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

Abstract

This invention provides a method for separating and purifying active substances contained in humic materials that can be used to promote plant growth. [Solution] A method for separating and purifying highly hydrophilic fulvic acid, comprising: (1) 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 separating them from unwanted residues such as humin; (2) adding acid to the obtained extract to precipitate and separate the humic acid contained in the extract; (3) treating the supernatant obtained in (2) with a hydrophobic substance adsorption resin to adsorb and separate hydrophobic fulvic acid; (4) treating the tranque obtained in (3) with a hydrophilic substance adsorption resin to adsorb and separate hydrophilic fulvic acid; and (5) adding aluminum chloride to the tranque obtained in (4) to adjust the pH from weakly acidic to neutral, thereby precipitating and separating 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 the use of the specific fulvic acid obtained by the method.

Background Art

[0002] Humic substances are brown to black organic substances formed by further undergoing biological and abiotic reactions of decomposition products and microbial metabolites generated during the process of decomposition of biomass such as plant remains, animal remains, and feces by microorganisms. The generation of such humic substances occurs not only in natural environments such as soil, wetlands, inland waters, and seawater but also in artificial processes such as composting. Since humic substances have physiological activity effects such as promoting plant growth, alleviating stress, and promoting nutrient absorption (Non-Patent Documents 1 to 3), many humic substance materials are commercially available. However, most of the commercially available humic substance materials are not made from humic substances generated in the organic matter decomposition process involving microorganisms as described above, but are made from weathered coal or peat, which are types of coal, and are treated with nitric acid or sulfuric acid. They are made from substances that are compositionally different from humic substances derived from microbial reactions. As a result of a meta-analysis of many papers reporting on the effects of the application of humic substances on plant growth (Non-Patent Document 4), the growth-promoting effect of humic substances derived from peat or peat is said to be lower 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, acid-insoluble), and fulvic acids (alkali-soluble, acid-soluble) based on their solubility in alkalis and acids. Some humic material products are commercially available as "humic acid" or "fulvic acid." Humic acid is easily recovered because it becomes insoluble and precipitates when the alkaline solution of the humic substance raw material is acidified. However, fulvic acid remains dissolved even when such an alkaline solution is acidified, making 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 Literature 5), which defines fulvic acid as the fraction adsorbed onto DAX-8 resin, a hydrophobic substance adsorption resin (Figure 1). However, most fulvic acid sold as a humic material on the market is not manufactured using these standard methods, and therefore differs significantly in composition from fulvic acid used in academic research.

[0004] The fulvic acid defined by the IHSS method only includes the fraction that adsorbs onto DAX-8 resin, i.e., hydrophobic fulvic acid. Therefore, fulvic acid that does not adsorb onto DAX-8 resin, i.e., hydrophilic fulvic acid, is excluded. However, in the field of water chemistry, it is known that fulvic acid that does not adsorb onto DAX-8 resin can be recovered as hydrophilic acid by adsorption onto XAD-4 resin, which is a hydrophilic substance adsorption resin (Non-Patent Literature 6), and this hydrophilic acid in water is sometimes called hydrophilic fulvic acid. In addition, a method is known in which fulvic acid can be recovered as a precipitate by adjusting the fulvic acid solution from weakly acidic to neutral (pH 4-7) rather than by adsorption onto DAX-8 resin (XAD-8 resin) (Non-Patent Literature 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 containing compounds such as sodium hydroxide, which is used for alkaline extraction in the conventional IHSS method. It is described that by such a method, the amount of by-product salts produced can be suppressed, and fulvic acid and humic acid can be extracted from humic substances simply and safely with an efficiency equal to or greater than that of the conventional method. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2018-127413 (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

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0008] The present invention aims to provide further active substances contained in humic materials that can be used to promote plant growth and other purposes. [Means for solving the problem]

[0009] The inventors compared the plant growth promoting effect of an extract containing humic substances such as fulvic acid and humic acid without isolation, produced according to the method described in Patent Document 1, with the plant growth promoting effect of humic acid and fulvic acid (hydrophobic fulvic acid) prepared according to the IHSS method, and found from the test results that the former humic substance contains substances other than humic acid and fulvic acid (hydrophobic fulvic acid) that have an active effect on plants (see "Test Example 1" below). The inventors further found that the fraction that does not adsorb to the hydrophobic substance adsorption resin used to isolate conventional fulvic acid (hydrophobic fulvic acid) contains a more hydrophilic "hydrophilic fulvic acid" and an even more hydrophilic "highly hydrophilic fulvic acid," and that these can be isolated and purified separately, and that both "hydrophilic fulvic acid" and "highly hydrophilic fulvic acid" have excellent plant-activating effects.

[0010] In other words, the present invention encompasses at least the following matters. [Section 1] (1) A step of immersing humic material raw materials 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 separating them from humin, which is an insoluble residue (hereinafter referred to as the "alkaline treatment step"). (2) A step of adding acid to the extract obtained by the alkali treatment step in (1) above, and precipitating and separating the humic acid contained in the extract (hereinafter referred to as the "acid treatment step"), (3) A step of treating the supernatant obtained in the acid treatment step of (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 liquid obtained from the hydrophobic fulvic acid separation step in (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 step to separate highly hydrophilic fulvic acid by adding aluminum chloride to the phosphate obtained in the hydrophilic fulvic acid separation step of (4) above and adjusting the pH from weakly acidic to neutral (hereinafter referred to as the "highly hydrophilic fulvic acid separation step"). A method for separating and purifying highly hydrophilic fulvic acid, including [the specified substance]. [Section 2] 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 polyvinylpolypyrrolidone resin. [Section 3] 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® XAD-4" or styrene-divinylbenzene resin. [Section 4] A highly hydrophilic fulvic acid obtained by the separation and purification method described in any one of items 1 to 3. [Section 5] A plant activator whose main component is the highly hydrophilic fulvic acid described in item 4. [Effects of the Invention]

[0011] According to the present invention, it becomes possible to separate and purify "highly hydrophilic fulvic acid" which has been contained in humic substances but has not been fully utilized so far. "Highly hydrophilic fulvic acid" can be used, for example, as a new humic substance material mainly composed of it, a plant activator (biostimulant), etc.

Brief Description of the Drawings

[0012] [Figure 1] Figure 1 shows the outline [A] of the separation and purification method of humic acid and fulvic acid (hydrophobic fulvic acid) by the IHSS method and more specific operations and conditions [B]. [Figure 2] Figure 2 shows the outline of the separation and purification method of highly hydrophilic fulvic acid according to the present invention. [Figure 3] Figure 3 shows the results of the root elongation test (Part 1) using Komatsuna seedlings in [Test Example 1]. [Figure 4] Figure 4 shows the reverse phase chromatogram in the reverse phase chromatography of hydrophilic fulvic acid and highly hydrophilic fulvic acid in [Test Example 3]. [Figure 5] Figure 5 shows the results of the root elongation test (Part 2) using Komatsuna seedlings in [Test Example 4] for the humic substance material "HS-2 (registered trademark) Pro" (which may be referred to as "HS-2" in this specification). The numerical values in the figure are relative values with distilled water (0 mg / L) set to 100. The alphabet in the figure indicates the significant difference at the 5% level, and the same symbol indicates no significant difference. [Figure 6] Figure 6 shows the results of the root elongation test (Part 2) using Komatsuna seedlings in [Test Example 4] for humic acid, hydrophobic fulvic acid, hydrophilic fulvic acid, and highly hydrophilic fulvic acid. The numerical values in the figure are relative values with distilled water (0 mg / L) set to 100. The alphabet in the figure indicates the significant difference at the 5% level, and the same symbol indicates no significant difference.

Modes for Carrying Out 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 (which may also be referred to as "the method of the present invention" in this specification) comprises the following steps (1) to (5): (1) A step of immersing humic material raw materials 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 separating them from humin, which is an insoluble residue (hereinafter referred to as the "alkaline treatment step"); (2) A step of adding acid to the extract obtained by the alkali treatment step in (1) above, and precipitating and separating the humic acid contained in the extract (hereinafter referred to as the "acid treatment step" in this specification); (3) A step of treating the supernatant obtained in the acid treatment step of (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 liquid obtained from the hydrophobic fulvic acid separation step in (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 step to precipitate and separate highly hydrophilic fulvic acid by adding aluminum chloride to the phosphate obtained in the hydrophilic fulvic acid separation step of (4) above and adjusting the pH from weakly acidic to neutral (hereinafter referred to as the "highly hydrophilic fulvic acid separation step").

[0014] Humic substances refer to aggregates of organic matter, such as high-molecular-weight compounds with irregular chemical structures, that are produced after the death of organisms (mainly plants) through chemical and microbiological processes involving organic substances that constituted the organism, such as lignin, polysaccharides, proteins, lipids, and nucleic acids. In this 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." As raw materials for humic substances, soil, compost, etc., that contain such humic substances can be used.

[0015] (1) Alkali treatment process The alkali treatment step in the method of the present invention can be carried out by treating the humic material raw material with an aqueous NaOH solution or an equivalent alkaline aqueous solution (e.g., an aqueous KOH solution) in accordance with the IHSS method (see Figure 1[B]), or by treating the humic material raw material with alkaline electrolyzed water instead of the above alkaline aqueous solution in accordance with the method described in Patent Document 1. By treating the humic material raw material with such an alkaline aqueous solution or alkaline electrolyzed water, fulvic acid (hydrophobic fulvic acid, hydrophilic fulvic acid, and highly hydrophilic fulvic acid) and humic acid contained in the raw material can be extracted, and the insoluble residue, humin, can be separated. The separation of humin can be carried out using general methods, for example, by centrifugation and / or filtration. The extract (supernatant) after the separation of humin is used for the next acid treatment step.

[0016] Further details of embodiments using alkaline electrolyzed water can be found in Patent Document 1. For example, the pH of the 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. In addition, the alkaline electrolyzed water into which the humic material raw material is immersed 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 after separating humin) obtained in the alkali treatment step, in accordance with the IHSS method (see Figure 1[B]). Such treatment causes the humic acid contained in the extract to precipitate. The precipitated humic acid can be recovered from the extract using general methods, for example, by centrifugation and / or filtration. The extract (supernatant) after separating the humic acid is used for the subsequent hydrophobic fulvic acid separation step.

[0018] In the acid treatment process, HCl is used in the IHSS method, but other equivalent inorganic acids or organic acids (such as citric acid) may also be used. When using an acid other than HCl, the concentration and amount of the acid (pH after addition) and the treatment time may be adjusted as appropriate to obtain an extract (supernatant) that is equivalent to or differs from that obtained when using HCl in accordance with the IHSS method, or within a range that does not pose a problem for implementing the present invention.

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

[0020] In the hydrophobic fulvic acid separation process, the IHSS method allows the use of "XAD-8" (Rohm & Haas, discontinued, methyl methacrylate) and its equivalent "Supelite® DAX-8" (Merck KGaA, discontinued), as well as polyvinylpolypyrrolidone resin (insoluble polyvinylpyrrolidone, polyvinylpyrrolidone crosslinked). The hydrophobic substance adsorption resin and processing conditions can be appropriately selected within a range that allows for the adsorption of hydrophobic fulvic acid while leaving hydrophilic fulvic acid and highly hydrophilic fulvic acid in the effluent.

[0021] Furthermore, the hydrophobic fulvic acid adsorbed on the resin can be desorbed (eluted) from the resin and recovered as desired. A suitable eluent can be used for desorbing the hydrophobic fulvic acid; for example, 0.1 M sodium hydroxide and distilled water can be used. The eluted fraction can, if necessary, be neutralized by passing it through a hydrogen-type cation exchange resin, such as Amberlite® IR120BH (Organo Corporation) or another strongly acidic cation exchange resin, and a purified hydrophobic fulvic acid product can be recovered from the resulting effluent.

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

[0023] As the "hydrophilic substance adsorption resin" in the hydrophilic fulvic acid separation process, "XAD-4" (Organo Corporation) described in Non-Patent Literature 6 can be used. However, other hydrophilic substance adsorption resins that can adsorb hydrophilic fulvic acid and leave highly hydrophilic fulvic acid in the quenchable liquid, such as styrene-divinylbenzene resin, can also be used, similar to "XAD-4". The processing conditions for the hydrophilic substance adsorption resin can be appropriately selected depending on the resin used.

[0024] Furthermore, the hydrophilic fulvic acid adsorbed onto the resin can be desorbed (eluted) and recovered from the resin as desired. A suitable eluent can be used for desorbing the hydrophilic fulvic acid; for example, 0.1 M sodium hydroxide and distilled water can be used. The eluted fraction can, if necessary, be neutralized by passing it through a hydrogen-type cation exchange resin, such as Amberlite® IR120BH (Organo Corporation) or another strongly acidic cation exchange resin, and a purified hydrophilic fulvic acid product can be recovered from the resulting effluent.

[0025] (5) Separation process of highly hydrophilic fulvic acid The highly hydrophilic fulvic acid separation step in the method of the present invention can be carried out by adding aluminum chloride to the passlime from the hydrophilic fulvic acid separation step and adjusting the pH from weakly acidic to neutral. Through such treatment, the highly hydrophilic fulvic acid contained in the passlime precipitates. The precipitated highly hydrophilic fulvic acid can be recovered from the passlime using general methods, for example, by centrifugation and / or filtration.

[0026] The amount of aluminum chloride added in the highly hydrophilic fulvic acid separation process can be appropriately adjusted while considering the amount (concentration) of highly hydrophilic fulvic acid present in the lapsed liquid. For example, 10 to 100 mL of a 0.1 to 1 M aqueous aluminum chloride solution can be added per liter of lapsed liquid.

[0027] The pH range in the highly hydrophilic fulvic acid separation process can be adjusted from weakly acidic to neutral, for example, within the range of pH 5.0 to 7.0. pH adjustment can be performed using general methods; the acidic phosphate obtained by adding aluminum chloride can be adjusted to a neutral pH by adding an appropriate amount of an alkaline aqueous solution of appropriate concentration.

[0028] The precipitated highly hydrophilic fulvic acid can be recovered from the phosphate using conventional methods, such as by 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 Inc.) or other strongly acidic cation exchange resins, for example by adding the hydrogen-type cation exchange resin to a suspension of the highly hydrophilic fulvic acid precipitate and shaking, thereby making the highly hydrophilic fulvic acid solubilized in water.

[0029] -Highly hydrophilic fulvic acid / plant activator- The highly hydrophilic fulvic acid according to the present invention is obtained by the separation and purification method for highly hydrophilic fulvic acid according to the present invention, that is, 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 composed of different components than hydrophilic fulvic acid. Humic substances are generally aggregates of organic matter such as high-molecular-weight compounds with inconsistent chemical structures, and it is difficult to identify the chemical structure and content of individual high-molecular-weight compounds. Therefore, it is permissible 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 general methods.

[0032] The uses of the highly hydrophilic fulvic acid according to the present invention are not particularly limited, but it is suitable for use as a plant activator that can be used for purposes similar to those of conventional humic material, such as promoting plant growth. Such plant activators can be manufactured by formulating highly hydrophilic fulvic acid as an aqueous solution or other dosage form using general methods. Plant activators can be used in various cultivation stages and fertilization methods, such as during the seedling stage (improving root development, suppressing excessive elongation, suppressing tillering, suppressing yellowing), during the transplanting stage (early establishment), during the growth stage (vegetative growth, reproductive growth, suppressing aging (increasing yield)), during periods of adverse weather (measures against drought, high temperature damage, insufficient sunlight, and excessive moisture), when physiological disorders occur (salt damage, yellowing, root rot, and gas damage), and by aerial spraying.

[0033] While highly hydrophilic fulvic acid is also contained in conventional humic substances prepared as a solution containing extracts of fulvic acid, humic acid, etc. (for example, by the method described in Patent Document 1), the plant activator according to the present invention has highly hydrophilic fulvic acid as its main component. That is, the content of highly hydrophilic fulvic acid in the plant activator is higher than the content in conventional humic substances (e.g., the content in HS-2 (aqueous solution) shown in Test Example 2 below, which is 7.8% by mass), for example, the content of highly hydrophilic fulvic acid is 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, etc. The content of highly hydrophilic fulvic acid can be appropriately adjusted depending on the dosage form of the plant activator (e.g., aqueous solution, freeze-dried product, etc.), taking into account the dilution ratio if it is used after dilution. It is also possible to produce humic substances with an increased content of highly hydrophilic fulvic acid by adding the highly hydrophilic fulvic acid according to the present invention to conventional humic substances.

[0034] Matters not explicitly stated herein can be addressed by considering the well-known practices or known technologies of the art to which the present invention pertains, and a person skilled in the art can implement the present invention without any problems. Furthermore, a person skilled in the art can appropriately modify the category or embodiment of the invention based on the matters described herein, to the extent that the effects and advantages of the present invention are not lost. [Examples]

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

[0036] For the "humic acid" test sample, we used humic acid 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 before use.

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

[0038] The effect of each of the above test products on the root length of komatsuna seedlings was tested using seed packs (seed growth bags) manufactured by Fujihei Kogyo Co., Ltd., as follows: After sowing 10 komatsuna seeds in the seed pack, 25 mL of aqueous solutions of the above test products at different concentrations was added, and the samples were left to stand in the dark at 25°C for 3 days. The root length was then measured, and the average root length of the germinated seeds was determined. Five seed packs were used for each concentration. As a control, the samples were also tested with distilled water instead of the aqueous solutions of the above test products.

[0039] The results are shown in Figure 3. It was found that when humic acid and hydrophobic fulvic acid were administered individually, the effect of promoting root elongation tended to be smaller compared to when unfractionated humic material was administered, and the effect of fulvic acid (hydrophobic fulvic acid) tended to be smaller than that of humic acid. From the results of this test, it was recognized that unfractionated humic material contains active substances other than humic acid and hydrophobic fulvic acid, and that further detailed analysis of such substances is necessary.

[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 after acid treatment of HS-2, and "hydrophobic fulvic acid" was recovered as the adsorbed fraction on the DAX-8 resin column of the supernatant after acid treatment of HS-2.

[0041] Furthermore, the liquid (non-adsorbed fraction) passed through the DAX-8 resin column as a result of the above treatment was recovered and passed through a column packed with the hydrophilic substance adsorption resin "Amberlite® XAD-4" (Merck KGaA) (hereinafter referred to as "XAD-4 resin"). The adsorbed fraction on the XAD-4 resin as a result of this treatment was desorbed with 0.1M sodium hydroxide and distilled water, and then neutralized by passing it through a hydrogen-type cation exchange resin column to be recovered as "hydrophilic fulvic acid".

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

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

[0044] [Test Example 3] Reverse-phase chromatography of hydrophilic fulvic acid and highly hydrophilic fulvic acid To examine the differences in the degree of 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. For reversed-phase chromatography, "COSMOSIL PBr" (Nacalai Tesque Co., Ltd., 4.6 mm ID × 250 mm) was used as the column, and a mixture of 0.2% formic acid and acetonitrile was used as the eluent, with gradient elution performed by increasing the acetonitrile concentration from 0% to 65% (Reference: 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 excitation wavelength 265 nm and emission wavelength 440 nm.

[0045] The results are shown in Figure 4. In all cases of fulvic acid, fluorescence detection revealed many peaks, indicating that fulvic acid itself is composed of many constituent components. In reverse-phase chromatography, substances with higher hydrophilicity elute faster (shorter retention time), while substances with higher hydrophobicity elute slower (longer retention time). As can be seen from the figure, hydrophilic fulvic acid that adsorbs to XAD-4 resin without adsorbing to DAX-8 resin elutes faster than hydrophobic fulvic acid that adsorbs to DAX-8 resin, indicating that it is more hydrophilic. Furthermore, highly hydrophilic fulvic acid that does not adsorb to XAD-4 resin elutes faster than hydrophilic fulvic acid, indicating that it is even more hydrophilic. From these results, it is clear that the "highly hydrophilic fulvic acid" obtained by the separation and purification method of the present invention is a collection (fraction) of substances that are more hydrophilic than "hydrophobic fulvic acid" and "hydrophilic fulvic acid".

[0046] [Test Example 4] Root elongation test using young Komatsuna plants (Part 2) Following the same procedure as in Test Example 1, the effect of HS-2 on the root length of young komatsuna plants was tested. The average root length for each concentration was determined, and a statistically significant difference (Tukey's method) was performed on the average values. The results are shown in Figure 5. Compared to the control without humic substance (0 mg / L), the addition of HS-2 at concentrations of 5-20 mg / L significantly promoted root elongation in young komatsuna plants.

[0047] Furthermore, using the same procedure as in Test Example 1, the effects of aqueous solutions of humic acid, hydrophobic fulvic acid, highly hydrophilic fulvic acid, and highly hydrophilic fulvic acid, prepared using the same procedure as in Test Examples 1 and 2, on the root length of young komatsuna plants were tested. The average root length for each concentration was determined, and a statistically significant difference in the average values ​​was tested. The results are shown in Figure 6. Hydrophobic fulvic acid did not show a significant effect on promoting root elongation in young komatsuna plants, but hydrophilic fulvic acid and highly hydrophilic fulvic acid showed a significant effect on promoting root elongation at concentrations of 1-2 mg / L, and tended to show a higher average root length than humic acid. From these results, it is clear that the "highly hydrophilic fulvic acid" obtained by the separation and purification method of the present invention has higher biological activity in terms of promoting plant root elongation than the "hydrophobic fulvic acid" that was conventionally manufactured and sold as a humic material under the name "fulvic acid." As shown in Test Example 2, "highly hydrophilic fulvic acid" is present in higher concentrations in humic material raw materials than "hydrophilic fulvic acid" which has a similar effect in promoting plant root elongation. Furthermore, the effect of promoting plant root elongation is exerted even at low concentrations of 1-2 mg / L. Therefore, "highly hydrophilic fulvic acid" can be utilized as a new humic material or biostimulant that is industrially useful.

Claims

1. (1) A step of immersing humic material raw materials 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 separating them from humin, which is an insoluble residue (hereinafter referred to as the "alkaline treatment step"). (2) Adding acid to the extract obtained by the alkali treatment step in (1) above, and precipitating and separating the humic acid contained in the extract (hereinafter referred to as the "acid treatment step") (3) A step of treating the supernatant obtained in the acid treatment step of (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 liquid obtained from the hydrophobic fulvic acid separation step in (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 step in which aluminum chloride is added to the phosphate obtained in the hydrophilic fulvic acid separation step of (4) above, and the pH is adjusted from weakly acidic to neutral, thereby precipitating and separating the highly hydrophilic fulvic acid (hereinafter referred to as the "highly hydrophilic fulvic acid separation step"). A method for separating and purifying highly hydrophilic fulvic acid, including [the specified substance].

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 "Supelite® DAX-8" or polyvinylpolypyrrolidone resin.

3. The separation and purification method according to claim 1 or 2, wherein the hydrophilic substance adsorbing resin used in the hydrophilic fulvic acid separation step is "Amberlite® XAD-4" or styrene-divinylbenzene resin.

4. A highly hydrophilic fulvic acid obtained by the separation and purification method described in any one of claims 1 to 3.

5. A plant activator comprising highly hydrophilic fulvic acid as the main component, as described in claim 4.

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