroblasts.
  • the fertilizer contains phosphoric acid.
  • the concentration of phosphoric acid contained in the fertilizer tends to increase.
  • the concentration of phosphate contained in the fertilizer can be, for example, 280 mM or higher, 300 mM or higher, or 320 mM or higher.
  • the fertilizer contains both peptide fragments derived from one or more selected from the group consisting of type I collagen, alpha-2-HS-glycoprotein, periostin, biglycan and SPARC, and phosphate. contains.
  • a method for producing phosphoric acid includes, for example, a calcium phosphate purification step and a calcium removal step.
  • steps for producing calcium phosphate include the following procedures. 1. An alkaline solution is added to the acid extract obtained in step 2 to neutralize it. This gives a precipitate. 2. An acid solution (such as aqueous hydrochloric acid) is added to the precipitate to resolubilize the precipitate. 3. Repeat 1 and 2 to purify the calcium phosphate.
  • calcium is removed by adding a chelating agent to the purified calcium phosphate solution.
  • 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 a solution of purified calcium phosphate.
  • 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 multicomponent thereof. mixtures.
  • the reagent to be added to the calcium phosphate solution may be added as powder or as a solution (aqueous solution, etc.). Since the above-described method can be applied to acidic solutions at room temperature, calcium can be easily removed.
  • Bone tissue contains inorganic components such as calcium and organic components such as collagen in addition to phosphoric acid.
  • phosphoric acid is dissolved in steps S11 and S12, and components other than phosphoric acid and calcium are removed in steps S13 and S14.
  • Step S13 and step S14 may be repeated, and the number of repetitions can be set appropriately.
  • step S15 sulfate, carbonate or bicarbonate is added to remove calcium. Calcium sulfate, calcium carbonate, or calcium hydrogen carbonate produced in step S15 may be used as agricultural fertilizers or industrial raw materials. Each step will be described in detail below.
  • step S11 bone tissue is pretreated.
  • Step S11 is an optional step and may not be performed. By pretreating the bone tissue, the amount of phosphoric acid contained in the acid extract can be increased. This step is the same step as step S1 described above, and the details are as described in section [1.5].
  • step S12 the bone tissue is acid treated. This step yields an acid extract. This step is the same step as the above-described step S2, and the description in Section [1.1] is incorporated for the acid treatment conditions.
  • step S13 the acid extract is neutralized with a base.
  • phosphate ions contained in the acid extract are precipitated as calcium phosphate, and components derived from bone impurities are contained in the supernatant. Therefore, the phosphoric acid can be purified by collecting the precipitate.
  • usable bases include sodium hydroxide and potassium hydroxide.
  • step S14 an acid is added to the calcium phosphate precipitate.
  • an acid is added to the calcium phosphate precipitate.
  • examples of acids that can be used include hydrochloric acid, nitric acid and formic acid.
  • Steps S13 and S14 may be performed repeatedly. By repeating these steps, the purity of phosphoric acid increases.
  • the number of times steps S13 and S14 are repeated may be, for example, 2 or more, 3 or more, 4 or more, or 5 or more. From the viewpoint of economy, the number of times steps S13 and S14 are repeated may be, for example, 10 times or less.
  • the washing treatment for washing away unnecessary components contained in the calcium phosphate precipitate with pure water may be performed as many times as necessary before proceeding to the step S15.
  • step S15 sulfate, carbonate or bicarbonate is added to the calcium phosphate solution. This causes calcium ions in solution to precipitate as calcium sulfate, calcium carbonate or calcium hydrogen carbonate. Precipitated calcium sulfate, calcium carbonate or calcium hydrogen carbonate is removed by centrifugation or the like.
  • sulfates include one or more selected from sodium sulfate, potassium sulfate, ammonium sulfate and magnesium sulfate.
  • 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.
  • sulfates, carbonates and hydrogen carbonates can be combined arbitrarily.
  • Adding sulfate instead of sulfuric acid in step S15 has the following advantages. ⁇ Because no liquid is added, the increase in volume of the reaction system can be reduced. ⁇ Since the reaction system does not become strongly acidic, only a small amount of base is required for neutralization. ⁇ High safety. ⁇ High removal rate of calcium.
  • the amount of sulfate, carbonate, or hydrogen carbonate to be added in step S15 can be appropriately set by those skilled in the art.
  • the amount of sulfate, carbonate, or hydrogencarbonate added is, for example, the concentration of sulfate, carbonate, or hydrogencarbonate in the reaction system is 0.2M or more, 0.4M or more, 0.6M or more, or 0.8M or more. Alternatively, the amount may be 1.0M or more. A larger amount of sulfate, carbonate, or hydrogen carbonate added can reduce residual calcium.
  • the upper limit of the amount of sulfate, carbonate, or hydrogencarbonate to be added may be, for example, an amount such that the concentration of sulfate in the reaction system is 3.0M or less, 2.0M or less, or 1.0M.
  • step S15 a small amount of sulfate ions, carbonate ions, or bicarbonate ions remain in the supernatant due to the solubility product.
  • the salt added in step S15 is preferably one or more selected from the group consisting of carbonates and hydrogencarbonates.
  • the supernatant after removing calcium is purified.
  • the supernatant may contain cations such as sodium ions and anions such as chloride ions that were contained in sulfate.
  • High-purity phosphoric acid can be obtained by adsorbing these ions on, for example, an ion-exchange resin.
  • carbonate ions or hydrogen carbonate ions are contained, the ions can be removed from the system by converting them to carbon dioxide by heating.
  • Example 1 Production of fertilizer containing decomposition products of bone tissue
  • Example 1-1 Preparation of acid extract with nitric acid and component analysis
  • the bone tissue was acid-treated with nitric acid according to the following procedure to obtain an acid extract.
  • Pork bones obtained from a slaughterhouse were finely pulverized with a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.).
  • 40 mL of nitric acid aqueous solution was added to 3 g of wet weight of pork bone, and the mixture was immersed at 20°C.
  • the concentration of the nitric acid aqueous solution was 0.3 mol/L, 0.5 mol/L, 0.75 mol/L or 1.0 mol/L.
  • the immersion time was 12 hours, 24 hours or 48 hours. 3.
  • the supernatant was collected and used as an acid extract.
  • Example 1-2 Preparation of acid extract with hydrochloric acid and component analysis
  • the bone tissue was acid-treated with hydrochloric acid according to the following procedure to obtain an acid extract.
  • Pork bones obtained from a slaughterhouse were finely pulverized with a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.).
  • 2. 35 mL of hydrochloric acid aqueous solution was added to a pig bone having a wet weight of 2 g, and the mixture was immersed at 20°C.
  • the concentration of the hydrochloric acid aqueous solution was 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 an acid extract.
  • Example 1-3 Preparation of acid extract with formic acid and component analysis
  • the bone tissue was acid-treated with formic acid according to the following procedure to obtain an acid extract.
  • Pork bones obtained from a slaughterhouse were finely pulverized with a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.).
  • 35 mL of formic acid aqueous solution was added to 2 g of wet weight of pork bone, and the mixture was immersed at 20°C.
  • the concentration of the formic acid aqueous solution was 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 an acid extract.
  • Example 1-4 Preparation of acid extract with sulfuric acid and component analysis
  • the bone tissue was acid-treated with sulfuric acid according to the following procedure to obtain an acid extract.
  • Pork bones obtained from a slaughterhouse were finely pulverized with a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.).
  • 35 mL of an aqueous sulfuric acid solution was added to 2 g of wet weight of pork bone, and the mixture was immersed at 20°C.
  • the concentration of the sulfuric acid aqueous solution was 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 an acid extract.
  • Table 2 shows the results of analyzing the total phosphorus of the obtained acid extract.
  • the component analysis was outsourced to Kurita Analysis Center Co., Ltd.
  • Table 2 shows the measurement results of total phosphorus (mg) converted to 1 g of wet weight of bone.
  • the analysis results were summarized for each acid for each concentration of nitric acid, hydrochloric acid, formic acid, or sulfuric acid and for each immersion time. It was shown that most of the phosphorus can be recovered in about 24 hours in any acid aqueous solution. Moreover, it was shown that phosphorus can be sufficiently recovered at an acid concentration of about 1 mol/L. Among the four acids examined, hydrochloric acid and sulfuric acid had the highest phosphorus recovery efficiencies. From Table 2, it was found that about 100 mg of phosphorus can be recovered from 1 g of bone tissue.
  • Table 3 shows the results of analyzing the total calcium of the obtained acid extract.
  • the component analysis was outsourced to Kurita Analysis Center Co., Ltd.
  • Table 3 shows the measurement results of total calcium (mg) converted to 1 g of wet weight of bone.
  • Concentrations of nitric acid, hydrochloric acid, formic acid and sulfuric acid and the analysis results for each acid were summarized for each immersion time. In the aqueous solutions of nitric acid, hydrochloric acid and formic acid, it was shown that most of the calcium could be recovered in about 24 hours. Also, in aqueous sulfuric acid, the liberated calcium forms insoluble calcium sulfate. Therefore, although the calcium concentration is low as an analysis result, calcium is actually eluted from the bone tissue.
  • Example 1-5 Preparation of protease-treated solution
  • the acid-treated bone tissue obtained in Example 1-2 was immersed in a treatment solution containing the following two kinds of proteases.
  • the conditions for protease treatment were protease concentration: 2% (w/w), temperature: 50°C, pH: optimum pH.
  • protease concentration 2% (w/w), temperature: 50°C, pH: optimum pH.
  • Protease 1 Neurase F3G (Amano Enzyme Co., Ltd., protease derived from Rhizopus niveus filamentous fungus)
  • Protease 2 pepsin (Sigma Aldrich, aspartic protease)
  • A represents the results of protease 1 treatment
  • B represents the results of protease 2 treatment.
  • Treatment times were 3.5 hours, 19 hours or 24 hours.
  • the results of visually confirming the transparency of the treatment liquid and the remaining bone tissue are as follows. ⁇ 3.5 hours later: Bone tissue was almost lost in all protease-treated systems. ⁇ 24 hours later: Bone tissue completely disappeared in all protease-treated systems, and the transparency of the protease-treated solution increased.
  • a protease-treated solution can be obtained by treating acid-treated bone tissue with a commercially available protease.
  • Example 1-6 Preparation of protease-treated solution and component analysis
  • a protease-treated solution was obtained by the following procedure. 1. The bone tissue was immersed in a 1 mol/L nitric acid aqueous solution or hydrochloric acid aqueous solution for 48 hours for acid treatment. 2. The resulting treated liquid was separated into an acid extract and a residue of bone tissue, and the acid extract was transferred to another container. 3. A 0.1 mol/L citrate buffer (pH 3.5) was added to the bone tissue residue. The amount added was 10 mL per 1 g of initial bone weight. 4. Actinidyne (a cysteine protease from kiwifruit) was pretreated.
  • the resulting protease-treated solution was passed through a 100 ⁇ m filter to obtain a filtrate from which fine insoluble matter was removed.
  • the results of component analysis of this filtrate are shown in Table 4 (values converted per 1 g of wet weight of bone).
  • the component analysis was outsourced to Kurita Analysis Center Co., Ltd.
  • Bone lysate A was prepared in one step without solution exchange between acid treatment and protease treatment.
  • the specific procedure is as follows. 1. The bone tissue was immersed in a 1 mol/L nitric acid aqueous solution, hydrochloric acid aqueous solution, formic acid aqueous solution or sulfuric acid aqueous solution for 48 hours for acid treatment. 2. A citrate buffer (pH 3.5) was added to the reaction system to a final concentration of 0.1 mol/L. 3. The optimum pH for the protease was adjusted with 5N NaOH and 5N HCl.
  • actinidine was adjusted to pH 3.5, pepsin to pH 3.0, and neurolase F3G to pH 3.0. 4.
  • Each protease was added to 1% (w/w).
  • the reaction conditions for the protease were 50° C. for 4 days.
  • Actinidyne (kiwifruit-derived cysteine protease) was pretreated at 25°C with a 20 mmol/L phosphate buffer (pH 6.5) containing 10 mmol/L dithiothreitol and 5 mmol/L ethylenediaminetetraacetic acid. Pretreatment for 90 minutes.
  • the obtained bone lysate A was passed through a 100 ⁇ m filter to obtain a filtrate from which fine insoluble matters were removed.
  • the results of measuring the calcium concentration of this filtrate are shown in Table 5 (values converted to 1 g of bone mass). Calcium was measured using a compact calcium ion meter LAQUAtwin-Ca-11 (Horiba Advanced Techno Co., Ltd.).
  • Example 1-8 Extraction of phosphoric acid
  • Crude phosphoric acid was obtained from bone tissue by the following procedure. 1. The bone tissue was immersed in a 1 mol/L nitric acid aqueous solution, hydrochloric acid aqueous solution, formic acid aqueous solution or sulfuric acid aqueous solution for 48 hours for acid treatment. 2. 0.15 volume of 5N NaOH was added to the reaction system, mixed well, and allowed to stand at room temperature for 1 hour. This gave a white solution. 3. The resulting solution was centrifuged at 10,000 g for 10 minutes at room temperature to remove the supernatant. The same amount of 1N HCl added in step 1 was added to the white precipitate and mixed well. This gave a clear solution. 4.
  • Na 2 SO 4 , K 2 SO 4 , MgSO 4 , (NH 4 ) 2 SO 4 , Na 2 CO 3 or NaHCO 3 is added in an amount equal to the calcium concentration contained in the reaction system and mixed well, It was allowed to stand at room temperature for 1 hour. This gave a white solution. 5. The resulting solution was centrifuged at 10,000 g for 10 minutes at room temperature to capture the supernatant. The resulting clear solution contains phosphoric acid.
  • Example 2 Growth evaluation and component analysis of plants cultivated with fertilizer containing bone tissue decomposition products]
  • material and method 1. Radish (Raphanus sativus L) seeds (Takii Seed Co., Ltd.) were sown on wet paper towels. The seeds were allowed to germinate by standing in the dark at 22° C. for 2 days. 2. The resulting plant was transplanted to a hydroponic urethane cube (2 cm x 2 cm x 2 cm). 3. A urethane cube was immersed in acid extract, protease-treated solution or bone solubilizing solution A and allowed to grow for 5 days.
  • the light conditions during this period were a photosynthetically effective photon flux density of about 150 ⁇ mol/m 2 ⁇ s, a light period of 16 hours, and a dark period of 8 hours.
  • a fluorescent lamp for growing cultivated plants Biolux A, NEC Lighting Co., Ltd.
  • Growth evaluation and component analysis of grown plants were performed. Specifically, it is as follows. ⁇ Aerial fresh weight: The plant body was cut at the boundary between the hypocotyl and the root, and the weight of the hypocotyl side was measured. Dry weight of above-ground part: The part of the plant body whose fresh weight was measured was dried in an oven at 80°C for 2 days. The weight was then measured.
  • ⁇ Aerial moisture content Calculated based on the difference between the fresh weight of the aerial part and the dry weight of the aerial part. •
  • Total polyphenol content 50 mg of cotyledons were crushed in a 90% methanol solution. Using the supernatant obtained by centrifugation, the total polyphenol content was measured by the Folin-Ciocalteu method.
  • Plants given protease-treated solution 1 and protease-treated solution 2 showed accelerated growth compared to the control. Similarly, the growth of the plants to which the acid extract was given was promoted as compared with the control (this point is as shown in (1)). Furthermore, the plant body given the bone solubilized solution A, which is a mixture of the protease-treated solution and the acid extract solution, showed a much higher growth-promoting effect than the control. From these results, it can be seen that both the acid extract and the protease-treated solution have the effect of promoting plant growth. Furthermore, it was found that the plant growth promoting effect of the bone solubilized solution A is higher than the growth promoting effect of the acid extract solution or the protease-treated solution alone.
  • the above-ground water content of the plant body increased by adding bone lysate A, and further increased by adding both bone lysate A and culture medium.
  • the total polyphenol content per cotyledon was increased by feeding bone lysate A and further increased by feeding both bone lysate A and culture medium. This is considered to be due to the effect of increased cotyledon weight as a result of accelerated plant growth.
  • Example 3 Plant body RNA expression analysis 1] [Extraction of total RNA from plants] 1. Radish (Raphanus sativus L) seeds (Takii Seed Co., Ltd.) were sown on wet paper towels. The seeds were allowed to germinate by standing at 22° C. in the dark at 100% humidity for 2 days. 2. The resulting plant body was transplanted to a urethane sponge. The urethane sponge is impregnated with either (1) water, (2) bone solubilizer A, (3) commercially available liquid fertilizer, or (4) a mixture of bone solubilizer A and commercially available liquid fertilizer. let me 3. Plants were grown at 22° C. for 1, 3 or 5 days.
  • the light conditions during this period were as follows: amount of light: about 100 ⁇ mol/m 2 ⁇ s, light period: 16 hours, dark period: 8 hours.
  • a fluorescent lamp for growing cultivated plants (Biolux A, NEC Lighting Co., Ltd.) was used as the light source. 4. About 0.1 g of leaf tissue and root tissue were collected from each grown plant. Immediately after collection, the tissue was placed in a mortar, liquid nitrogen was added, and the tissue was ground in a frozen state. 5.
  • Total RNA was extracted from ground tissue 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 fluorometer (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.
  • Table 6 shows the results. 13.2 to 88.2 ⁇ g of total RNA was extracted from about 0.1 g of plant leaves from the start of culture to 5 days. From about 0.1 g of plant roots, 8.4-43.8 ⁇ g of total RNA was extracted. A 260 /A 280 was about 2.0, confirming that there was no problem with the purity of RNA.
  • RNA sequencing library and RNA sequence analysis by next-generation sequencer Preparation of a cDNA library from total RNA extracted from sprouts and analysis of gene expression using a next-generation sequencer were entrusted to Agenta Corporation. The analysis procedure is as follows. 1. The quality of extracted total RNA (degree of RNA degradation) was assessed by BioAnalyzer (Agilent Technologies). 2. PolyA-mRNA was enriched from the extracted total RNA using beads coupled with polyT oligo DNA. 3. A cDNA library was generated using reverse transcriptase. 4. The base sequence of cDNA was determined using a next-generation sequencer (DNBSEQ-G400, MGI tech).
  • RNAseq analysis Based on this, the base sequences of expressed mRNAs were comprehensively determined (RNAseq analysis). Assembly of the sprout gene was performed by Stringtie software. At this time, reference was made to the entire sprout genome base sequence (http://radish.kazusa.or.jp) published in the database of the Kazusa DNA Research Institute.
  • Target genes include glutathione S-transferase ⁇ 19 (GSTU19), catalase 2 (CAT2), auxin transporter-like protein 2-1 (LAX2-1), glutathione S-transferase 12 (GST12) and calmodulin 5 (CaM5). It was adopted. The specific procedure is as follows. 1. Leaves of plants grown by feeding with (1) water, (2) bone solubilizing solution A, (3) commercially available liquid fertilizer, or (4) mixture of bone solubilizing solution A and commercially available liquid fertilizer and roots, total RNA was extracted. 2.
  • RT reaction 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. Quantitative PCR was performed on the target gene. For quantitative PCR, a specific primer pair, TB Green Premix Ex Taq II (Takara Bio Inc.), with 2.0 ng of cDNA as template was used. Thermal Cycler Dice Real Time System TP850 (Takara Bio Inc.) was used as an instrument for quantitative PCR.
  • each target gene was normalized to the expression level of the actin gene.
  • the nucleotide sequences of the primer pairs used for amplification of the target gene and actin gene are shown in Table 7 (SEQ ID NOs: 1 to 12 from top to bottom).
  • Example 4 Improvement of phosphoric acid extraction rate by pretreatment
  • pretreatment of bone tissue increased the amount of phosphoric acid contained in the acid extract.
  • an acid extract was prepared by the following procedure, and the amount of phosphoric acid contained was quantified. 1. Pork bones obtained from a slaughterhouse were finely pulverized with a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.). 2. 5 g of pulverized pork bone was subjected to one of the following pretreatments (1) to (5).
  • microwave irradiation is a preferred embodiment because it can be completed in a short period of time, does not require heating, can be applied to large bone tissue, and can further improve the extraction rate of phosphoric acid.
  • Example 5 Analysis of protein components contained in acid extract
  • the protein components contained in the acid extract were analyzed by the following procedure. 1. Pork bones obtained from a slaughterhouse were finely pulverized with a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.). 2. 30 mL of hydrochloric acid (1 mol/L) was added to a wet weight of 2 g of pork bone, and the mixture was immersed in the mixture at 20° C. for 48 hours while shaking (100 rpm). In this way the bone tissue was decalcified. 4. The supernatant was collected and used as an acid extract. 5. The obtained acid extracts were respectively designated as (1) undiluted acid extract, (2) 1/2 diluted acid extract, and (3) 1/4 diluted acid extract. 6.
  • Example 6 Preparation of protease-treated solution and protein component analysis
  • Example 6-1 Preparation of protease-treated solution
  • a protease-treated solution was obtained by the following procedure. 1. The bone tissue was immersed in a 1 mol/L hydrochloric acid aqueous solution for 48 hours for acid treatment. 2. The resulting treated liquid was separated into an acid extract and a residue of bone tissue, and the acid extract was transferred to another container. 3. A 0.1 mol/L citrate buffer (pH 3.5) was added to the bone tissue residue. The amount added was 10 mL per 1 g of initial bone weight. 4. The obtained bone tissue residue was immersed in a treatment liquid containing the following three kinds of proteases.
  • protease treatment The conditions for protease treatment were protease concentration: 2% (w/w), temperature: 50°C, pH: optimum pH.
  • protease concentration 2% (w/w), temperature: 50°C, pH: optimum pH.
  • ⁇ Pro Leather FG-F from Amano Enzyme Co., Ltd., Bacillus sp.
  • Protease P “Amano” 3G from Aspergillus melleus, Amano Enzyme Co., Ltd.
  • Protease M “Amano” SD From Aspergillus oryzae, Amano Enzyme Co., Ltd.
  • FIG. 15 shows the appearance of the treatment liquid.
  • Examples 1-5 neutral F3G and pepsin
  • 1-6 actinidyne
  • FIG. 15 shows the appearance of the treatment liquid.
  • the results of Examples 1-5 neutral F3G and pepsin
  • 1-6 actinidyne
  • the protease examined in this example also decomposed the bone decomposition residue, and a protease-treated solution was obtained. Precipitation is observed in the protease-treated solution obtained in this example, and this is considered to be a precipitate of calcium salt due to the near-neutrality of the solution.
  • Example 6-2 described later, it can be said that the protein contained in the bone decomposition residue was sufficiently degraded by the protease treatment.
  • Example 6-2 Analysis of protein components contained in protease-treated solution
  • a protease-treated solution was prepared in the same manner as in Examples 1-5, 1-6 and 6-1 using the following proteases.
  • ⁇ Pepsin Sigma Aldrich, aspartic protease
  • ⁇ Peptidase R (Amano Enzyme Co., Ltd., derived from the genus Rhizopus)
  • Protease P “Amano” 3G from Aspergillus melleus, Amano Enzyme Co., Ltd.)
  • Pro Leather FG-F from Amano Enzyme Co., Ltd., Bacillus sp.
  • ⁇ Neurase F3G derived from Rhizopus niveus, Amano Enzyme Co., Ltd.
  • ⁇ Protease M “Amano” SD (from Aspergillus oryzae, Amano Enzyme Co., Ltd.)
  • Actinidine from kiwifruit, cyste
  • the resulting protease-treated solution was diluted 10-fold (lane A) or 5-fold (lane B) and electrophoresed using a 16% polyacrylamide gel.
  • the migrated proteins were silver-stained.
  • a solution containing only the enzyme (lane C) was also subjected to electrophoresis.
  • Example 7 Recovery of phosphoric acid from bone tissue
  • Phosphoric acid was recovered as a precipitate from the acid extract by the following procedure. The recovery of phosphoric acid was also calculated.
  • Pork bones obtained from a slaughterhouse were finely pulverized with a mill (IKA TUBE MILL 100, IKA JAPAN Co., Ltd.).
  • the crushed pork bone was immersed in a 1N nitric acid aqueous solution or a 1N hydrochloric acid aqueous solution for 48 hours. 3. The supernatant was collected to obtain an acid extract. 4.
  • the content of phosphoric acid in the acid extract using nitric acid was 24.6 mg. This corresponds to 34.7% of the wet weight of pork bone (71 mg) used in step 2.
  • Example 8 Purification of phosphoric acid by addition of sulfate
  • Example 8-1 Examination of phosphate recovery rate in supernatant
  • Calcium was removed as calcium sulfate precipitate from the redissolved solution of the phosphate precipitate obtained in Example 7 by the following procedure. 1.
  • the acid used in step 2 of Example 7 was a 1N nitric acid aqueous solution or a 1N hydrochloric acid aqueous solution.
  • the amount of 5N aqueous sodium hydroxide solution added in step 4 of Example 7 was 100 ⁇ L. 2.
  • Sulfuric acid or sulphate sodium sulphate, potassium sulphate, ammonium sulphate or magnesium sulphate
  • the amount of sulfuric acid or sulfate added was such that the final concentration was 0.4M, 0.6M, 0.8M or 1.0M.
  • the calcium sulfate precipitate that formed 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.
  • Phosphoric acid recovery was also based on the phosphoric acid content in the precipitate formed by adding an aqueous sodium hydroxide solution to the acid extract (precipitate obtained in step 5 of Example 7) before adding sulfuric acid or a sulfate salt. calculated the rate.
  • Table 9 shows the results. It was found that if the concentration of sulfate is appropriately set, the same level of phosphoric acid recovery can be achieved with sulfate as with sulfuric acid. Also, at a low concentration of 0.4 M, the phosphoric acid concentration obtained by adding sulfate tended to be slightly higher than the phosphoric acid concentration obtained by adding sulfuric acid. Importantly, not only does the addition of sulfate allow for a smaller system volume than the addition of sulfuric acid, but the concentration of phosphoric acid obtained is as much as 1.5 times higher. In this respect, the method according to one embodiment of the invention is a useful method for purifying phosphoric acid. Furthermore, when the technique is put into practical use, it is preferable that the amount of sulfuric acid or sulfate added is small.
  • sulfate rather than sulfuric acid for calcium precipitation has the following advantages.
  • ⁇ Phosphate concentration is about 1.5 times higher.
  • ⁇ Because no liquid is added the increase in volume of the reaction system can be reduced.
  • ⁇ Since the reaction system does not become strongly acidic, only a small amount of base is required for neutralization. ⁇ High safety.
  • Example 8-2 Examination of ability to remove calcium from supernatant
  • the calcium content in the supernatant obtained in step 4 of Example 8-1 was measured.
  • LAQUAtwin-Ca-11 was used for the measurement.
  • the calcium residual rate was determined based on the calcium content in the precipitate formed by adding an aqueous sodium hydroxide solution to the acid extract (precipitate obtained in step 5 of Example 7) before adding sulfuric acid or sulfate. Calculated.
  • Table 11 shows the calculated relative ratio (%) of calcium amount (mg)/phosphoric acid amount (mg) based on the results of Tables 9 and 10.
  • Phosphate can also be recovered by adding sulfuric acid, but it was shown that the amount of residual calcium was greater than that of the experimental system with the addition of sulfate at any concentration.
  • the amount of residual calcium was significantly lower than in the experimental system to which sulfuric acid was added, indicating that the recovery of phosphoric acid was relatively high.
  • the residual amount of calcium can be reduced to 0.2% by weight without reducing the recovery of phosphoric acid. Therefore, it can be said that it is more preferable to use sulfate than sulfuric acid for purifying phosphoric acid extracted from bone tissue.
  • Example 8-3 Examination of phosphate leakage rate to precipitate
  • the phosphoric acid content in the calcium sulfate precipitate obtained in step 3 of Example 8-1 was measured by the following procedure. 1. The calcium sulfate precipitate was redissolved by adding EDTA (pH 7.4) and 5N sodium hydroxide. 2. The phosphoric acid content in the resulting solution was measured. A Malachite Green Phosphate Assay Kit (BioAssay Systems) was used for the measurement. Also, the phosphoric acid loss was determined based on the phosphoric acid content in the precipitate formed by adding aqueous sodium hydroxide solution to the acid extract (precipitate obtained in step 5 of Example 7) before adding sulfuric acid or sulfate. calculated the rate.
  • Table 12 shows the results. As can be seen from the table, the phosphate content in the calcium sulphate precipitate could be reduced to the same extent as the sulfuric acid by adjusting the sulphate concentration accordingly. If the sulfate concentration was 0.4M, the leaked phosphoric acid could be sufficiently reduced.
  • Example 8-4 Examination of total phosphoric acid recovery rate
  • the total amount of the phosphate recovery rate in the supernatant examined in Example 8-1 and the phosphate leakage rate to the precipitate examined in Example 8-3 was examined.
  • Phosphoric acid contained in the precipitate has the potential to be re-recovered by washing the precipitate.
  • phosphoric acid contained in the precipitate can be recovered by washing the precipitate (calcium sulfate) with pure water. Therefore, the higher the total phosphoric acid content, the higher the potential amount of phosphoric acid that can be recovered.
  • Example 9 Plant body RNA expression analysis 2
  • Preparation of cDNA library and gene expression analysis by next-generation sequencer A procedure similar to that of Example 3 was followed.
  • FIG. 18 shows the results of analysis of expression-variable genes in leaves of sprouts cultivated for 5 days with bone lysate B or a commercially available liquid fertilizer. There were 1,568 genes with more than two-fold increase in FPKM values. There were 1,654 genes whose FPKM values decreased by more than half. Therefore, the expression levels of a total of 3,222 genes varied. As a result of the GO enrichment analysis, 65 genes related to growth control, 44 genes related to photosynthesis, and 44 genes related to photosynthesis, and light harvesting in photosynthetic system I were found to be variable in expression levels. 26 genes were included.
  • Fig. 19 shows the analysis results of expression variation genes in the roots of sprouts cultivated for 5 days with a mixture of bone lysate B and a commercially available liquid fertilizer or only with a commercially available liquid fertilizer.
  • genes with varying expression levels included 17 genes related to nitrate assimilation, 16 genes related to root hair elongation, and 12 genes related to nitrate uptake. It was
  • Fig. 20 shows the analysis results of expression variation genes in sprout leaves cultivated for 5 days with a mixed solution of bone lysate B and a commercially available liquid fertilizer or only with a commercially available liquid fertilizer.
  • 32 genes related to response to abscisic acid (a plant hormone), 13 genes related to root hair elongation, and 13 genes related to the promotion of germination were found to be variable in expression levels. It contained 6 genes that
  • Example 10 Examination of calcium ion removal efficiency by addition of sulfate
  • Calcium ions were removed as a calcium carbonate precipitate by adding sulfate to the redissolved solution of the phosphate precipitate.
  • Calcium removal efficiency was examined by measuring the calcium ion content in the supernatant after removing the precipitate.
  • the specific procedure is as follows. 1. In the same manner as in Example 7, a redissolved solution of phosphoric acid precipitate was prepared. The acid used in step 2 of Example 7 was 1N aqueous hydrochloric acid. The amount of 5N aqueous sodium hydroxide solution added in step 4 of Example 7 was 100 ⁇ L. 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 0.4M.
  • the calcium sulfate precipitate that formed was removed by centrifugation.
  • the weight (mg) of calcium ions contained in the supernatant was measured. Calcium ions were measured under precision 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 methasulfonic 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
  • the injection volume was 30 ⁇ L.
  • Calcium ion concentration was measured by measuring electric conductivity ( ⁇ S) and obtaining a regression equation from the area of the standard substance. From the calcium ion concentration, the calcium ion content per sample (30 ⁇ L) was determined.
  • the calcium ions contained in the bone tissue contribute to a decrease in the recovery of phosphoric acid. This is because calcium ions combine with phosphate ions to form calcium phosphate precipitates in the neutral range. Therefore, it is desirable to remove calcium ions by precipitation, and it has been shown to be successfully removed by addition of sulfate.
  • Potassium ions, magnesium ions or ammonium ions contained in sulfates are components necessary for plant growth. Therefore, when potassium sulfate, magnesium sulfate or ammonium sulfate is used as a sulfate, the fertilizer can contain the components necessary for plant growth. Alternatively, even if the base added in step 4 of Example 7 is changed from sodium hydroxide to potassium hydroxide, the fertilizer can contain components necessary for plant growth. By adopting such a manufacturing method, a fertilizer with increased value can be manufactured.
  • Example 11 Purification of phosphoric acid by addition of carbonate
  • Calcium was removed as calcium carbonate precipitate from the redissolved phosphate precipitate obtained in Example 7 by the following procedure. 1.
  • a redissolved solution of phosphoric acid precipitate was prepared.
  • the acid used in step 1 of Example 7 was a 1N nitric acid aqueous solution or a 1N hydrochloric acid aqueous solution.
  • the amount of 5N aqueous sodium hydroxide solution added in step 4 of Example 7 was 100 ⁇ L.
  • Carbonate solid sodium bicarbonate or sodium carbonate
  • the amount of sodium bicarbonate added was such that the final concentration was 0.4M.
  • the amount of sodium carbonate added was such that the final concentration was 0.6M. 3.
  • the calcium carbonate precipitate that formed was removed by centrifugation. 4.
  • the concentration (ppm) of phosphate ions contained in the supernatant was measured. Phosphate ions were measured under precision measurement conditions using a high performance ion chromatography IC-8100EX (Tosoh Corporation) connected to a TSKgel SuperIC-Anion HS (4.6 mm ID ⁇ 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 ⁇ L.
  • the phosphate ion concentration was measured by measuring the electric conductivity ( ⁇ S) and determining the regression equation from the area of the standard substance. From the phosphate ion concentration, the phosphate ion content per sample (30 ⁇ L) was determined.
  • Example 12 Removal of cations contained in sulfate
  • Example 12-1 Removal of sodium ions or potassium ions
  • the cations (sodium ions or potassium ions) contained in the sulfate added to remove calcium ions were removed by the following procedure. Specifically, a strong cation exchange gel was used to adsorb sodium ions or potassium ions. 1. TSKgel SP-TOYOPEARL 650M gel (Tosoh Corporation) was decanted with pure water and packed in a microspin column (GE Healthcare). 2. An appropriate amount of the supernatant after addition of sulfate and removal of calcium sulfate was added to the upper layer of the gel. 3.
  • the supernatant was passed through the gel by centrifugation in a tabletop centrifuge.
  • the flow-through fraction that passed through the gel was collected. 3.
  • the content (mg) of sodium ions or potassium ions contained in the flow-through fraction was measured.
  • LAQUAtwin-Na-11 (Horiba Advanced Techno Co., Ltd.) was used to measure sodium ions.
  • LAQUAtwin-K-11 was used to measure potassium ions.
  • Example 12-2 Removal of magnesium ions or ammonium ions
  • the cations (magnesium ions or ammonium ions) contained in the sulfate added to remove calcium ions were removed by the following procedure. Specifically, a strong cation exchange gel was used to adsorb magnesium ions or ammonium ions. 1. TSKgel SP-TOYOPEARL 650M gel (Tosoh Corporation) was decanted with pure water and packed in a microspin column (GE Healthcare). 2. An appropriate amount of the supernatant after addition of sulfate and removal of calcium sulfate was added to the upper layer of the gel. 3. The supernatant was passed through the gel by centrifugation in a tabletop centrifuge.
  • the flow-through fraction that passed through the gel was collected. 3.
  • the content ( ⁇ g) of magnesium ions, ammonium ions or calcium ions contained in the flow-through fraction was measured.
  • the ion concentration was measured under precision 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 methasulfonic 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
  • the injection volume was 30 ⁇ L.
  • Calcium ion concentration was measured by measuring electric conductivity ( ⁇ S) and obtaining a regression equation from the area of the standard substance. From the ion concentration, the ion content per sample (30 ⁇ L) was determined.
  • Example 13 Purification of phosphoric acid from commercially available bone meal
  • Phosphoric acid was recovered by solubilizing commercially available steamed bone meal (Omiya Green Service Co., Ltd.) according to the following procedure. 1. 10 mL of sulfuric acid (1N), hydrochloric acid (1N) or nitric acid (1N) was added to 1 g of steamed bone meal and shaken at 25° C. for 19 hours. 2. The resulting acid extract was separated into a supernatant and a precipitate using a centrifuge. 3. 0.1 mL of aqueous NaOH (5N) was added to 0.5 mL of supernatant and shaken for 1 hour. 4.
  • the reaction solution was separated into supernatant and precipitate by centrifugation. 5. A suitable amount of hydrochloric acid (1N) was added to the precipitate to dissolve the precipitate. 6. Steps 3-5 were repeated multiple times. The precipitate was then dissolved in 0.5 mL of hydrochloric acid (0.4N). As a result, a precipitate solution enriched with phosphoric acid was obtained. 7. The anion content in the precipitate solution obtained was measured. The measurement was performed under precision measurement conditions using a high performance ion chromatography IC-8100EX (Tosoh Corporation) connected to a TSKgel SuperIC-Anion HS (4.6 mm ID ⁇ 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
  • the injection volume was 30 ⁇ L.
  • the anion concentration was measured by measuring the electric conductivity ( ⁇ S) and determining the regression equation from the area of the standard substance. Anion content per sample (30 ⁇ L) was determined from the anion concentration.
  • the results of this example show that phosphoric acid can be extracted from commercially available bone powder (bone tissue heat-treated under pressure).
  • the method for producing phosphoric acid according to one embodiment of the present invention is a sustainable technology with lower energy costs and less environmental destruction than the method of recovering phosphoric acid from phosphate rock or dried sewage sludge. It has been proven.
  • Example 14 Production of protease-treated liquid from commercially available bone meal
  • Commercially available steamed bone meal (Omiya Green Service Co., Ltd.) was solubilized by the following procedure to produce a protease-treated solution.
  • 10 mL of sulfuric acid (1N), hydrochloric acid (1N) or nitric acid (1N) was added to 1 g of steamed bone meal and shaken at 25° C. for 24 hours.
  • the resulting acid extract was separated into a supernatant (acid extract) and a precipitate (bone residue) using a centrifuge and recovered. 3.
  • a protease-treated solution was prepared from the precipitate according to S5 of FIG. Neurase F3G (Amano Enzyme Co., Ltd.) or Papain W-40 (Amano Enzyme Co., Ltd.) was used as the protease, and the procedure was the same as in Example 1-5. Specifically, it was as follows.
  • steamed bone meal is a mixture of pork and chicken bones heat-treated under high pressure.
  • a protease-treated solution could be prepared by the method for producing a fertilizer according to one embodiment of the present invention.
  • the protease-treated solution itself can be used as a fertilizer, or can be used as a raw material for the bone solubilizing solution B.
  • Liquid fertilizer is expected to have a rapid plant growth effect that cannot be expected from solid bone meal. Liquid fertilizers can also be applied to hydroponics and foliar application.
  • Example 15 Measurement of peptide concentration in acid extract made from commercially available bone meal
  • An acid extract was prepared from commercially available steamed bone meal (Omiya Green Service Co., Ltd.) by the following procedure. The peptide concentration contained in the acid extract was measured. 1. 10 mL of sulfuric acid (1N), hydrochloric acid (1N) or nitric acid (1N) was added to 1 g of steamed bone meal and shaken at 25° C. for 24 hours. 2. The resulting acid extract was separated into a supernatant and a precipitate using a centrifuge. The supernatant was recovered as an acid extract. 3.
  • a 0.4 mol/L potassium hydroxide aqueous solution was added to 0.5 mL of the supernatant to precipitate discrete calcium. 4.
  • the peptide concentration in the supernatant obtained by removing the precipitate from the reaction solution obtained in step 3 was measured.
  • a protein assay BCA kit (product number 297-73101, Fujifilm Wako) was used for the measurement. The measurement method followed the product manual. Absorbance was measured at 540 nm. The higher the absorbance, the higher the peptide concentration. 5.
  • a precipitate was recovered from the reaction solution obtained in step 3. At this time, only the system treated with hydrochloric acid or nitric acid in step 1 was recovered. 6. The precipitate was completely dissolved with 1N hydrochloric acid.
  • step 6 after the first and third iterations were completed, the peptide concentration was measured in the supernatant after the precipitate was removed from the reaction.
  • a protein assay BCA kit (product number 297-73101, Fujifilm Wako) was used for the measurement. The measurement method followed the product manual. Absorbance was measured at 540 nm. The higher the absorbance, the higher the peptide concentration.
  • Example 5 it was shown that the acid extract contained almost no protein components that were stained with the silver staining kit (see also Figure 14). For this reason, in Example 5, it was presumed that the peptide bonds of proteins were cleaved to form low-molecular-weight peptides and amino acids. This example supports this presumption.
  • Peptides become impurities in the production of phosphate. It was found that most of the peptides as impurities could be removed by repeating the calcium phosphate dissolution-precipitation process (steps S13 and S14 in FIG. 3). As shown in Table 19, repeating the dissolution-precipitation process three times reduced the peptide concentration by 1/100. This result suggested that peptides could be successfully removed to increase phosphate concentration, especially from acid extracts treated with hydrochloric acid or nitric acid.
  • the present invention can be used for the production of phosphoric acid.

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