Highly hydrophilic fulvic acid, its separation and purification method, and uses of highly hydrophilic fulvic acid

Abstract

The present invention provides a method for separating and purifying active substances contained in humic substances that can be used to promote plant growth, etc. [Solution] A method for separating and purifying highly hydrophilic fulvic acid, comprising: (1) a step of extracting fulvic acid and humic acid into alkaline aqueous solution or alkaline electrolyzed water by immersing a humic substance raw material in the alkaline aqueous solution or alkaline electrolyzed water, and separating the fulvic acid from the unwanted residue, humin; (2) a step of adding an acid to the obtained extract to precipitate and separate the humic acid contained in the extract; (3) a step of treating the supernatant obtained by (2) with a hydrophobic substance adsorption resin to adsorb and separate the hydrophobic fulvic acid; (4) a step of treating the effluent from (3) with a hydrophilic substance adsorption resin to adsorb and separate the hydrophilic fulvic acid; and (5) a step of adding aluminum chloride to the effluent from (4) and adjusting the pH from weakly acidic to neutral, thereby precipitating and separating the highly hydrophilic fulvic acid (highly hydrophilic fulvic acid separation step).

Description

[Technical Field]

[0001] The present invention relates to a method for separating and purifying a specific fulvic acid (highly hydrophilic fulvic acid) contained in humic substances, and to uses of the specific fulvic acid obtained by the method. [Background technology]

[0002] Humic substances are brown to black organic substances produced by further biotic and abiotic reactions of decomposition products and microbial metabolic products produced during the microbial decomposition of biological materials such as plant remains, animal remains, and feces. Humic substances are produced not only in natural environments such as soil, wetlands, inland water, and seawater, but also in artificial processes such as composting. Humic substances have physiologically active effects, such as promoting plant growth, alleviating stress, and promoting nutrient absorption (Non-Patent Documents 1-3), and many humic substances are commercially available. However, most commercially available humic substances are not made from humic substances produced during the microbial decomposition process described above. Instead, they are produced by treating weathered coal or lignite, a type of coal, with nitric acid or sulfuric acid, which are different in composition from humic substances derived from microbial reactions. A meta-analysis of numerous published papers on the effects of humic substance application on plant growth (Non-Patent Document 4) found that the growth-promoting effect of humic substances derived from lignite and peat was less effective than that of humic substances extracted from compost or soil.

[0003] Humic substances have long been classified into humins (alkali-insoluble), humic acids (alkali-soluble and acid-insoluble), and fulvic acids (alkali-soluble and acid-soluble) based on their solubility in alkali and acid. Some humic substances are commercially available as "humic acid" or "fulvic acid." Humic acids are easily recovered because they are insoluble and precipitate when the alkaline solution of the humic substance raw material is acidified. However, fulvic acids remain dissolved even when the alkaline solution is acidified, making their recovery difficult. The International Humic Substances Society (IHSS, https: / / humic-substances.org), the only international organization specializing in humic substances, has established a standard method (IHSS method) (Non-Patent Document 5), which defines fulvic acid as the fraction adsorbed by DAX-8 resin, a hydrophobic adsorption resin (Figure 1). However, most of the fulvic acid available on the market as a humic material is not produced using these standard methods, and its components differ significantly from those of fulvic acid in academic research.

[0004] The IHSS method defines fulvic acid as only the fraction that adsorbs to DAX-8 resin, i.e., hydrophobic fulvic acid, and therefore excludes fulvic acid that does not adsorb to DAX-8 resin, i.e., hydrophilic fulvic acid. However, in the field of water quality chemistry, it is known that fulvic acid that does not adsorb to DAX-8 resin can be recovered as a hydrophilic acid by adsorption to XAD-4 resin, a hydrophilic substance adsorption resin (Non-Patent Document 6). This hydrophilic acid in water is sometimes called hydrophilic fulvic acid. Another method is known in which fulvic acid is recovered as a precipitate by adjusting the fulvic acid solution from weakly acidic to neutral (pH 4-7) rather than by adsorption to DAX-8 resin (Non-Patent Document 7).

[0005] On the other hand, Patent Document 1 discloses a method for extracting fulvic acid and humic acid contained in humic substances, in which alkaline electrolyzed water is used instead of the alkaline aqueous solution in which compounds such as sodium hydroxide are dissolved, which is used for alkaline extraction in the conventional IHSS method.The document describes that such a method reduces the amount of by-product salts produced, and enables easy and safe extraction of fulvic acid and humic acid from humic substances with efficiency equal to or higher than conventional methods. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2018-127413 A (Patent No. 6653858) [Non-patent literature]

[0007] [Non-Patent Document 1] Canellas, LP, Olivares, FL, Aguiar, NO, Jones, DL, Nebbioso, A., Mazzei, P. and Piccolo, A. (2015): Humic and fulvic acids as biostimulants in horticulture. Sci. Hort., 196, 15-27 [Non-patent document 2] Olk, DC, Dinnes, DL, Scoresby, JR, Callaway, CR and Darlington, JW (2018): Humic products in agriculture: potential benefits and research challenges-a review. J. Soil Sediment, 18, 2881-2891 [Non-patent document 3] Jindo, K., Olivares, FL, da Paixao Malcher, DJ, Sanchez-Monedero, MA, Kempenaar, C. and Canellas, LP (2020): From lab to field: Role of humic substances under open-field and greenhouse conditions as biostimulant and biocontrol agent. Front. Plant Sci., 11, 426

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Non-licensed Document 7

[0008] An object of the present invention is to provide a further active substance contained in a humic substance that can be used for promoting plant growth, etc. [Means for solving the problem]

[0009] The present inventors conducted a test comparing the plant growth-promoting effect of a humic substance material produced according to the method described in Patent Document 1, i.e., an extract containing humic substances such as fulvic acid and humic acid without isolation, with the plant growth-promoting effect of humic acid and fulvic acid (hydrophobic fulvic acid) prepared according to the IHSS method alone. They found that the former humic substance material contains substances that have plant-activating effects in addition to humic acid and fulvic acid (hydrophobic fulvic acid) (see "Test Example 1" below). The present inventors further discovered that the fraction that does not adsorb to a conventional hydrophobic substance adsorption resin used to isolate fulvic acid (hydrophobic fulvic acid) contains a more hydrophilic "hydrophilic fulvic acid" and an even more hydrophilic "highly hydrophilic fulvic acid," which can be isolated and purified separately, and that both the "hydrophilic fulvic acid" and the "highly hydrophilic fulvic acid" have excellent plant-activating effects.

[0010] That is, the present invention includes at least the following: [Section 1] (1) A process of soaking a humic substance raw material in an alkaline aqueous solution or alkaline electrolyzed water to extract fulvic acid and humic acid into the alkaline aqueous solution or alkaline electrolyzed water and separate them from the insoluble residue, humin (hereinafter referred to as the "alkali treatment process"); (2) a step of adding an acid to the extract obtained by the alkali treatment step (1) to precipitate and separate the humic acid contained in the extract (hereinafter referred to as the "acid treatment step"); (3) A step of treating the supernatant obtained by the acid treatment step (2) with a hydrophobic substance adsorption resin to adsorb and separate hydrophobic fulvic acid (hereinafter referred to as the "hydrophobic fulvic acid separation step"); (4) a step of treating the permeate from the hydrophobic fulvic acid separation step (3) with a hydrophilic substance adsorption resin to adsorb and separate hydrophilic fulvic acid (hereinafter referred to as the "hydrophilic fulvic acid separation step"); and (5) A process of adding aluminum chloride to the effluent from the hydrophilic fulvic acid separation process (4) and adjusting the pH from weakly acidic to neutral to precipitate and separate highly hydrophilic fulvic acid (hereinafter referred to as the "highly hydrophilic fulvic acid separation process"); A method for separating and purifying highly hydrophilic fulvic acid, comprising: [Section 2] Item 1. The method for separating and purifying highly hydrophilic fulvic acid according to Item 1, wherein the hydrophobic substance adsorption resin used in the hydrophobic fulvic acid separation step is "Supelite (registered trademark) DAX-8" or polyvinyl polypyrrolidone resin. [Section 3] Item 1 or 2, the separation and purification method according to Item 1 or 2, wherein the hydrophilic substance adsorption resin used in the hydrophilic fulvic acid separation step is "Amberlite (registered trademark) XAD-4" or a styrene-divinylbenzene resin. [Section 4] Item 4. A highly hydrophilic fulvic acid obtained by the separation and purification method according to any one of Items 1 to 3. [Section 5] Item 5. A plant activator containing the highly hydrophilic fulvic acid according to item 4 as a main component. [Effects of the Invention]

[0011] The present invention makes it possible to separate and purify "highly hydrophilic fulvic acid," which is contained in humic substances but has not been fully utilized until now. "Highly hydrophilic fulvic acid" can be used, for example, to create new humic substances containing it as a main component, or as a plant activator (biostimulant). [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1 shows an outline of the IHSS method for separating and purifying humic acid and fulvic acid (hydrophobic fulvic acid) [A] and more specific procedures and conditions [B]. [Figure 2] FIG. 2 shows an outline of the method for separating and purifying highly hydrophilic fulvic acid according to the present invention. [Figure 3] FIG. 3 shows the results of [Test Example 1] Root elongation test (part 1) using Komatsuna seedlings. [Figure 4] FIG. 4 shows reversed-phase chromatograms of hydrophilic fulvic acid and highly hydrophilic fulvic acid in [Test Example 3]. [Figure 5] Figure 5 shows the results of the humic substance "HS-2 (registered trademark) Pro" (sometimes referred to as "HS-2" in this specification) in [Test Example 4] Root elongation test (part 2) using Komatsuna seedlings. The values ​​in the figure are relative values, with distilled water (0 mg / L) set at 100. The letters in the figure indicate significant differences at the 5% level, and the same symbols indicate no significant differences. [Figure 6] Figure 6 shows the results of the root elongation test (part 2) using Komatsuna seedlings in [Test Example 4], using humic acid, hydrophobic fulvic acid, hydrophilic fulvic acid, and highly hydrophilic fulvic acid. The values ​​in the figure are relative values, with distilled water (0 mg / L) set at 100. The letters in the figure indicate significant differences at the 5% level, and the same symbol indicates no significant differences. DETAILED DESCRIPTION OF THE INVENTION

[0013] -Method for separating and purifying highly hydrophilic fulvic acid- The method for separating and purifying highly hydrophilic fulvic acid according to the present invention (sometimes referred to as the "method of the present invention" in this specification) comprises the following steps (1) to (5): (1) A process of soaking a humic substance raw material in an alkaline aqueous solution or alkaline electrolyzed water to extract fulvic acid and humic acid into the alkaline aqueous solution or alkaline electrolyzed water and separate them from the insoluble residue, humin (referred to as the "alkali treatment process" in this specification); (2) a step of adding an acid to the extract obtained by the alkali treatment step (1) to precipitate and separate the humic acid contained in the extract (referred to as the "acid treatment step" in this specification); (3) a step of treating the supernatant obtained in the acid treatment step (2) with a hydrophobic substance adsorption resin to adsorb and separate hydrophobic fulvic acid (referred to as the "hydrophobic fulvic acid separation step" in this specification); (4) a step of treating the permeate from the hydrophobic fulvic acid separation step (3) with a hydrophilic substance adsorption resin to adsorb and separate hydrophilic fulvic acid (referred to as the "hydrophilic fulvic acid separation step" in this specification); and (5) A process in which aluminum chloride is added to the effluent from the hydrophilic fulvic acid separation process (4) and the pH is adjusted from weakly acidic to neutral, thereby precipitating and separating highly hydrophilic fulvic acid (referred to as the "highly hydrophilic fulvic acid separation process" in this specification).

[0014] Humic substances are aggregates of organic matter, such as polymeric compounds with variable chemical structures, that are produced when the organic matter that constituted an organism (mainly a plant) after its death, such as lignin, polysaccharides, proteins, lipids, and nucleic acids, is subjected to chemical and microbiological action. In the present invention, "humin," "humic acid," "hydrophobic fulvic acid," "hydrophilic fulvic acid," and "highly hydrophilic fulvic acid" are all aggregates (fractions) of organic matter that correspond to "humic substances." Soil, compost, and the like that contain such humic substances can be used as humic substance raw materials.

[0015] (1) Alkali treatment process The alkaline treatment step in the method of the present invention can be carried out by treating the humic material with an NaOH aqueous solution or an equivalent alkaline aqueous solution (e.g., a KOH aqueous solution) according to the IHSS method (see Figure 1[B]), or by treating the humic material with alkaline electrolyzed water instead of the alkaline aqueous solution according to the method described in Patent Document 1. By treating the humic material with such an alkaline aqueous solution or alkaline electrolyzed water, fulvic acids (hydrophobic fulvic acid, hydrophilic fulvic acid, and highly hydrophilic fulvic acid) and humic acid contained in the material can be extracted, and the insoluble residue, humin, can be separated. Humin can be separated using common techniques, such as centrifugation and / or filtration. The extract (supernatant) from which humin has been separated is used for the subsequent acid treatment step.

[0016] For details of embodiments using alkaline electrolyzed water, refer to Patent Document 1. For example, the pH of alkaline electrolyzed water is preferably 10.0 to 13.0, more preferably 11.0 to 13.0, and particularly preferably 12.0 to 13.0. The alkaline electrolyzed water in which the humic substance raw material is soaked is preferably heated to 40 to 70°C.

[0017] (2) Acid treatment process The acid treatment step in the method of the present invention can be carried out by adding acid to the extract (supernatant from which humin has been separated) obtained in the alkali treatment step, in accordance with the IHSS method (see Figure 1 [B]). This treatment precipitates the humic acid contained in the extract. The precipitated humic acid can be recovered from the extract using common techniques, such as centrifugation and / or filtration. The extract (supernatant) from which humic acid has been separated is used in the subsequent hydrophobic fulvic acid separation step.

[0018] In the IHSS method, the acid used in the acid treatment step is HCl, but other equivalent inorganic acids or organic acids (such as citric acid) can also be used. When an acid other than HCl is used, the conditions such as the concentration and amount of the acid (pH after addition) and the treatment time can be appropriately adjusted so as to obtain an extract (supernatant) equivalent to that obtained when HCl is used according to the IHSS method, or a different extract (supernatant) within a range that does not pose a problem in carrying out the present invention.

[0019] (3) Hydrophobic fulvic acid separation process The hydrophobic fulvic acid separation step in the method of the present invention can be carried out by treating the supernatant obtained from the acid treatment step with a hydrophobic substance adsorption resin, in accordance with the IHSS method (see Figure 1 [B]). By such treatment, the "hydrophobic fulvic acid" among the fulvic acids contained in the supernatant is adsorbed onto the resin and can be separated from the supernatant. The permeate after separation of the hydrophobic fulvic acid is used for the subsequent hydrophilic fulvic acid separation step.

[0020] In the IHSS method, the "hydrophobic substance adsorption resin" used in the hydrophobic fulvic acid separation process can be "XAD-8" (Rohm and Haas, discontinued, methyl methacrylate ester) or its equivalent, "Supelite® DAX-8" (Merck, discontinued), as well as polyvinyl polypyrrolidone resin (insoluble polyvinylpyrrolidone, crosslinked polyvinylpyrrolidone). The hydrophobic substance adsorption resin and treatment conditions can be appropriately selected within a range that allows the hydrophobic fulvic acid to be adsorbed and the hydrophilic and highly hydrophilic fulvic acids to remain in the effluent.

[0021] If desired, the hydrophobic fulvic acid adsorbed on the resin can be recovered by desorption (elution) from the resin. A suitable eluent, such as 0.1 M sodium hydroxide and distilled water, can be used to desorb the hydrophobic fulvic acid. If necessary, the eluted fraction can be neutralized by passing it through a hydrogen-type cation exchange resin, such as Amberlite® IR120BH (Organo Corporation) or other strongly acidic cation exchange resin, and purified hydrophobic fulvic acid can be recovered from the effluent.

[0022] (4) Hydrophilic fulvic acid separation process The hydrophilic fulvic acid separation step in the method of the present invention can be carried out by treating the permeate from the hydrophobic fulvic acid separation step in the method of the present invention with a hydrophilic substance adsorption resin, which has not been used in the IHSS method (see Figure 1 [B]). By such treatment, the "hydrophilic fulvic acid" contained in the permeate is adsorbed onto the resin and can be separated from the permeate. The permeate from which the hydrophilic fulvic acid has been separated is used for the subsequent highly hydrophilic fulvic acid separation step.

[0023] As the "hydrophilic substance adsorption resin" in the hydrophilic fulvic acid separation step, "XAD-4" (Organo Corporation) described in Non-Patent Document 6 can be used, but other hydrophilic substance adsorption resins that can adsorb hydrophilic fulvic acid and leave highly hydrophilic fulvic acid in the passing liquid, such as styrene-divinylbenzene resins like "XAD-4," can also be used. The treatment conditions for the hydrophilic substance adsorption resin can be appropriately selected depending on the resin used.

[0024] If desired, the hydrophilic fulvic acid adsorbed on the resin can be recovered by desorption (elution) from the resin. A suitable eluent, such as 0.1 M sodium hydroxide and distilled water, can be used to desorb the hydrophilic fulvic acid. If necessary, the eluted fraction can be neutralized by passing it through a hydrogen-type cation exchange resin, such as Amberlite® IR120BH (Organo Corporation) or other strongly acidic cation exchange resin, and purified hydrophilic fulvic acid can be recovered from the effluent.

[0025] (5) Highly hydrophilic fulvic acid separation process The highly hydrophilic fulvic acid separation step in the method of the present invention can be carried out by adding aluminum chloride to the permeate from the hydrophilic fulvic acid separation step and adjusting the pH from weakly acidic to neutral. This treatment causes the highly hydrophilic fulvic acid contained in the permeate to precipitate. The precipitated highly hydrophilic fulvic acid can be recovered from the permeate using common techniques, such as centrifugation and / or filtration.

[0026] The amount of aluminum chloride added in the highly hydrophilic fulvic acid separation step can be appropriately adjusted taking into consideration the amount (concentration) of highly hydrophilic fulvic acid present in the permeate, and for example, 10 to 100 mL of a 0.1 to 1 M aluminum chloride aqueous solution can be added per 1 L of the permeate.

[0027] The pH range in the highly hydrophilic fulvic acid separation step can be adjusted to a range from weakly acidic to neutral, for example, pH 5.0 to 7.0. The pH can be adjusted using a common method, and the pH can be adjusted from weakly acidic to neutral by adding an appropriate amount of an alkaline aqueous solution of an appropriate concentration to the permeate that has been made acidic by adding aluminum chloride.

[0028] The precipitated highly hydrophilic fulvic acid can be recovered from the permeate using common techniques, such as centrifugation and / or filtration. The recovered highly hydrophilic fulvic acid can be neutralized and purified by treatment with a hydrogen-type cation exchange resin, such as Amberlite® IR120BH (Organo Corporation) or other strongly acidic cation exchange resin, for example, by adding the hydrogen-type cation exchange resin to a suspension of the highly hydrophilic fulvic acid precipitate and shaking, resulting in the highly hydrophilic fulvic acid being solubilized in water.

[0029] -Highly hydrophilic fulvic acid / plant activator- The highly hydrophilic fulvic acid according to the present invention is obtained by the method for separating and purifying a highly hydrophilic fulvic acid according to the present invention, i.e., it is a substance (fraction) separated and recovered as a precipitate in the highly hydrophilic fulvic acid separation step, and is further purified as necessary.

[0030] As shown in Test Example 3 below, highly hydrophilic fulvic acid is an aggregate (fraction) of organic matter that is composed of constituent components different from those of hydrophilic fulvic acid. Humic substances are generally aggregates of organic matter, such as polymeric compounds, whose chemical structures are not uniform, and it is difficult to identify the chemical structures and contents of individual polymeric compounds. Therefore, it is acceptable to define the highly hydrophilic fulvic acid of the present invention as that obtained by the separation and purification method of the present invention.

[0031] Highly hydrophilic fulvic acid can be prepared as an aqueous solution, or as a dried or freeze-dried product. Drying or freeze-drying of highly hydrophilic fulvic acid can be carried out using a common method.

[0032] The highly hydrophilic fulvic acid of the present invention is not particularly limited in its applications, but is suitable for applications similar to those of conventional humic substances, such as as a plant activator that can be used to promote plant growth. Such plant activators can be produced by formulating the highly hydrophilic fulvic acid into an aqueous solution or other formulation using common techniques. The plant activator can be used in various cultivation stages and fertilization methods, such as the seedling stage (improving root establishment, inhibiting spindly growth, inhibiting tillering, and inhibiting yellowing), the planting stage (early rooting), the growth stage (vegetative growth, reproductive growth, and inhibiting aging (increased yield)), periods of unfavorable weather (countermeasures against drought, high-temperature damage, insufficient sunlight, and excessive humidity), when physiological disorders occur (salt damage, yellowing, root rot, and gas damage), and aerial spraying.

[0033] Highly hydrophilic fulvic acid is also contained in conventional humic substances prepared as solutions containing extracts of fulvic acid, humic acid, etc. (e.g., by the method described in Patent Document 1). However, the plant activator of the present invention is a plant activator containing highly hydrophilic fulvic acid as a main component, i.e., a plant activator containing a higher content of highly hydrophilic fulvic acid than that contained in conventional humic substances (e.g., the 7.8% by mass content in HS-2 (aqueous solution) shown in Test Example 2 below), e.g., a plant activator containing 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, or 50% by mass or more. The content of highly hydrophilic fulvic acid can be appropriately adjusted depending on the formulation of the plant activator (e.g., aqueous solution, lyophilized product, etc.) and, if diluted, the dilution rate. It is also possible to produce a humic substance with an increased content of highly hydrophilic fulvic acid by adding the highly hydrophilic fulvic acid of the present invention to a conventional humic substance.

[0034] Matters not explicitly stated in this specification can be understood by reference to well-known, commonly used, or publicly known techniques in the field to which the present invention pertains, and those skilled in the art can implement the present invention without any problems. Furthermore, those skilled in the art can appropriately change the categories and embodiments of the invention based on the matters described in this specification, as long as the effects of the present invention are not lost. [Example]

[0035] [Test Example 1] Root elongation test using Komatsuna seedlings (Part 1) The "unfractionated" test sample used was the humic substance material "HS-2 (registered trademark) Pro" (K2 Communications Co., Ltd.) (hereinafter referred to as "HS-2"). This product is a solution containing extracts of fulvic acid, humic acid, etc., obtained by soaking humic substance raw materials (composted thinned wood) in alkaline electrolyzed water and separating them from humin, according to the method described in Patent Document 1.

[0036] The test sample for "humic acid" was obtained by acid treatment of HS-2. Specifically, dilute hydrochloric acid was added to HS-2 to adjust the pH to 1.0, and the precipitated humic acid was washed with dilute hydrochloric acid and distilled water.

[0037] The test sample for "hydrophobic fulvic acid" was recovered from the supernatant obtained by acid treatment in the preparation of the above-mentioned "humic acid" using the hydrophobic substance adsorption resin "Supelite (registered trademark) DAX-8" (Merck, hereinafter referred to as "DAX-8 resin"). Specifically, the supernatant was passed through a column packed with DAX-8 resin, and the adsorbed fraction was desorbed with 0.1 M sodium hydroxide and distilled water. It was then neutralized by passing it through a hydrogen-type cation exchange resin column "Amberlite (registered trademark) IR120BH" (Organo Corporation), and the recovered hydrophobic fulvic acid was used.

[0038] The effect of each of the above test products on the root length of young komatsuna plants was tested using seed packs (seed growth bags) manufactured by Fujidaira Kogyo Co., Ltd., as follows. After sowing 10 komatsuna seeds in a seed pack, 25 mL of a solution of the above test products at various concentrations was added. The seed packs were then left in the dark at 25°C for three days, after which the root length was measured and the average root length of the germinated seeds was calculated. Five seed packs were tested for each concentration. As a control, a test was also conducted in which distilled water was used instead of the test product solution.

[0039] The results are shown in Figure 3. It was found that when humic acid or hydrophobic fulvic acid was administered alone, the root elongation-promoting effect tended to be smaller than when unfractionated humic substances were administered, and that the effect of fulvic acid (hydrophobic fulvic acid) tended to be smaller than that of humic acid. The results of this test suggest that unfractionated humic substances may contain active substances other than humic acid and hydrophobic fulvic acid, and it was recognized that such substances need to be analyzed in more detail.

[0040] [Test Example 2] Separation and purification of hydrophilic fulvic acid and highly hydrophilic fulvic acid Using the same procedure as in Test Example 1, "humic acid" was recovered as a precipitate resulting from the acid treatment of HS-2, and "hydrophobic fulvic acid" was recovered as the supernatant fraction adsorbed onto a DAX-8 resin column from the acid treatment of HS-2.

[0041] The liquid passing through the DAX-8 resin column (non-adsorbed fraction) was collected and passed through a column packed with Amberlite® XAD-4 (Merck) hydrophilic substance adsorption resin (hereafter referred to as "XAD-4 resin"). The fraction adsorbed onto the XAD-4 resin from this treatment was desorbed with 0.1 M sodium hydroxide and distilled water, then neutralized by passing through a hydrogen cation exchange resin column and collected as "hydrophilic fulvic acid."

[0042] Furthermore, the liquid that passed through the XAD-4 resin column (non-adsorbed fraction) after the above treatment was collected, and 50 mL of 0.1 M aqueous aluminum chloride solution was added to 1 L of the passed liquid, and then the pH was adjusted to 5.0 with 1 M sodium hydroxide and allowed to stand overnight. The precipitate resulting from this treatment was collected by centrifugation, washed with distilled water, suspended in distilled water, and solubilized by shaking overnight with a hydrogen cation exchange resin, and the solubilized product was collected as "highly hydrophilic fulvic acid."

[0043] The freeze-dried weight of each recovered material using the above procedure was 47.9% humic acid, 10.2% hydrophobic fulvic acid, 1.7% hydrophilic fulvic acid, and 7.8% highly hydrophilic fulvic acid, relative to the freeze-dried weight of HS-2 (unfractionated humic material).

[0044] [Test Example 3] Reversed-phase chromatography of hydrophilic fulvic acid and highly hydrophilic fulvic acid To examine the differences in the hydrophobicity / hydrophilicity of the hydrophobic, hydrophilic, and highly hydrophilic fulvic acids obtained in Test Example 2, reversed-phase chromatography was performed using a high-performance liquid chromatograph. Reverse-phase chromatography was performed using a COSMOSIL PBr column (Nacalai Tesque, Inc., 4.6 mm ID x 250 mm) with a gradient elution of a mixture of 0.2% formic acid and acetonitrile, increasing the acetonitrile concentration from 0% to 65% (see Aoyama, M. (2024): Separation and spectroscopic characterization of soil fulvic acid constituents by hydrophilic interaction chromatography and reversed-phase high-performance liquid chromatography with π-π interactions. Soil Sci. Plant Nutr. (Published online)). Peak detection was performed by absorbance at 265 nm and fluorescence at an excitation wavelength of 265 nm and an emission wavelength of 440 nm.

[0045] The results are shown in Figure 4. Fluorescence detection of all fulvic acids revealed multiple peaks, indicating that the fulvic acid itself is composed of many components. In reversed-phase chromatography, more hydrophilic substances eluted earlier (shorter retention time), while more hydrophobic substances eluted later (longer retention time). As can be seen from the figure, hydrophilic fulvic acids adsorbed to XAD-4 resin but not DAX-8 resin eluted earlier than hydrophobic fulvic acids adsorbed to DAX-8 resin, indicating their higher hydrophilicity. Furthermore, highly hydrophilic fulvic acids that did not adsorb to XAD-4 resin eluted earlier than hydrophilic fulvic acids, indicating their even higher hydrophilicity. These results clearly demonstrate that the "highly hydrophilic fulvic acid" obtained by the separation and purification method of the present invention is distinct from "hydrophobic fulvic acid" and "hydrophilic fulvic acid" and is a collection (fraction) of substances with higher hydrophilicity than them.

[0046] [Test Example 4] Root elongation test using Komatsuna seedlings (Part 2) The effect of HS-2 on the root length of komatsuna seedlings was tested using the same procedure as in Test Example 1. The average root length for each concentration was calculated, and a test for significance of the average values ​​(Tukey's method) was performed. The results are shown in Figure 5. Compared to the case where no humic substances were added (0 mg / L), the addition of HS-2 at concentrations of 5 to 20 mg / L significantly promoted root elongation of komatsuna seedlings.

[0047] In addition, using the same procedures as in Test Example 1, we tested the effects of aqueous solutions of humic acid, hydrophobic fulvic acid, highly hydrophilic fulvic acid, and highly hydrophilic fulvic acid prepared using the same procedures as in Test Examples 1 and 2 on the root length of young komatsuna plants. The average root length at each concentration was calculated and a significant difference between the average values ​​was tested. The results are shown in Figure 6. While hydrophobic fulvic acid did not show any significant effect on promoting root elongation in young komatsuna plants, hydrophilic fulvic acid and highly hydrophilic fulvic acid showed significant root elongation-promoting effects at concentrations of 1-2 mg / L, and the average root length tended to be higher than that of humic acid. These results clearly demonstrate that the "highly hydrophilic fulvic acid" obtained by the separation and purification method of the present invention has greater biological activity in promoting plant root elongation than the "hydrophobic fulvic acid" that has traditionally been manufactured and sold as a humic substance material under the name "fulvic acid." As shown in Test Example 2, "highly hydrophilic fulvic acid" is contained in humic material in greater amounts than "hydrophilic fulvic acid," which has the same effect of promoting plant root elongation. Furthermore, the effect of promoting plant root elongation is exerted even at a low concentration of 1 to 2 mg / L. Therefore, "highly hydrophilic fulvic acid" can be used as a new industrially useful humic material or biostimulant.

Claims

1. (1) A process of immersing a humic substance raw material in an alkaline aqueous solution or alkaline electrolyzed water to extract fulvic acid and humic acid into the alkaline aqueous solution or alkaline electrolyzed water and separate them from the insoluble residue, humin (hereinafter referred to as the "alkali treatment process"); (2) A step of adding an acid to the extract obtained by the alkali treatment step (1) to precipitate and separate the humic acid contained in the extract (hereinafter referred to as the "acid treatment step"); (3) A step of treating the supernatant obtained by the acid treatment step (2) with a hydrophobic substance adsorption resin to adsorb and separate hydrophobic fulvic acid (hereinafter referred to as the "hydrophobic fulvic acid separation step"). (4) A process of treating the effluent from the hydrophobic fulvic acid separation process of (3) with a hydrophilic substance adsorption resin to adsorb and separate hydrophilic fulvic acid (hereinafter referred to as the "hydrophilic fulvic acid separation process"); and (5) A process of adding aluminum chloride to the effluent from the hydrophilic fulvic acid separation process (4) and adjusting the pH from weakly acidic to neutral to precipitate and separate highly hydrophilic fulvic acid (hereinafter referred to as the "highly hydrophilic fulvic acid separation process"). A method for separating and purifying highly hydrophilic fulvic acid, comprising:

2. 2. The method for separating and purifying highly hydrophilic fulvic acid according to claim 1, wherein the hydrophobic substance adsorption resin used in the hydrophobic fulvic acid separation step is a polyvinyl polypyrrolidone resin.

3. The separation and purification method according to claim 1 or 2, wherein the hydrophilic substance adsorption resin used in the hydrophilic fulvic acid separation step is a styrene-divinylbenzene resin.

4. A fraction separated and recovered as a precipitate by the highly hydrophilic fulvic acid separation step of the separation and purification method according to claim 1 (hereinafter referred to as "highly hydrophilic fulvic acid fraction"), or a purified product thereof.

5. A plant activator containing the purified highly hydrophilic fulvic acid fraction according to claim 4 in an amount of 10% by mass or more.

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