JP2008013523A - Method for improving growth of field crop, plant or seed - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method which improves growth of field crop, plant or seed, simultaneously enhances plant stems and trunks, increases the amount of harvest of field crop and improves control of disease damage caused by plant pathogenic microbe. <P>SOLUTION: A material containing γ-polyglutamic acid (γ-PGA, H isomer) and/or its salt, γ-polyglutamate hydrogel, a fermentation broth containing γ-PGA, its salt and/or γ-polyglutamate hydrogel or their mixture is applied to a field crop, plant or seed or a field for growing a field crop, plant or seed. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to γ-polyglutamic acid (“γ-PGA”, H form), its salt (γ-polyglutamate), γ-polyglutamate hydrogel, and / or γ-PGA, its salt and / or γ-polyglutamate. Fermentation broth containing hydrogels with soil wetting, water retention, calcium and magnesium solubilization, crop, plant and seed growth stimulation, anti-phytopathogenicity (pathogenicity to plants) and / or antiviral function Concerning combination and cooperation effects.

In actual plant disease control sites, synthetic antifungal compounds are mainly used as fungicides (antifungal agents). The use of a broad spectrum of synthetic fungicides impairs biological control in nature and puts wildlife, farm workers and consumers at risk. A number of pathogens are involved in many plant diseases, particularly those related to soil, for example, Pythium sp., Rhizoctonia solani and Fusarium solani are involved in root rot of beans.

  At present and in the near future, it will be mainstream to use existing fungicides selectively in the control of actual plant diseases. In general, the disinfectant can be selectively used in consideration of application amount or application frequency. There is an interest in the possibility of reliable and selective control by using both biological and chemical means.

Agricultural diseases range from rare occurrences to epidemics. Grain powdery mildew is a frequent disease and the damage is enormous. Black sigatoka is a frequent and frequent disease of bananas. In view of the incidence of horny spot disease ( Rhizoctonia solani ) in temperate cereals and crops of global value, agents designed to control keratosis may be said to be commercially successful. In general, septoria and powdery mildew are considered to be related to the most important fungus among cereal pathogens currently controlled by fungicides. There are also pathogens that cannot be effectively controlled with fungicides and are associated with serious crop losses. For example, Gaeumannomyces in Sclerotinia and grasses in legumes, include fusarium in maize. Other major pathogens include Pyricularia grisea in rice, Erysiphe graminis and Septoria tritici in temperate cereals , Ventura inaequalis in fruits of fruit trees, and Sclerotinia sclerotiorum in legumes.

  Phytoalexin has been most widely studied as a natural antifungal agent, but now chitinase, glucan, chitin-binding lectin, zeamatin, thionine, and ribosome-inactivating protein are also considered important regulators of fungal invasion. It has been. Biotrophic pathogens invade living cells, while necrotic pathogens form colonies in tissues after the pathogen invasion.

  D-amino acids are components of microbial cell walls (see Non-Patent Document 1); lipopeptides (see Non-Patent Document 2); antibiotics (see Non-Patent Document 3); capsules and toxins (see Non-Patent Document 4). It is. It is believed that D-amino acids in antibiotics are generated from L-amino acids via changes to L-amino acid stereochemically unstable intermediates (such as cyclic dipeptides). It is presumed that a dehydroamino acid conjugate generated from the corresponding L-amino acid is stereospecifically converted to the D-isomer in vivo during the production of the antibiotic. Amino acid racemization is thought to proceed by a similar mechanism.

  Most peptide antibiotics produced by Aspergillus are active against gram positive bacteria. However, there are compounds that are active only against gram-negative bacteria, and compounds that are effective agents against fungi and yeasts, such as bacillomycin and mycobacillin. Mycobacillin is a cyclic peptide antibiotic having 13 residues of 7 amino acids (see Non-Patent Document 5). Mycobacillin contains 6 residues of D-amino acid and 7 residues of L-amino acid in the molecular structure, and 6 residues of D-amino acid are composed of 2 residues of D-glutamic acid and 4 residues of D-aspartic acid.

Non-herbicidal fungicides are usually multi-site inhibitors that elicit effects by disrupting multiple biochemical processes. This is accomplished by the bactericidal agent binding to a functional group such as a thiol group that is common to many enzymes. Substances that inhibit sterol biosynthesis are very effective cereal disease control agents. This is a herbicidal agent that is resistant, curative and radically controlled. Sterols are functional components important for maintaining the properties of cell membranes and are present in all eukaryotes. In fungi, sterol biosynthesis occurs de novo from acetyl-CoA, which produces the main sterol in the majority of fungi. The synthetic pathway of ergosterol is characteristic of many fungi (such as Ascomycetes , Deuteromysetes , Basidomycetes, etc.). The main sterol in cereal powdery mildew is 24-methylcholesterol. Ergosterol plays a unique role in maintaining membrane function, and when ergosterol availability decreases, membrane breakdown and electrolyte leakage occur.

Surfactin (see Non-Patent Document 6) is a cyclic depsipeptide produced by Bacillus subtilis and Bacillus natto, and has a β-hydroxy fatty acid and a 7-residue amino acid containing 2 residues of D-leucine. Surfactin exhibits potent antifungal activity and potent antitumor activity against Ehrlich ascites tumor cells and inhibits the formation of fibrin clots. Amphipathic lipopeptide-based surfactin interacts physicochemically with the outer layer of the lipid bilayer, greatly altering the permeability of the ion channel and leading to membrane tissue destruction. Surfactin also inhibits the proton-ATPase activity of viral enzymes. This enzyme activity is necessary when a virus enters a cell (see Non-Patent Document 7). The inhibition of surfactin proton-ATPase activity is shown by the action of pamiracidin, a surfactin analog, on gastric H ⁺ , K ⁺ -ATPase (see Non-Patent Document 8). Surfactin's antiviral activity can be attributed to a broad spectrum of viruses (Semliki Forest Fever Virus, Herpes Simplex Virus, Sud Herpes Virus, Vesicular Stomatitis Virus, Monkey Immunodeficiency Virus, Cat Calicivirus, Mouse Encephalomyocarditis Virus, Envelope Virus, (Refer to non-patent document 9).

Iturins (see Non-Patent Document 10) are antifungal lipopeptides and are produced by Bacillus subtilis strains. Iturin contains a cyclic heptapeptide containing 3 D-amino acids and 4 L-α amino acids and a lipophilic β-amino acid having an aliphatic side chain containing 14 to 16 carbon atoms. Iturin exhibits a broad spectrum of inhibition, both in vitro and in vivo, against fungi, yeast and bacteria having phytopathogenic properties (see Non-Patent Document 11 and Non-Patent Document 12). Iturin is amphiphilic because it has a polar peptide moiety, and its mechanism of action is related to interaction with the target membrane. An equimolar complex is formed when there is a strong interaction between iturin and cholesterol. Iturin also reacts with ergosterol. The interaction between iturin and the sterols of phytopathogenic cell membranes effectively changes membrane permeability and membrane lipid composition. This expands the K ⁺ ion release channel and loses various compounds in the cell. As a result, the filaments of the cells are degraded, and germination of new spore cells is inhibited.

Bacillus subtilis is listed on the GRAS list of the US FDA as a microorganism for producing animal feed grade digestive enzymes (proteases, carbohydrases, lipases, etc.).
Schleifer KH And Kandler O., 1972, "Bacterial cell wall peptidoglycan types and their taxonomic implications", Bacteriolo. Rev., 36: 407-477 Asselineau J., 1966, "Bacterial lipids", Harmann, paris Bycroft BW, 1969, `` Structural Relationships of Microbial Peptides '', Nature (London), 224: 595-597 Pigeon Field (Hatfield) GM, 1975, “Toxins of higher fungi”. Lloydia, 38: 36-55 Sengupta S., Banerjee AB, Bose SK, 1971, “γ-glutamyl D- or L-peptide bonds in mycobacillin (cyclic peptide antibiotics)”, Biochem. J., 121: 839-846 Arima K., Kakinums A., Tamura, G., 1968, “Surfactin, a crystalline peptide lipid surfactin produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation”, Biochem. Biophys. Res .Commun., 31: 488-494 Carrasco L., 1994, “Animal virus and macromolecular entry into cells”, FEBS Lett., 350: 151-154 Naruse N., Tenmyo O., Kobaru S., 1990, “Pamiracidin, a novel antiviral antibiotic complex: production, isolation, chemical properties, structure and biological activity”, J. Antibiot. Japan, 43: 267-280 Vollenbroich D., Paul G., Ozel M., Vater J., 1997, "Surfactin, an antimycoplasma of lipopeptide antibiotics derived from Bacillus subtilis and its application to cell culture," Appl. Environ. Microbiol., 63: 44-49 Peypoux F., Guinand M., Michel G., Delcambe L., Das BC, Lederer E., 1978, “Structure of iturin A, a peptide lipid antibiotic derived from Bacillus subtilis”, Biochemistry, 17: 3992-3996 Namai T., Hatakeda K., Asano T., 1985, "Identification of bacteria producing substances with antifungal activity against many important phytopathogenic fungi," Tohoku J. Agric. Res., 36: 1- 7 Gueldner RC, Reiley CC, Pusey PL, Costello CE, Arrendale RF, Cox RH, Himmelsbach DS, Crumley FG, Cutler HG, 1987, `` An antifungal peptide in the biological control of Bacillus subtilis peach blight Isolation and identification of iturin ", J. Agric. Food Chem., 36: 366-370

Disinfectants used throughout the world are used to control diseases caused by as few as 12 fungi. Although many germicides are relatively non-toxic to mammals, some fungicides such as mercury-containing compounds are very toxic and will cause disaster if not used properly. Also, the application of certain fungicides sometimes increased the disease caused by other uncontrollable pathogens. For example, some fungicides used to control peanut spot disease increased the number of peanut sclerotia ( Sclerotium rolfsii ), or the application of benomyl caused rye keratosis (by Rhizoctonia solani) . This is an example of an increased incidence of strawberry fruit rot (a type of Rhizopus ) and cowpy wet nuclei ( Pythium aphanidermatium ). In addition, if two or three types of fungicides having different specificities are used, the same effect as that obtained when a poison that acts over a wide range is used can be obtained. Plant growth hormone is well known as an antagonist of fungal diseases. Auxin is particularly active against wilt fungi by acting on the cell wall structure. Other growth regulators, such as auxin transport inhibitors and gibberellin biosynthesis inhibitors, also reduce the severity of Fusarium and Verticillium wilt disease in tomatoes and cotton. The antagonist activity of chlormequat chloride, a biosynthesis inhibitor, against Pherpotrichoides is not due to a direct action on fungal activity, but is thought to be due to an improvement in stem strength obtained as a result of applying this growth inhibitor. Cytokinin kinetin has an antagonist activity spectrum against fungal pathogens (such as those belonging to Alternaria spp. And Erysiphales ), and the mechanism of action is thought to be due to a decrease in the degradation rate of proteins and nucleic acids induced by the pathogen.

According to the study by the present inventors, γ-PGA, a salt thereof, that is, γ-polyglutamate (Na ⁺ form, K ⁺ form, NH ₄ ⁺ form, Mg ⁺⁺ form and Ca ⁺⁺ form), γ-polyglutamate Hydrogels (prepared from γ-polyglutamate Na ⁺ form, K ⁺ form, NH ₄ ⁺ form, Mg ⁺⁺ form and Ca ⁺⁺ form) and / or γ-PGA, its salts and / or γ-polyglutamate hydrogels The contained fermentation broth is not only non-toxic to the human body, but also has biodegradability, and its final degradation product is an environmentally friendly substance (glutamic acid), and has the following multi-functionality. It turns out to have. In other words, it has high water absorption and water retention capacity, and excellent release properties that provide a long-lasting effect, and further detoxifies by including toxic heavy metal ions by chelation, and coordinates with calcium and magnesium. It has been found that a ionic complex is formed to improve nutrient bioavailability and also exhibits excellent anti-phytopathogenic activity. Due to the combined and cooperative effects of these functions, γ-PGA, its salts and / or γ-polyglutamate hydrogels protect crops and other plants and seeds from phytopathogenic effects and stimulate their growth. Obviously, it can be used to advantage in soil improvement. A method that aggregates the nutrient supply action to plants, the action of adjusting the pH and water activity of the soil, and the combined bactericidal action to prevent various symptoms and plant diseases caused by pathogenic bacteria growing in the soil is appropriate. Yes, a better choice.

The present invention relates to a method for improving the growth of crops, plants or seeds, simultaneously strengthening the stems and stems of plants, increasing the yield of crops, and improving the suppression of diseases caused by plant pathogens. Is γ-PGA and / or a salt thereof (Na ⁺ form, K ⁺ form, NH ₄ ⁺ form, Ca ⁺⁺ form or Mg ⁺⁺ form), γ-polyglutamate hydrogel, or γ-PGA, a salt thereof and / or Or applying a material containing a fermentation broth containing γ-polyglutamate hydrogel, or a mixture thereof, to a crop, a plant or seed, or a field where a crop, plant or seed is grown.

γ-PGA, γ-polyglutamate (Na ⁺ isomer, K ⁺ isomer, NH ₄ ⁺ isomer, Mg ⁺⁺ isomer and Ca ⁺⁺ isomer) and γ-polyglutamate hydrogel (γ-polyglutamate Na ⁺ isomer, K ⁺ isomer) , NH ₄ ⁺ form, Mg ⁺⁺ form and Ca ⁺⁺ form) have extremely high water absorption and water binding ability, so that they can effectively hold water and slowly release the held water for a long time. It can act continuously over a wide range. This property is important for farms, especially in dry land and areas with dry and warm and hot climatic conditions. The high water holding capacity mainly improves the water activity of the soil and promotes the growth of microorganisms, and also promotes the transport of nutrients necessary for growth to the seeds and roots of plants.

In addition, γ-PGA and γ-polyglutamate (Na ⁺ isomer, K ⁺ isomer, NH ₄ ⁺ isomer, Mg ⁺⁺ isomer, and Ca ⁺⁺ isomer) can be produced from L-glutamic acid by a submerged fermentation process. (H. Kubota et al., 1993, “Production of poly (γ-glutamic acid) by Bacillus subtilis F-2-01”, Biosci. Biotech. Biochem, 57 (7), 1212-1213 and Y. Ogata, 1997, “Jar. “Effective production of γ-polyglutamic acid by Bacillus subtilis (natto) in internal fermentation”, Biosci. Biotech. Biochem., 61 (10), 1684-1687). γ-PGA and γ-polyglutamate are excellent in water absorption, and their polyanionic properties are attributed to the metal ions Ca ⁺⁺ , Mg ⁺⁺ , Mn ⁺⁺ , Zn ⁺⁺ , Se ⁺⁺⁺⁺ and Cr ^{+++ in} aqueous systems. Researches are being conducted on the use of solubilizing and stabilizing. In particular, γ-PGA and γ-polyglutamate (Na ⁺ form, K ⁺ form and NH ₄ ⁺ form) easily react with calcium salts and magnesium salts under neutral conditions (Ho, GH, 2005, “-Polyglutamic acid produced by Bacillus subtilis var. Natto: Structural characteristics and its industrial application”, Bioindustry, 16 Vol., No.3, 72-182), produces stable water-soluble γ-polyglutamate calcium and γ-polyglutamate magnesium bodies, ionic complexes of γ-polyglutamate calcium and γ-polyglutamate magnesium body, .ca ⁺⁺ and Mg ⁺⁺ supply nutrients necessary for Ca ⁺⁺ and Mg ⁺⁺ seed growth is easily accessible to the seed, very effectively to the roots of plants growing transport As a result, the overall growth of plant seeds, plant roots, cereals and other plants is improved.

Two possible mechanisms for metal adsorption onto gamma-PGA, i.e., (1) metal interacts directly and (2) of the ion and the carboxyl site COO ^- heavy metals versus movable by electrostatic potential field generated by a group ion Retention is involved. In addition to interaction with carboxylate groups, amide bonds can also provide weak interaction sites. In addition to the conformation structure and ionization of γ-PGA, it is also important to know the type of hydrolyzed metal species present in the aqueous solution. Since a variety of different chemical species are generated, there may be differences in the adsorption capacity for metal ions.

The molecular structures of γ-PGA and γ-polyglutamate (Na ⁺ isomer, K ⁺ isomer, NH ₄ ⁺ isomer, Ca ⁺⁺ isomer and Mg ⁺⁺ ) are shown in FIG. Further, ¹ H-NMR, ¹ C-NMR and FT-IR spectra of some of these are shown in FIGS. The spectral data and analytical data are summarized in Table 1. FIG. 5 shows a pH titration curve.

  γ-PGA is a polymer of glutamic acid having a polymerization degree of 1,000 to 20,000, and consists only of γ-peptide bonds between glutamic units. γ-PGA contains a terminal amine and a number of α-carboxylic acid groups. This polymer is generally encapsulated in several conformational states, ie, α-helix, random coil, β-sheet, helix coil transition region, and environmental conditions such as pH, ionic strength, and other cationic species. Exists as an association. Usually, circular dichroism (CD) is used to measure the abundance of the helix as a function of spectral intensity at 222 nm. The helix coil transition occurs in a homogeneous aqueous solution from about pH 3-5, and γ-PGA is in an unbound form and is bound as the pH is shifted to 5-7. The transition from random coil to encapsulated aggregate occurs when a large-scale conformational change of γ-PGA forms a chelate with a divalent or higher metal ion.

  Most proteins have one hydrogen bond formed per 3.6 amino acid residues, whereas γ-PGA can form four hydrogen bonds per three consecutive glutamic units (Rydon HN, 1964, “Polypeptide, Part X, Optical Rotatory Dispersion of Poly (γ-D-glutamic acid)”, J. Chem. Soc., 1928-1933). Therefore, γ-PGA has particularly excellent hydrophilicity. By adopting such a conformation, γ-PGA also plays an important role as a carrier and stimulant for many other biological functions such as anti-phytopathogenic activity. Combining the above properties, γ-PGA and its salts and / or γ-polyglutamate hydrogel can be used for soil preparation or soil improvement for promoting the growth of agricultural crops and for agricultural use to control phytopathogenic fungi. It can also be used as a biocide.

In one embodiment of the present invention, the γ-polyglutamate hydrogel comprises a γ-polyglutamate Na ⁺ form, a γ-polyglutamate K ⁺ form, a γ-polyglutamate NH ₄ ⁺ form, a γ-polyglutamate Mg ⁺⁺ form, γ-polyglutamate Ca ⁺⁺ or a mixture thereof is converted into diglycerol polyglycidyl ether, polyglycerol polyglycidyl ether, sorbitol polyglycidyl ether, polyoxyethylene sorbitol polyglycidyl ether, polysorbitol polyglycidyl ether, polyethylene glycol diglycidyl ether Alternatively, it is prepared by crosslinking with a mixture thereof. In another embodiment of the present invention, the γ-polyglutamate hydrogel comprises a γ-polyglutamate Na ⁺ form, a γ-polyglutamate K ⁺ form, a γ-polyglutamate NH ₄ ⁺ form, and a γ-polyglutamate Mg ⁺⁺ form. , Γ-polyglutamate Ca ⁺⁺ or a mixture thereof is prepared by irradiating with gamma rays or electron beam and crosslinking.

  According to the present invention, a material containing a fermentation broth comprising γ-PGA and / or a salt thereof, γ-polyglutamate hydrogel, γ-PGA, a salt thereof and / or γ-polyglutamate hydrogel or a mixture thereof is Agents, soil preparation / improvement wetting agents, growth stimulants for spraying on plant leaves, chelating agents for removing heavy metals present in crops, plants or seeds, and / or soluble calcium and / or Used as a complexing agent to form magnesium. When the material of the present invention is applied to seeds, the material is coated on the seeds.

  Furthermore, the above-mentioned materials can be dissolved in a polar solvent (such as ethanol or methanol) or water, and the pH is adjusted to 5.0 to 8.0. The concentration of γ-PGA and / or its salt in the polar solvent or water is 0.001 wt% to 15 wt%. Further, in the above-mentioned material, the ratio of glutamic acid and / or glutamate in D form to glutamic acid and / or glutamate in L form is 90%: 10% to 10%: 90%, preferably 65%: 35% to 35%. : 65%.

A commercially available target amount of γ-PGA and its salt, ie, γ-polyglutamate (Na ⁺ isomer, K ⁺ isomer, NH ₄ ⁺ isomer, Ca ⁺⁺ isomer, and Mg ⁺⁺ isomer) are produced by Bacillus subtilis by a submerged fermentation process. Natto (Naruse N., Tenmyo O., Kobaru S., 1990, “Pamiracidin, a novel antiviral antibiotic complex: production, isolation, chemical properties, structure and biological activity”, J. Antibiot. Japan, 43: 267-280) or Bacillus licheniformis (Vollenbroich D., Paul G., Ozel M., Vater J., 1997, "Antimycoplasma of Surfactin, a lipopeptide antibiotic derived from Bacillus subtilis." And application to cell culture ", Appl. Environ. Microbiol., 63: 44-49) and can be produced using L-glutamic acid and glucose as the main feedstock. The microbial culture medium contains an appropriate amount of a carbon source, a nitrogen source, various inorganic minerals, and other nutrients. Usually, the amount of L-glutamic acid is 3-12%, glucose is used in a concentration range of 5-12% and citric acid is used in a concentration of 0.2-2% as part of the carbon source. Peptone and ammonium sulfate (or urea or NH ₃ ) are used as nitrogen sources, yeast extract and biotin are used as nutrient sources, and Mn ⁺⁺ , Mg ⁺⁺ and NaCl are used as mineral sources. The culture is maintained at 30-40 ° C. with appropriate aeration and agitation, and the pH is maintained at 6-7.5 using urea solution, NH ₃ or sodium hydroxide solution. The culture time is usually 48 to 84 hours. γ-PGA and its salts, that is, γ-polyglutamate are accumulated extracellularly.

γ-PGA and its salts, ie, γ-polyglutamate (Na ⁺ form, K ⁺ form, NH ₄ ⁺ form, Ca ⁺⁺ form and Mg ⁺⁺ form) are usually extracted from fermentation broth by a series of procedures. The procedure includes a step of separating cells by ultracentrifugation or pressure filtration, and adding 3 to 4 times ethanol to precipitate γ-PGA or a salt thereof. The precipitate is redissolved in water and γ-PGA and its salt are precipitated anew using ethanol. This dissolution-precipitation step is repeated several times to recover pure γ-PGA and its salts.

Usually, γ-PGA and salts thereof, that is, γ-polyglutamate (Na ⁺ isomer, K ⁺ isomer, NH ₄ ⁺ isomer, Ca ⁺⁺ isomer, and Mg ⁺⁺ isomer) are suitable solvents such as water, ethanol, and methanol. And the pH is adjusted to 5.0-7.5. An appropriately selected polyfunctional chemical cross-linking agent such as polyglycerol polyglycidyl ether, sorbitol polyglycidyl ether, polyethylene glycol diglycidyl ether, trimethylolpropane triacrylate is added to this solution with constant stirring. The amount added varies depending on the type of crosslinking agent and the quality required for the hydrogel, but is in the range of 0.01 to 20% of the weight of γ-PGA or a salt thereof. Normally, the gelation reaction is completed in 1 to 4 hours at a reaction temperature of 50 to 120 ° C., although it varies depending on the equipment and conditions used. Next, the produced hydrogel was freeze-dried, and dried crosslinked γ-PGA or a salt thereof, that is, γ-polyglutamate hydrogel (Na ⁺ form, K ⁺ form, NH ₄ ⁺ form, Ca ⁺⁺ form and Mg ⁺⁺ form). Of γ-polyglutamate). It is super water-absorbing, water-insoluble, and produces a colorless and transparent biodegradable hydrogel when fully swollen in water.

Γ-PGA having a molecular weight of 5,000 to 90,000 or a salt thereof, that is, γ-polyglutamate (Na ⁺ form, K ⁺ form, NH ₄ ⁺ form, Ca ⁺⁺ form and Mg ⁺⁺ form) is selected. It can be produced by controlled acid hydrolysis under specific reaction conditions (pH, temperature, reaction time, γ-PGA concentration). pH can be adjusted to 2.5 to 6.5 with a suitable acid such as HCl or H ₂ SO _4, other organic acids. The temperature of hydrolysis can be performed in the range of 50-100 degreeC. The reaction time is 0.5 to 5 hours, and the concentration of γ-PGA having a molecular weight of 1 × 10 ⁶ or more can be set to any necessary concentration. Select high-purity, low to medium molecular weight γ-PGA or a salt thereof, that is, γ-polyglutamate (Na ⁺ , K ⁺ , NH ₄ ⁺ , Ca ⁺⁺ and Mg ⁺⁺ ) In order to produce it, it is necessary to carry out further purification and drying by dialysis or membrane filtration after completion of the reaction. The acid hydrolysis rate is faster as the pH is lower, the temperature is higher, and the concentration of γ-PGA is higher. γ-polyglutamate salt reacts selected γ-PGA with selected metal ions (Na ⁺ , K ⁺ , NH ₄ ⁺ , Ca ⁺⁺ and Mg ⁺⁺ ) basic hydroxide solutions or oxides The pH can be adjusted to a desired value of 5.0 to 7.2 as necessary.

  Hereinafter, the present invention will be described in more detail with reference to experimental examples. The present invention can be achieved by the following experimental examples, but the scope of the present invention is not limited by these experimental examples.

Experimental example 1
300 L of culture broth containing 0.5% yeast extract, 1.5% peptone, 0.3% urea, 0.2% K ₂ HPO ₄ , 10% L-monosodium glutamate, 8% glucose (pH 6.8) ) Was prepared and added to a 600 L fermentor, followed by steam sterilization according to standard procedures. Next, Bacillus subtilis was inoculated and the pH was adjusted with a 10% NaOH solution. When continuously fermented at 37 ° C. for 96 hours, the γ-PGA content of the culture broth reached 40 g / L. 15 grams of this culture broth was sampled and transferred to each of three sample bottles with 50 mL caps. Subsequently, 600 μL of glycerol-based or sorbitol-based polyglycidyl ether was added to each culture broth-containing sample bottle and capped. Each reaction mixture was reacted in a shaking incubator at 60 ° C. for 24 hours. The rotation speed of the incubator was moderate. Next, the reaction mixture was collected from a 50 mL sample bottle and immersed in a sufficient amount of water at 4 ° C. overnight. A hydrogel formed after hydration swelling. Subsequently, the obtained hydrogel was filtered through an 80 mesh metal screen to remove moisture and dried. The weight of the swollen hydrogel with no apparent free water was measured and recorded. The gel was then re-immersed in a sufficient amount of water overnight at 4 ° C. using the same beaker. The same procedure was repeated for 5 consecutive days, and the water absorption obtained was shown in Table 2.

Determination of water absorption of γ-polyglutamate hydrogel:
A weighed dry hydrogel sample (W ₁ ) was soaked in an excess of water, left in water and allowed to swell overnight to maximize hydration. The hydrated hydrogel was filtered using an 80-mesh metal screen to remove free water, and the water was removed and dried. The weight of the dried hydrogel was then measured (W ₂ ). The amount of water absorption (W) is represented by the difference: W = W ₂ −W ₁ .
Water absorption: X = W / W ₁ = (W ₂ −W ₁ ) / W ₁

Experimental example 2
According to the method of Experimental Example 1, another experiment was performed using a sample of 5% γ-PGA sodium solution and diglycerol polyglycidyl ether as a polyglycidyl-based crosslinking compound. The pH of the sample was adjusted to the pH shown in Table 3. The obtained reaction mixture was placed on a culture shaker. The rotation speed of the culture shaker was moderate. The reaction was carried out continuously at 60 ° C. for 24 hours. After completion of the reaction, the water absorption was determined and the results are shown in Table 3.

Experimental example 3
According to the method of Experimental Example 1, another experiment was performed using a sample of 5% γ-PGA sodium solution and diglycerol polyglycidyl ether as a polyglycidyl-based crosslinking compound. The pH of each solution was adjusted to 6.0. The crosslinking reaction was carried out by changing the amount of diglycerol polyglycidyl ether used. The reaction was carried out continuously at 60 ° C. for 24 hours. The water absorption rate of each sample at each hydration time was determined, and the results are shown in Table 4.

Experimental Example 4
The experiment was performed according to the method of Experimental Example 1. In the same manner as in Experimental Example 1, Bacillus subtilis was inoculated and grown in the culture. Culture broth samples were collected from the fermentor at different growth times and used in this experiment. Diglycerol polyglycidyl ether was used as a crosslinking agent. The pH of the solution was adjusted to 6.0. The reaction was carried out continuously at 60 ° C. for 24 hours as in Experimental Example 1. Table 5 shows the water absorption rate of the sample at each incubation time.

Experimental Example 5
Since γ-polyglutamate has high solubility around neutral pH and good pH buffering ability (pH 4 to 7.0) shown in the following pH titration curve (ie, FIG. 5B), seed It can be advantageously used in soil preparation for promoting root and plant growth.

Experimental Example 6
For γ-polyglutamate (Na ⁺ form) and γ-polyglutamate hydrogel (prepared from γ-polyglutamate Na ⁺ form), efficacy tests for growth or inhibition of growth of populations of agricultural pathogens were carried out. The test followed standard potato dextrose agar (PDA disc). The ability to inhibit the growth of pathogenic bacteria was measured. The concentration of was used in the inhibition test .gamma.-polyglutamate (in Na ⁺ form) and (prepared from .gamma.-polyglutamate in Na ⁺ form) .gamma.-polyglutamate hydrogel was 1% to 5%.

Preparation of pathogen sample solution:
The selected pathogen sample was inoculated in the center of a plain potato dextrose agar (PDA) disc and incubated (25 ° C.) for 3 to 9 days depending on the type of the pathogen before use. A sample with a diameter of 4 mm was collected from a PDA disk on which the pathogenic bacteria had grown on the entire surface with a sterilized 4 mm diameter perforator, placed in the center of a new PDA disk, and stored as a spare sample in an incubator (25 ° C.).

Preparation of 10% γ-polyglutamate (Na ⁺ form) solution sample:
3 g of a γ-polyglutamate (Na ⁺ form) sample was added to a 200 mL Erlenmeyer flask, and 27 mL of sterilized water was added to prepare a 10-fold diluted sample solution. Next, the sample-containing flask was shaken with a reciprocating shaker at 200 rpm and 30 ° C. for 1 hour. Subsequently, the flask was incubated in a 60 ° C. water bath, and after the temperature reached 60 ° C., the flask was maintained for another 30 minutes before use.

Preparation of 50% γ-polyglutamate (Na ⁺ form) fermentation broth sample:
A fresh fermentation broth sample (50 mL) was added to a sterile flask, and 50 mL of sterile water was added and mixed well. After ultracentrifugation at 10,000 rpm for 30 minutes, cells were separated. The top clear solution was then passed through a 0.4 μm microfiltration membrane, which was used as a 50% fermentation broth solution.

  In order to test the effectiveness of the sample at each concentration, 100 mL of PDA medium contained 100 ppm neomycin sulfate to prevent contamination by environmental microbiota. A disc of PDA medium containing no other than 100 ppm neomycin sulfate was used as a control. 100 mL of PDA medium was evenly dispensed onto five Petridiscs (diameter 9 cm), and after solidification, a 4 mm pathogen sample piece was inoculated in the center of each PDA Petridisc. Subsequently, the surface which inoculated the pathogen sample was faced down, and it incubated at 25 degreeC. The test was performed in 5 series. The growth diameter (mm) at each concentration of the sample until pathogens grew on the entire control disk was recorded.

Experiment on inhibition of growth of pathogenic bacteria by counterculture using nutrient agar (NA):
Pathogenic bacteria were prepared at a concentration of 10 ⁷ to ⁸ cfu / mL. 0.1 mL of this preparation was transferred to a Petridisc NA and applied evenly. Next, two pieces of filter paper having a diameter of 1.0 cm including test samples of various concentrations were placed. The experiment was performed in triplicate. As a control, filter paper containing no test sample was used. Petri discs with NA were incubated at 25 ° C. for 2-4 days and the diameter of the growth area was recorded.

Each of γ-polyglutamate (Na ⁺ form), γ-polyglutamate hydrogel (prepared from γ-polyglutamate Na ⁺ form) and γ-polyglutamate (Na ⁺ form) fermentation broth has growth inhibitory ability against agricultural pathogens. Tested. The results are shown in Tables 6, 7, 8, 9 and 10, respectively.

Experimental Example 7 Study on the growth of Diana watermelon conducted in an outdoor field of Silo farm:
An outdoor field having an area of 1000 M ² (10 M × 100 M) was divided into two equal parts by a trough having a width of 20 cm and a height of 25 cm (1 section: 5 M × 100 M). Each compartment was designated as lot A and lot B. Lot A was used for control and lot B was used for experiments. Two Diana watermelon seedlings (one week old) were planted at intervals of 1 m in each compartment. Periodic fertilizers and irrigation follow standard programs and procedures, and Section A contains Taiwan, Fattilizer, Organic No. 39 (12-18-12) for 3 times irrigation using, the compartment B, the irrigation liquid used containing a large amount of 3.5% γ-PGA (Na ⁺ form) containing gamma-PGA fermentation broth, 0. Irrigation was performed at a supply rate of 75 kg / 500 M ² . The γ-PGA fermentation broth was diluted about 300 times. Irrigation was performed three times at intervals of 20 days in both sections A and B, and the same amount of liquid was supplied to both sections using an automatically controlled water pump. The results of harvesting and evaluating Diana watermelon after 60 days are shown in Table 11.

Experimental Example 8 A study on the growth of Acacia peppers on an outdoor farm at Chia-Yi Farm:
A study in an outdoor field was conducted in the same manner as in Experimental Example 7 using a seedling (1 week old) of Amato pepper instead of Diana watermelon. Table 12 shows the results of harvesting and evaluating Amato pepper 60 days after the planting.

Experiment 9 A study on the growth of pearl eel carried out at an outdoor farm at Taichung Agricultural Station:
An outdoor farm study similar to Experimental Example 7 was carried out using a herbaceous sea bream, which has long been known in the Orient, instead of Diana watermelon. We used seedlings (1 week old) of staghorn. First, the soil was fertilized using a 2% soluble magnesium enriched organic fertilizer champion (Champion) 280 (12-8-10). After planting the young seedlings, two holes with a diameter of 1.5 inches and a depth of 10 cm were made in a place 20 cm away from both sides of the seedling. This hole was used when adding fertilizer and broth for γ-PGA fermentation containing 3.5% γ-PGA (Na ⁺ form). Two holes were provided to face both sides of the seedling. After seedlings were planted, fertilizer was given twice more at 24 day intervals. Additional fertilizers include Taiwan, Fattilizer, Organic No. 39 (12-18-12) (60 g / hole) and 300-fold diluted 3.5% γ-PGA (Na ⁺ form) -containing γ-PGA fermentation broth (500 mL) were used. After 96 days, the herbaceous seaweed was harvested and the roots were collected and washed. The results of evaluation on fresh roots and leaves are shown in Table 13.

(A) γ-PGA (H form), (B) γ-polyglutamate K ⁺ form, γ-polyglutamate Na ⁺ form and γ-polyglutamate NH ₄ ⁺ form, (C) γ-polyglutamate Ca ⁺⁺ form And γ-polyglutamate Mg ⁺⁺ form (M (I) = K ⁺ , Na ⁺ , NH ₄ ⁺ , M (II) = Ca ⁺⁺ or Mg ⁺⁺ ). 400 MHz ¹ H of various (A) γ-polyglutamate Na ⁺ isomers, (B) γ-polyglutamate K ⁺ isomers, and (C) γ-polyglutamate NH ₄ ⁺ isomers in D ₂ O at neutral pH and 30 ° C. -NMR spectrum. Chemical shift is measured in ppm from internal standard. X represents an impurity peak. Various (A) γ-polyglutamate K ⁺ isomers, (B) γ-polyglutamate Na ⁺ isomers, (C) γ-polyglutamate Ca ⁺⁺ isomers and (D) in D ₂ O at pH neutral and temperature 30 ° C. The ¹³ C-NMR spectrum of γ-polyglutamate Mg ⁺⁺ form is shown. Chemical shift is measured in ppm from internal standard. Infrared (FT-IR) absorption spectra of (A) γ-polyglutamate Na ⁺ form and (B) γ-polyglutamate NH ₄ ⁺ form by KBr pellets are shown. (A) 10% γ-PGA and 0.2N NaOH, (B) 2% γ-PGA and Ca (OH) ₂ and (C) 4% γ-PGA and 5N NH ₄ OH at 25 ° C. Indicates.

Claims (11)

  A method for improving the growth of crops, plants or seeds, simultaneously strengthening the stems and stems of plants, increasing the yield of crops, and improving the suppression of diseases caused by plant pathogens, comprising γ-polyglutamic acid (“Γ-PGA”, H form) and / or a salt thereof, γ-polyglutamate hydrogel, γ-PGA, a salt thereof and / or a fermentation broth containing γ-polyglutamate hydrogel, or a material containing a mixture thereof Applying to a crop, plant or seed, or a field in which the crop, plant or seed is grown. The salt is a γ-polyglutamate Na ⁺ isomer, a γ-polyglutamate K ⁺ isomer, a γ-polyglutamate NH ₄ ⁺ isomer, a γ-polyglutamate Mg ⁺⁺ isomer or a γ-polyglutamate Ca ⁺⁺ isomer. Item 2. The method according to Item 1. The γ-polyglutamate hydrogel is composed of a γ-polyglutamate Na ⁺ body, a γ-polyglutamate K ⁺ body, a γ-polyglutamate NH ₄ ⁺ body, a γ-polyglutamate Mg ⁺⁺ body, and a γ-polyglutamate Ca ⁺⁺ body. Or a mixture thereof is cross-linked with diglycerol polyglycidyl ether, polyglycerol polyglycidyl ether, sorbitol polyglycidyl ether, polyoxyethylene sorbitol polyglycidyl ether, polysorbitol polyglycidyl ether, polyethylene glycol diglycidyl ether or a mixture thereof. The method of claim 1, which is prepared. The γ-polyglutamate hydrogel is composed of a γ-polyglutamate Na ⁺ body, a γ-polyglutamate K ⁺ body, a γ-polyglutamate NH ₄ ⁺ body, a γ-polyglutamate Mg ⁺⁺ body, and a γ-polyglutamate Ca ⁺⁺ body. The method according to claim 1, which is prepared by irradiating gamma rays or an electron beam to a mixture thereof and crosslinking.   The material grows a biocide, a soil preparation / improvement wetting agent, a growth stimulator for spraying on plant leaves or for use in irrigating crop fields or plant fields, crops, plants or seeds. The method according to claim 1, wherein the method is used as a chelating agent for removing heavy metals present in the field to be produced and / or as a complexing agent for producing soluble calcium and / or magnesium.   6. The method of claim 5, wherein the material is coated on seeds.   The method according to claim 1, wherein the material is dissolved in a polar solvent or water and the pH is adjusted to 5.0 to 8.0.   The method according to claim 7, wherein the concentration of γ-PGA and / or a salt thereof is 0.001% to 15%.   The method according to claim 7, wherein the concentration of γ-polyglutamate hydrogel is 0.001% to 10%.   The method according to claim 1, wherein the ratio of the D-form glutamic acid and / or glutamate to the L-form glutamic acid and / or glutamate is 90%: 10% to 10%: 90%.   11. The method of claim 10, wherein the ratio is from 65%: 35% to 35%: 65%.

Images