TWI323157B - Method for enhancing the growth of crops, plants, or seeds, and soil renovation - Google Patents

Description

1323157 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to γ-polyglycolic acid ("γ-PGA", Η form), its salt (丫-poly面amine), γ-polymer A glutamate hydrogel and/or a fermentation broth comprising γ-PGA, a salt thereof and/or a γ-polyamine hydrate hydrogel to moisturize the soil, retain moisture, dissolve calcium and magnesium, and stimulate Combination and synergy of crop, plant and seed growth and resistance to plant pathogens and/or antiviral functions. • [Prior Art] In actual plant disease control, synthetic antifungal compounds are the main fungicides used. The widespread use of synthetic fungicides has resulted in reduced control of natural organisms' and has caused harm to wildlife, farm workers and consumers. For many plant diseases, especially those related to bandits, • may involve pathogen complexes, such as Pythium sp., Rhizoctonia solani (four) fa ί0/section ·) and Fusarium solani 〇/<3«Ζ·) Soybean root rot. • The selective use of conventional fungicides appears to be the primary means of continuous plant disease control, both now and in the near future. In general, the fungicide can be selectively used in an application amount or frequency of application. The possibility of using both chemical and biological procedures to achieve reliable selective control is of interest. Crop diseases range from self-reported diseases to epidemic diseases. Cereal powdery mildew is a frequently occurring and serious disease. Black spot disease (Sigatoka) is a frequently occurring and destructive disease associated with camphor. 107342.doc / incidence rate of temperate cereals and global high-value crops for sharp eye spots (R. urticae) indicates that the drug designed to control the disease is commercially viable. It is generally believed that Septoria and mycosis are associated with most of the important zoonotic pathogens currently controlled by fungicides. There are several pathogens that are not effectively controlled by fungicides, but examples of their association with severe crop losses are Sclerotinia infected with beans, Gaeumannomyces infected with mites, and Fusarium infected with corn. . Other major pathogens include rice blast fungus (10), temperate cereals of wheat powdery mildew (Erysiphe gramirtiQ anti-leaf 栝i$)%, Sept〇riatritici, and good-dyed jujube tree crowns of black scab (z called (10)... ), and Sclerotinia sclerotiorum infected with beans. The most widely studied natural antifungal agent is phytoalexin. However, chitin-to-glucosamine, glycogen-binding lectin, zeamatin, thiopurine, and ribosome-inactivated proteins are now considered important regulators of fungal invasion. Living trophic pathogens invade living cells, while dead body trophic organisms colonize invaded tissues. D-amino acids have been found to be microbial cell walls (see schleifei Κ H. and Kandler 0., 1972, Peptidoglycan types of bacterial cell walls and their taxonomic implications, Bacteriolo. Rev. 36: 407-477), lipopeptides ( See Asselineau J., 1966, The bacterial lipids, Harmann, Paris), antibiotics (see Bycroft B_W., 1969,

Structural relationships in microbial peptides, Nature (London), 224: 595-597), capsules and toxins (see Hatfield G. M., 1975, Toxins of higher fungi, ZJo Shaoshan 38: 36-55). 107342.doc 1323157 It is assumed that the D_amino acid in the antibiotic is formed from the L-amino acid after the L-amino acid is cleaved into a stereochemically unstable intermediate such as a cyclic dipeptide. The combined form of the dehydroamino acid derived from the corresponding L-amino acid during the formation of the antibiotic can be stereospecifically converted into the D• isomer in vivo. The racemization of the amino acid can be carried out via a similar mechanism.

Most peptide antibiotics produced by bacilli have anti-Gram-positive bacterial activity. However, some compounds are almost exclusively active against Gram-negative bacteria' while some other compounds such as bacillomycin and mycobaci Uin are effective anti-fungal and yeast agents. Mycobacterin is a cyclic peptide antibiotic containing 13 residues of 7 different amino acids (see Sengupta S., Banerjee AB, and B〇se s κ 1971, γ-Glutamyl and D- or L-peptide linkages In mycobacilhn, a cyclic peptide antibiotic, Biochem. J., 121: 839-846). There are 6 amide acids in the molecular structure, including 2 D-glutamic acid and 4 D-aspartic acid, and 7 other amine acids.

Non-systemic fungicides are typically multi-site inhibitors that initiate reactions by disrupting several biochemical processes. This is achieved by its ability to bind to chemical groups (e.g., thiol moieties) common to many enzymes. A substance that inhibits sterol biotin is a highly effective crop disease control agent. It is systemic and provides control over & treatment and eradication. Sterols are important functional components that maintain cell membrane integrity and are present in all eukaryotic cells. In eubacteria, sterol biosynthesis is recombined from B 61 coenzyme A in most fungi to produce primary sterols. The synthetic pathway to ergosterol is characteristic of most fungi (eg, Asc〇 mycetes, Beauveria bassiana (1) coffee noodles (4) I07342.doc 1323157 and Basidomycetes). In powdery mildew, the main sterol is 24-methyl cholesterol. Ergosterol plays an important role in maintaining membrane function, and reducing the availability of ergosterol leads to membrane damage and electrolyte leakage. Surfactant (see Arima K., Kakinums A. and Tamura, G., 1968, Surfactin, a Crystalline Peptidelipid Surfactant

Produced by Bacillus subtilis: Isolation, Characterization and Its Inhibition of Fibrin Clot Formation, Biochem. φ &〇;?;^.7^5 <::0 with 眺31:488-494) for Bacillus subtilis (heart ( ;"/1^ subtilis) and 萆 萆 i Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba Ba - leucine, which has potent antifungal activity, antitumor activity, against Ehrlich ascite cancer cells and inhibition of fibrin clot formation. Amphoteric lipopeptide surfactant and lipid bilayer membrane outer layer The physicochemical interaction causes severe ion channel permeability changes leading to destruction of the membrane system. Surfactin also inhibits the proteolytic activity of proton-ATPase, which is required for some viruses to enter cells (see Carrasco L., 1994,

Entry of animal viruses and macromolecules into cells, FEBS 3 5 0: 151-154, as evidenced by inhibition of the gastric H+, K+-ATPase by the surfactant analog pumilacidin (see

Naruse N., Tenmyo 〇. and Kobaru S., 1990, Pumilacidin, a complex of new antiviral antibiotics: Production, isolation, chemical properties, structure and biological activity, J.

Japan, 43:267-280). The antiviral activity of surfactin against a wide range of different viruses has been demonstrated (see Vollenbroich D., Paul G., Ozel Μ. 107342.doc 1323157 and Vater J., 1997, Antimycoplasma properties and application on cell cultures of surfactin, a lipopeptide antibiotic 63.44-49) > Includes Semiski forest virus, simple sore virus, suid herpes virus ' simian immunodeficiency virus, feline calicivirus, murine encephalomyocarditis virus, enveloped virus, retrovirus and so on.

Iturin (see Peypoux F., Guinand M., Michel G., Delcambe L., Das C·C· and Lederer E·, 1978, Structure of

Iturin A, a peptidolipid antibiotic from Bacillus subtilis, valence 17:3992-3996) is an antifungal lipopeptide produced by a Bacillus subtilis strain containing a ring comprising 3 D-amino acids and 4 L-alpha amino acids Heptapeptide and a lipophilic β-amino acid having an aliphatic side chain of 14 to 16 carbon atoms. Isectudin has a wide range of inhibitory effects on various plant pathogenic fungi, yeasts and bacteria in vitro and in vivo (see Namai T., Hatakeda Κ. and Asano T., 1985, Identification of a bacterium which INFORMATION substances having Antifungal activity against many important phytopathogenic fungi, Tohoku J. Agric. Res., 36:1-7 and Gueldner RC, Reiley C. C,, Pusey PL3 Costello C. E·, Arrendale R. F·, Cox RH, Himmelsbach D. S_, Crumley F_G. and Cutler H. G., 1987, Isolation and identification of iturin as antifungal peptides in biological control of peach brown rot with Bacillus subtilis, J. Agric. Food C/zem., 36:3 66 -3 70). The polar peptide moiety confers ipadrimin amphotericity 107342.doc -10·characteristics' and the mode of action involves interaction with the target membrane. The strong interaction between the avermectin and cholesterol causes the formation of an isomeric complex. Isectudin also reacts with ergosterol. The interaction between these putecins and the sterols of the phytopathogenic cell membrane effectively changes the membrane permeability and the mass composition 'thus causing amplification of the κ+ ion release channel and loss of various cellular contents' resulting in cells Decomposition of microfilaments and inhibition of sprouting of new fines. 4 According to the US Food and Drug Administration (U.S. FDA) classification, Bacillus subtilis species are classified under the list of microbial GRAS that can be used to produce animal feed grade digestive enzymes, including proteinases 'carbohydrates and lipases. Most of the fungicides used worldwide are used only to control diseases caused by 12 fungi. Although most fungicides are relatively non-toxic to mammals, some fungicides, such as aqueous silver compounds, are extremely toxic and can cause human hazards when not used properly. The use of some fungicides has led to an increase in the number of diseases caused by other uncontrolled pathogens. For example, some fungicides used to control peanut leaf spot disease increase the occurrence of peanut stem rot (C. albicans r〇//iZ〇), and the application of benzim (ben〇myl) leads to rye Sharp eye spot caused by Rhizoctonia solani, strawberry fruit rot disease (Rhizopus) and cowpea wet stalk rot (Pythium aphanidermatium) Anti-tactactants with different specificities are close to the effects achieved by broad-spectrum agents. Plant growth hormone is known as an antagonist of fungal diseases. Because auxin acts on cell wall structure, it is especially active against blight. Other growth regulators (such as auxin transport inhibitors and gibberellin 107342.doc 1323157 biosynthesis inhibitors) also reduce the severity of fusarium and Verticillium blight in tomato and cotton. Antagonist/tongue of chlormequat chloride against Pherp〇trich〇ides may be due to the application of this growth blocker, rather than by stem strength Direct action on fungal activity. Cytokinin has the antagonistic activity of antifungal pathogens (including streptococcus (1) heart (7) and members of Erysiphales), possibly by reducing pathogens The rate of degradation of the induced protein and nucleic acid is achieved. SUMMARY OF THE INVENTION Our research shows that in addition to its non-toxic, biodegradable and degradable environmentally friendly end products (glutamic acid), its salt (ie γ-poly glutamate) (Na+, κ+, NH4+, ++ and Ca++ forms), polyamidomate hydrogels (prepared from Na+, κ+, NH/, 6++ and Ca++ forms of polyamidomate) and / or a hydrolyzed fermentation broth containing multiple functions, its salts and / or γ-poly glutamate

Sex. Because of all these combinations and synergistic functionalities, gamma poly glutamate hydrogels are clearly used to renew soils to stimulate the growth of crops and other plants and seeds for disease seed growth and to protect them from plants.

真原107342.doc The present invention relates to a method for enhancing the growth of crops, plants or seeds while enhancing plant stems and stems, increasing crop yield and improving inhibition of plant pathogenic diseases. The method comprises crops, plants or seeds or for use in crops, plants or seeds. Field application of growing crops, plants or seeds containing γ_Ρ (3Α and/or its salt (Na+, K+ < NH4, Ca++ or Mg++ form), γ·poly glutamate hydrogel, containing Y-PGA, its salt And/or γ_poly glutamate hydrogel fermentation broth or a substance thereof; • Y-pGA, γ-poly glutamate (Na+, κ+, NH4+, Mg++, and Ca++) Form) and Y-poly glutamate hydrogel (prepared from γ-poly glutamate in the form of Na+, K+, NH4+, Mg++ and Ca++) with exceptionally strong water absorption and binding ability, and can effectively maintain And slowly release the retained water for long-term efficacy, which is important for agricultural fields and especially for dry land or areas under warm/hot weather conditions. High water retention can effectively improve soil for microbial growth Water activity and also contribute to growth Required nutrient transport to the plant roots or seeds. Lu Further, γ-PGA and γ- polyglutamic acid salts (Na +, Κ +, NH4 + 'Mg ++ and

Ca++ form) can be produced from L_glutamic acid via an immersion fermentation process (see Kubota H. et al., 1993, Production of poly Y_giutamic acid by

Bacillus subtilis F-2-01, Biosci. Biotech. Biochem, 57 (7) 1212-1213 and Ogata Y. et al' 1997, Efficient production 〇f γ-polyglutamic acid by Bacillus subtilis (natto) in jar fermentation, Biosci. Biotech. Biochem., 61 (i〇) 1684-1687) » γ-PGA and γ-poly glutamate have excellent water absorption properties and explore their polyanionic properties for dissolution and stabilization in aqueous systems. .doc -13- 1323157

Ca++, Mg++, Mn++, Zn++, Se++++, and Cr+++ metal ions e Y_PGA and γ-polyaluminate (Na+, K+, and NH4+ forms) are particularly susceptible to reaction with pharmaceutically or salted salts under neutral conditions (see Ho, G). H., 2005, γ-Polyglutamic acid produced by Bacillus subtilis var. natto: Structural characteristics and its industrial application, Bioindustry, Vol. 16, No. 3, 172-182) to form a water-soluble and stable 丫_聚面Anionic complex of calcium amide or gamma-poly-glycerate beta gamma-poly glutamate and gamma gamma glutamate & a ready-to-use Ca ion and Mg ++ ion for seed growth And more efficiently transported to the roots of growing plants, resulting in a comprehensive enhancement of the growth of plant seeds, plant roots, crops and other plants. 'The metal absorption on γ_Ρ (3Α involves two possible mechanisms: (1) the metal interacts directly with the 'carboxy-position of the carboxyl group', and (2) keeps moving by the electrostatic potential field generated by the COO· group Form heavy metal relative ions. In addition to interaction with carboxylate, the indole bond can also provide a weak interaction site. In addition to Lu γ·ρ (3Α configuration and ionization, understand the presence in aqueous solution The type of hydrolyzed metal species is also important. The formation of various species can lead to different absorption capacities for metal ions. Figure 1 shows γ-PGA and γ-poly glutamate (Na+, κ+, ΝίΪ4+, Ca++) And the molecular structure of Mg form, Figures 2, 3 and 4 show typical 1hjmr, t-NMR and FT-IR spectra respectively. Table summarizes the spectral data and analytical data. Figure $ shows the pH-titration curve. I07342.doc 14 1323157

Table 1 Item H Na+ K+ nh4+ Ca* Mg"4 a.'H-NMR (400MHz, D20, 30°〇 Chemical shift in ppm: aCH 3.98 4.00 3.68 4.18 4.08 PCH2 1.98, 1.80 1.99, 1.80 1.68, 1.48 2.16, 1.93 2.05, 7CH2 2.19 2.19 1.93 2.38 1.88 2.31 b. ,3C-NMR (67.9MHz, D20, 30°C) Chemical shift in ppm; aCH 56.43 62.21 62.21 62.10 β〇Η2 31.61 35.16 36.17 35.11 YCH2 34.01 39.74 39.68 39.60 CO 182.21 182.11 182.16 182.12 coo· 182.69 185.46 185.82 185.16 a. FT-IR absorption (KBr), cm·1 c=o, stretching of indoleamine I, Ν·Η bending 1739 1643 1643 1622 1654 guanamine π, Stretching 1585 c=o, symmetric stretching 1454 1402 1395 1412 1411 CN, stretching 1162 1131 1139 1116 1089 NH, oop bending 698 707 685 669 616 0-H, stretching 3449 3436 3443 3415 3402 b. Thermal analysis: hydration Water 0 10% 42% 20% 40% Dehydration temperature, °C 109. 139. 110 122 Tm - °c 206 160 193,238 219 160. Td > °c 209.8 340 341 223 335.7 331.8 γ-PGA is 1,000 a gathering of up to 20,000 A glutamic acid polymer, and is formed only by a γ-peptide bond between the glutamic acid moieties. The γ-PGA contains a terminal amine and a plurality of α-carboxylic acid groups. The polymer is usually present in several configuration states: --helix, random coil, β-sheet, spiral-curled transition region, and enveloped aggregation, depending on environmental conditions, such as pH, ions Strength and other cationic substances. The presence of the helical form is usually measured by the spectral magnitude function of the circular dichroism, "CD" at 22 nm. The free form of gamma-PGA undergoes a spiro-coil transition in a homogeneous aqueous solution of about pH 3-5 and is converted to a bonded form at a higher pH 5-7. When chelated with certain monovalent and some higher valence metal ions, the transition from random coiling to encapsulation occurs via a sharp conformational change of γ·Ρ(}. With each found in most proteins 3.6 Only one hydrogen bond per unit of amino acid residues, γ-PGA can form four types of hydrogen bonds in every three consecutive glutamic acid moieties (see Ryd〇n Η Ν., 1964,

Polypeptides, Part X, The optical rotary dispersion of poly γ-D-glutamic acid, 乂 C/zew. *SW., 1928-1933), and therefore have exceptionally strong hydrophilicity. Its conformational state also plays an important role in the carrier and stimuli of many other biological functions, including anti-phytopathogenic activity. Combining all of the above characteristics 'γ-PGA and its salts and/or 丫-polyamine hydrochloride hydrogels can be used for soil § weeks or soil renewal to aid crop growth, and at the same time as agricultural for plant pathogen control Biocide. In one embodiment of the present invention, the γ-poly glutamate hydrogel is derived from a Na+ form of γ-poly glutamate, a K+ form of γ-polyamine, a nh4+ form of γ-polygluten Amine, Mg++ form of γ-poly glutamate, Ca++ form of γ·polyfonite, or a mixture thereof with diglycerin polyglycidyl linkage, polyglycerol polyglycidyl ether, sorbitol polyglycidyl ether , polyoxyethylene sorbitol polyglycidyl linkage, polysorbate polyglycidyl ether or polyethylene glycol diglycidyl mystery or a mixture thereof is prepared by cross-linking β in another embodiment of the invention 'γ-polygluten Amine hydrochloride hydrogels are from the form of polyamidomate in Na+ form, γ-poly glutamate in the form of Κ+, γ-poly glutamate in the form of ΝΗ4+, Mg++ form 107342.doc •16- 1323157 The γ-poly glutamate, the gamma-polyamine hydrochloride in the form of Ca++ or a mixture thereof is prepared by crosslinking by irradiation with a ray or an electron beam. According to the present invention, a substance containing γ-PGA and/or a salt thereof, a γ-poly glutamate hydrogel, a γ-PGA, a salt thereof and/or a γ-poly glutamate hydrogel or a mixture thereof It can be used as a biocide, a moisturizer for soil regulation and renewal, a growth stimulator for spraying on plant leaves, and a chelate for removing heavy metals present in crops, plants or seeds. Mixtures and / or used to form soluble calcium and / or magnesium complexes. When the substance of the present invention described above is applied to a seed, it is coated on the seed. Further, the substance described above may be dissolved in a polar solvent such as ethanol or methanol or water, and the pH is adjusted to be in the range of from 5.0 to 8.0. The concentration of γ-PGA and/or its salt in the polar solvent or water is in the range of from 1% by weight to 15% by weight of 〇. Further, the substance described above has 90%: 1% to 10%: 90%, preferably 65%: 35% to 35%: 65. /. The ratio of D-form glutamic acid and/or face amine salt to L-form face acid and/or face amine salt. • [Embodiment] Industrial use of γ-PGA and its salt γ_polyglutamate (Na+, Nh4+, Ca++ and Mg++ forms) can be used as B. subtilis by using L_glutamic acid and glucose as main raw materials. , Bacillus subtilis nat. var. natto) (see Naruse N., Tenmyo 〇· and Kobaru S., 1990,

Pumilacidin, a complex 〇f new antiviral antibiotics: Production, isolation, chemical properties, structure and biological activity,/· (10), 43:267-280) or Bacillus licheniformis (仏/zc/zaz/oMb) (see v〇llenbr 〇ich D,paid 107342.doc . i7. 1323157 G., Ozel Μ. and Vater J., 1997, Antimycoplasma properties

And application on cell cultures of surfactin, a lipopeptide antibiotic from Bacillus subtilis, Appl. Environ. Microbiol., 63:44·49) produced in an immersion fermentation process. The microbial culture medium contains appropriate sources of gorges, gas, inorganic minerals and other nutrients. A concentration of β 5·12% glucose and a 0.2 to 2% citric acid in the range of 3 to 12% of L_ face acid are generally used as a partial carbon source. Peptone and ammonium sulphate (or urea or NH3) are used as a nitrogen source. Yeast extract and biotin are used as a source of nutrition. Mn + +, Mg 〇 and Naa were used as mineral sources. The culture is maintained at a temperature of (10) at its proper aeration and agitation, and the pH is maintained at 6-75 by using a urea solution, NH3 or sodium hydroxide solution. The culture time usually lasts for 48 to 84 hours. The PGA and its salt γ-poly glutamate accumulate outside the cell. Usually, γ_ρ(}^ potted sleep, 丫polyaluminate _, Κ+, ΝΗ/, c broad and Mg, formula) are extracted from the fermentation broth by a series of procedures, including ultracentrifugation or pressurization. The transition was to separate the cells, followed by the addition of 3 to 4 times ethanol to precipitate the sputum-PGA and its salts. The sinking material is redissolved in water, and another part of ethanol is used to (4) heart PGA and its salt. The dissolution and sedimentation steps were repeated several times to recover pure γ-PGA and its salts. The γ-PGA and its salts, γ-polyamidomate (Na+, d ++ and hydrazine, formula) are usually dissolved in a suitable solvent (for example, water 'ethanol or 4' to adjust the pH to a range from 5.0 to 7.5. · $探*夕, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 3107342.doc 1323157 methacrylic acid is added to the solution at a dose rate in the range of γ_ρ〇Α and its salt weight 〇1 to 20%, depending on the crosslinking agent The type and quality of the desired hydrogel. Depending on the equipment and conditions used, the gelation reaction can be completed in 1 to 4 hours at a reaction temperature of 50 to 120 C. Kneeling to produce dry cross-linked y_pga and its salt, γ-polyaluminate hydrocoagulum (prepared from gamma-poly glutamate in the form of Na+, K+, NH4+, Ca++ and Mg++) with Excellent water absorption capacity, non-φ water-soluble, and form a colorless, transparent and biodegradable hydrogel when fully expanded in water. γ-PGA having a molecular weight in the range of 5, 〇〇〇 to 900,000 and its salt γ-polybutamine are produced by controlled acidic hydrolysis under the selected pH, temperature, reaction time and reaction conditions of the enthalpy concentration. Acid salt (forms of Na+, -K+, NH4+, Ca++ and Mg++). The pH can be adjusted from 2.5 to 6.5 with a suitable acidulant such as HCb h2S〇4 or other organic acid, and the hydrolysis temperature can be controlled at 50 to 1 〇〇. The reaction time in the range of 〇 is 〇5 to 5 hours, and the concentration of y-pga having a molecular weight of 1x 1 〇6 and higher may be any suitable degree as needed. After the reaction is completed, it is necessary to further dialysis or membrane. Filtration and drying to produce the desired high purity small molecular weight and medium molecular weight γ-PGA and its salt γ-polyfaced aminate (Na+, γ, nh4+, Ca++ and Mg++ forms). The acid hydrolysis rate is faster at higher temperatures and higher γ_Ρ〇Α concentrations, by using an alkaline hydroxide solution of the selected γ-PGA with selected metal ions (Na+, κ+, ΜΗ/, Ca++, and Mg++) or Oxidation reaction to produce γ-t faceamine k salt, and adjust pH to desired as needed 7.2. Experimental Example l07342.doc -19. To further explain the present invention in detail, experimental examples are presented below to demonstrate that the present invention can be used to achieve the subject matter. However, the scope of the present invention is not limited by such experimental examples. Preparation of 0.5% yeast extract, 15% peptone, 〇3% urea, 〇2〇/〇KzHPO4, l〇〇/〇L-monosodium glutamate, 8% glucose and pH 6 8 3 [ The culture was added and added to a 600 L fermentation tank and then steam sterilized according to standard procedures. Bacillus subtilis was then inoculated and the pH was controlled with a 丨〇% Na〇H solution. Continue to vinegar for 96 hours at 37C. The γ-pGA content in the culture solution reached 40 g/Ι. Aliquots of 15 grams of culture were taken and transferred to each of the three 50 ml vials with lids. Next, take 6 μl of glycerol-based or sorbitol-based polyglycidyl ether and transfer it to a vial containing the culture solution and cover. The reaction mixture was then brought to 60 in a medium speed rotating shaker incubator. The armpit reaction was carried out for 24 hours. The reacted mixture is then removed from a sample vial, and soaked in sufficient water at 4 ° C overnight. A hydrogel is formed after hydration and expansion. The hydrogel was then filtered through a 8 mesh metal screen and drained to dryness. The weight of the expanded hydrogel without significant free water was measured and recorded. Soak the gel overnight in a sufficient amount of water at 4 °c in the same beaker. The same procedure was repeated for 5 consecutive days. The measured water absorption rate is shown in Table 2. Determination of water absorption of gamma-poly glutamate hydrogel: The weighed dry hydrogel sample (W) was soaked in excess water and left in water to expand overnight to achieve the highest hydration reaction. The hydrazine hydrogel was exchanged with 8 mesh metal to remove free water and drained to dryness. Then weigh dry 107342.doc •20· 1323157 dry water gel (W2). The amount of water absorption (W) is defined as the following difference: W = W2 - W,. Water absorption rate, x=w/w1=(w2-w))/w1 Table 2: Water absorption rate of γ-poly glutamate hydrogel (Na+) prepared from different fermentation agents by fermentation broth Binding reaction time, hourly water absorption, X remarks diglycerin polyglycidime mystery 24 4450 3 dimensional polyglycerol polyglycidime mystery 24 4560 3 dimensional polyoxyethylene sorbitol polyglycidyl ether 24 4480 3D experimental example 2 According to experimental example 1 In the other method, a sample of 5% γ-PGA nano solution and diglycerin polyglycidyl hydrazine were used as a polyglycidyl cross-linking compound in another set of experiments. The pH was further adjusted to the values shown in Table 3. The reaction mixture was placed in a medium speed rotating culture shaker. The reaction was allowed to continue at 60 ° C for 24 hours. After the completion of the reaction, the water absorption rate was measured, and the results are shown in Table 3. Table 3: Water absorption of γ-polyamine hydrochloride hydrogel (Na+ form) produced at different pH values Water absorption (X) Remarks 4 435 3D 5 610 3D 6 3450 3D 7 4550 3D Experimental Example 3 According to the method shown in Experimental Example 1, a sample of 5% γ-PGA nano solution and diglycerin polyglycidyl ether were used in another set of experiments. The solution was adjusted to pH 6.0. Different amounts of diglycerol polyglycidyl ether were used for the crosslinking reaction. 107342.doc •21 · 1323157 The reaction was continued for 24 hours at 6 (TC). The water absorption of the test article, the crusting of which is shown in Table 4. Table 4. γ-poly glutamate hydrogel under 4C ( Na+ person rate ~ eight) different expansion and water hydration various hydration time sample

Expansion/hydration time, hours 24 48 72 450 1250 2350 459 1103 2200 .........2090

Water absorption, X 96 120 4050 4150 4100 4280 4010 4120 Experimental Example 4 Bacillus subtilis was inoculated according to the method shown in Experiment m, and cultured in the same manner as shown in Example t. The culture broth samples were taken from the post for use in this set of experiments at different growth times. Diglycerin polyglycidol g was used as a crosslinking agent. The solution was adjusted to ρΗ6.〇β to continue the reaction for 24 hours. The same method was carried out as in Experiment (4). The results of water absorption at different times of time are shown in Table 5. Table 5: Water absorption of γ-poly glutamine g brine gel (Na+ form) manufactured from microbial cultures at 4 ° C at different fermentation times

Experimental Example 5 High solubility of γ-poly glutamate at neutral pH and near neutral pH, 2 Good pH buffering capacity (in the range of pH 4 to 7.0) can be as shown in the following figure (ie Fig. 5B) t 107342. The PH-titration curve of doc • 22- is known to contribute to the growth of seeds, roots and plants in soil regulation.

Experimental Example 6 Study of γ-poly glutamate (Na+ form) and γ_poly glutamate hydrogel (made from Na form of 7_poly glutamate) against agricultural pathogen growth or inhibition of agricultural pathogens The effectiveness. The standard potato dextrose agar method (PDA Petri dish) was then carried out. Measure the inhibition of pathogen growth. Concentrations of polyglutamate (Na+ form) and 丫·poly glutamate hydrogel (made from Na+ form of 丫_poly glutamate) in the range of 1% to 5% were used in the inhibition study. Prepare a pathogen sample solution: Inoculate the selected pathogen sample in the center of a pure potato dextrose agar ("PDA") culture dish, and then incubate for 3 to 9 days at 25 °C depending on the type of pathogen. . A 4 mm diameter sample was taken from the fully grown pathogen pDA culture with a 4 mm sterile perforator and placed in the center of the new PDA dish' and stored at 25. (: as a source of spare samples in the incubator. Prepare a sample of 10% γ-poly glutamate (Na + form) solution: Transfer 3 g of γ-poly glutamate (Na+ form) sample to 2 〇〇 ml cone Shape 107342.doc • 23- 1323157 flask, and add 27 ml of sterile water to prepare a 1 〇 diluted sample solution. Then at 3 (TC, use a reciprocating shaker at 200 rpm for 2 hours. The flask was placed in a 6 (TC water bath for further incubation and maintained for an additional 30 minutes after the temperature reached 60 ° C. Preparation of 50% γ·poly glutamate (Na + form) fermentation broth sample: Transfer 50 ml of fresh culture sample to a sterile flask and add 5 μs of sterile water, mix well, and centrifuge at 10,000 rpm for 3 minutes to separate the cells. Then pass the supernatant through a 0.4 μηη microfiltration membrane. 5 〇% fermentation broth solution. To test the potency of each sample, prepare 1 〇〇ml 1> 〇8 medium containing 100 ppm neomycin (ne〇mycin) sulfate to prevent any environmental microbial contamination To contain only 1 〇〇ppm neomycin sulfuric acid The culture medium was cultured as a control group. 100 ml of PDA medium was equally distributed into five 9 cm diameter culture dishes. After solidification, a 4-claw pathogen sample piece was inoculated into the center of each PDA culture dish. The sample was incubated face down at 25 C. Five multiple replicates were used. The growth diameter (mm) of each sample concentration was recorded until the control culture completely grew pathogens. Lu Yang was known (ΝΑ '') for 峙 culture Inhibition of pathogenic bacteria: Preparation of pathogenic bacteria with a concentration of 1〇7 8 cfu/ml, 0.1 ml was transferred to each of the two. The dishes were evenly distributed. Next, two tablets containing different concentrations were placed. 1 _ 〇 £; 111 diameter filter paper. Use the test repeat group. It will not contain the test pattern. The paper is used as a control. The NA culture is incubated for 2-4 days at 25 ° C. Record the growth area Diameter. '5-tested by polyglutamate (Na+ form)' γ-poly glutamic acid 107342.doc -24- 1323157 brine gel (prepared from Na+ form of γ-poly glutamate) and γ- Polyglutamate (Na+ form) fermentation broth Inhibition of growth of pathogens. The results are shown in Tables 6, 7, 8, 9 and 10. Table 6: Pathogen growth inhibition by γ-poly glutamate (Na+ form)

Inhibition of 48 hours mycelial growth, γ-poly glutamate (Na+ form) concentration tested pathogen cultured in PDA degree, molecular weight = 500k Dalton fungus species: Sclerotium rolfsii 0% 0.5 % 白绢菌0% 1.0% Rhizoctonia 15-25% 0.5% solani Rhizoctonia solani 30-50% 1.0% Fusarium 15-25% 0.5% oxysporum Anocctochilum Tip Fusarium oxysporum 15-25% 1.0% Anocctochilum Phytophthora 0% 1.0% capsici Pythium 0% 1.0% aphanidermatum Pythium 0% 1.0% myriotylum Bacterial species: Bacteria (Ralstonia > 50% 0.5% solanacearum) Erwinia carotovora 15-25% 0.5% (Erwinia carotovora) Carrot g. Escherichia coli 30-50% 1.0% Table 7: From γ-poly glutamate (Na+ form) inhibition of pathogen growth inhibition by pathogen growth induced by fermentation broth for 48 hours Mycelium growth, concentration in PDA γ-poly glutamate (Na+ form) fermentation broth: fungus species: white绢 bacteria 1% white fungus 15-25% 5% Rhizoctonia solani 0% 1% Rhizoctonia solani 15-25% 5% 107342.doc 25· 1323157 Fusarium oxysporum Fsp. Niveum Phytophthora capsici Rot fungus ginger soft rot 10-20% 0% 0% 0% 5% 5% 5% 5% Bacterial species: R. solanacearum 0% 1% R. solanacearum 30-50% 5% Carrot soft rot Evangelion 0% 5% Table 8: Inhibition of growth regions within 48 hours of pathogens tested by pathogen growth inhibition by γ-polyglycinate (Na+ form) fermentation broth (cultured on filter paper with sputum on nutrient agar) - Concentration of polyglutamate (Na+ form) fermentation broth: Bacillus subtilis 0.6-1.0 cm 5% E. coli 0.0 5% Table 9: γ-poly glutamate hydrogel (made from Na+ form of γ-poly glutamate

Preparation of the pathogen induced by pathogen growth inhibition 48 hours mycelial growth inhibition, cultured in PDA γ-poly glutamate hydrogel (Na + form) concentration fungal species: white fungus 51-75% 1 % Rhizoctonia solani 25-50% 1% Fusarium oxysporum 10-25% 1% Phytophthora capsici 25-50% 1% Pythium genus 25-50% 1% ginger soft rot 25-50% 1 % Table 10: Growth inhibition of pathogens tested by γ-poly glutamate hydrogel (prepared from γ-poly glutamate in NaH h form) for 48 hours, on nutrient agar Concentration inhibition zone* (radius) of γ-poly glutamate hydrogel (Na+ form) for the culture of cockroaches Bacterial species: Ralstonia solanum > 15 mm 1% Erwinia carotovora 10-15 mm 1% 107342. Doc -26· 1323157 Note: Suppression area*=(suppressed area of treated filter paper)_(Blank filter paper or PGA disc area, 〇·5 cm) Experimental example 7 Diana in the open farmland of Xiluo Farm (Diana) Watermelon Growth Study: Divide 1000 m2 (1〇ΜχΙΟΟ M) of open farmland into 2 by 20 cm wide x 25 cm high ditch 5 Μχ 1 〇〇 Μ equal plots. Designate the parcel as a parcel and parcel. The plot was used for the control group and the plot β was used for the experimental group. Diana watermelon plants of the age of 丨 were planted at a distance of 1 m between the two plots. Use conventional fertilizers and irrigation in accordance with standard plans and procedures, use Taiwan Fertilizer Organic Fertilizer No. 39 (12-18-12), and irrigate plot A three times, with 〇·75 kg/500 Dose rate of M2 For the plot 3, an irrigation solution containing γ-PGA fermentation broth containing 3,5% y-PGA (Na+ form) was used, and the γ-PGA fermentation broth was diluted about 300 times. Three irrigation sessions were carried out with a period of 20 days. Block A and Block B are simultaneously irrigated with automatic control of water chestnut, and the same amount of irrigation liquid is applied to Block A and Block B. Diana watermelon was harvested at the end of 6 days and the results were evaluated and shown in Table 11. Table Π. Effect of γ-PGA fermentation broth containing 3.5% Y-PGA (Na+ form) on the growth of 黛 Anna watermelon. Average size of horizontal horizontal diameter during harvest* cm Relative production % during the same period Appearance quality plot A (control) 15 21 100% — Parcel B (test) 25 26 125% Increase % 100%x(BA)/ A 66.7% 30% 25% Note: Randomly take the average size of 10 Diana watermelon samples. Experimental example 8 107342.doc •27- 1323157 Growth of sweet pepper in open farmland of Chiayi Farm: In the experimental example 7 In a similar open field study, a 1 week old sweet pepper plant was used instead of Diana watermelon. Sweet peppers were harvested 60 days after planting. § The results were evaluated and shown in Table 12. Table 12. Effect of γ-PGA fermentation broth containing 3.5% Y-PGA (Na+ form) on the growth of sweet pepper. Average size of appearance horizontal diameter * cm Average sweetness of slurry Brix0 per 100 Μ2 level per ~ yield, % plot A (control) smooth / bright 8.3 cm 9.3 100% ~ plot B (test) smooth / bright 10.2 cm 10.7 122% — increase % 100o/WB-AVA 23.8% 15.1% 22% — Note: *-·- Randomly take the average size of 1 sample of sweet pepper. Experimental Example 9 Astragalus in the open farmland of the agricultural station in σ

Growth study of Membranaceus): In the open field study similar to that described in Experimental Example 7, the ancient Eastern medicinal herbaceous scutellaria was used instead of Diana watermelon. A 1 week old scutellaria juvenile strain was used. First, fertilize the soil with 2% soluble magnesium organic fertilizer champion 280 (12-8-10). After planting the young plants, dip two 1.5 mm diameters on both sides of the plant 2〇em. A small hole of 1 cm depth is used to add additional fertilizer at a later stage and contains 3.5% Y_PGA (Na+ form) fermentation broth. The two wells are located on the opposite side of the plant, and the additional fertilizer is added twice at 24 day intervals after planting the young plants. For each addition of fertilizer, 60 g of Taiwan Fertilizer Organic Fertilizer No. 39 (121812) and 500 ml of 300-fold diluted I07342.doc • 28-1323157 γ- containing 35% Y_pGA (Na+ form) were used per well. PGA fermentation broth. After 96 days, the yellow eucalyptus was harvested and roots were collected and washed. Fresh roots and leaves were evaluated and the results are shown in Table 13. Table 13. Effect of γ-PGA fermentation broth containing 3.5% Y-PGA (Na+ form) on the growth of Scutellaria baicalensis Georgi.

Average leaf length * cm Average root length cm Average main root diameter * cm Average number of small roots * Main root color plot Α (control) 11.5 18.5 1.45 10 Bright white plot B (test) 16.6 26,8 2.17 15 Bright white increase % 100 %χ(Β-Α)/Α 44.3% 44.8% 49.6% 50% [Simplified illustration] Figure 1 shows (A) y-PGA (H form), (B) K+ form of γ-poly glutamate , Na+ form of γ-poly glutamate and ΝΗ4+ form of γ-poly glutamate, and (C) Ca++ form of γ-poly glutamate and Mg++ form of γ-polyamine structure. M(I) = K+, Na+ or ΝΗ4+, M(II) = Ca++ or Mg++.

Figure 2 shows (A) Na+ form of γ-poly glutamate, (B) K+ form of γ-poly glutamate, and (C) NH4+ at a neutral pH of D20 and at a temperature of 30 °C. 400 MHz W-NMR spectrum of the form of gamma-polyamine. The chemical shift relative to the internal reference is measured in ppm units. X indicates an impurity peak. Figure 3 shows (A) K+ form of γ-poly glutamate, (B) Na+ form of γ-poly glutamate, (C) Ca++ at a neutral pH of D20 and at a temperature of 30 °C. 13C-NMR spectrum of the form of γ-poly glutamate and (D) Mg++ form of γ-poly glutamate. The chemical shift relative to the internal reference is measured in ppm units. Figure 4 shows the infrared (FT-IR) absorption spectrum of KBr particles in (A) Na+ form of γ-poly glutamate and (Β)ΝΗ4 + form of γ-poly glutamate. Figure 5 shows titration of 10% γ-PGA with 0_2 N NaOH at 25 ° C, (Β) 107342.doc -29- 1323157 titration of 2% γ-PGA with Ca(OH) 2 and (C) 5 N NH4OH titration of the pH-titration curve of 4% γ-PGA.

107342.doc •30·

Claims (1)

1323157 Patent application No. 095124883 Replacement of Chinese patent application scope (February 1999) p 丨本丨10, the scope of application for patent: f7 year wy day repair φ original • 1. A method to improve the inhibition of plant pathogenic diseases, The method comprises applying γ-poly glutamic acid ("γ-PGA", Η form) and/or its salt, γ-poly to the crops, plants or seeds or fields in which the crops, plants or seeds are grown. A glutamate hydrogel, a material comprising a γ-PGA, a salt thereof and/or a fermentation broth of a γ-poly glutamate hydrogel or a mixture thereof. 2. The method of claim 1, wherein the salt is a Na+ form of γ-poly glutamate, an I Κ+ form of γ-poly glutamate, a Νη4+ form of γ-poly glutamate, a Mg++ form Γ-poly glutamate or gamma·poly glutamate in the form of Ca + +. 3 _ The method of claim 1 wherein the γ-poly glutamate hydrogel is derived from the Na+ form of γ·poly glutamate, the K+ form of γ-poly glutamate, and the NH 4+ form of γ _Polylaminate, Mg++ form of γ-poly glutamate, Ca++ form. γ_Water glutamate, or a mixture thereof with diglycerin polyglycidyl ether, polyglycerol polyglycidyl ether, sorbitol Polyglycidyl ether polyoxyethylene kaempferol is produced by crosslinking glycidyl ether, polysorbate polyglycidyl ether or polyethylene glycol # diglycidyl ether or a mixture thereof. 4. The method according to the present invention, wherein the γ-poly glutamate hydrogel is derived from γ-polyamidomate of 弋, γ-poly glutamate in the form of κ+, and γ in the form of NH4+ Polyglutamate, gamma-poly glutamate in the form of Mg++, gamma polythylamine in the form of Ca++ or mixtures thereof are prepared by cross-linking by gamma irradiation or electron beam irradiation. Such as. The method of claim 1, wherein the substance is used as a biocide. 7. The method of claim 5, wherein the substance is coated on the seeds. The method of claim 1, wherein the substance is dissolved in a polar solvent or water 107342-990209.doc 1323157, and the pH is adjusted to be in the range of 5. 〇 to 8. 。. 8. The method of claim 7, wherein the concentration of 丫- and/or its salt is in the range of 〇〇〇1% to 15%. 9. The method of claim 7, wherein the concentration of the gamma-polyamine hydrochloride hydrogel is in the range of 0.001% to 10%. 10. A method of enhancing the growth of crops, plants or seeds, while enhancing plant stems and stems, and increasing crop yields, the method comprising applying γ_ to the crops, plants or seeds or fields in which the crops, plants or seeds are grown a polyaminate hydrogel, a substance comprising a γ-poly glutamate hydrogel vinegar culture solution or a mixture thereof, wherein the 7_poly glutamate hydrogel is from a Na+ form of γ· Polyaminate, κ+ form of poly(tetra) acid salt, 4+ form of γ-polyammonium salt, Mg, formula 丫 poly_(tetra), γ-polyamine, or The mixture is prepared by crosslinking with diglycerin polyglycidyl hydrazine, polyglycerol polyglycidol, sorbitol polyglycidol, polyoxyethylene sorbitol polyglycidyl ether, polysorbate polyglycidyl ether or mixtures thereof. 〃 11. The method of claim 10, wherein the substance is used for soil conditioning and regeneration (IV) 1 for spraying on plant leaves or for stimulating growth stimuli of the crop or plant field, for removing the presence An integrator of heavy metals in the field of growing such crops, plants or seeds and/or a complexing agent for forming soluble calcium and/or magnesium. 12. The method of claim 11, wherein the substance (4) is coated on the seeds. η. The method of claim η, wherein the substance f is dissolved in a polar solvent or water and the pH is adjusted to a range of from 5. 〇 to 8 。. The method of claim 13, wherein the concentration of the γ-poly glutamate hydrogel is in the range of 0.001% to 10%. 15. The method of claim 11, wherein the substance further comprises γ-poly glutamic acid and/or a salt thereof. 16. The method of claim 15, wherein the salt is a Na+ form of γ-poly glutamate, a Κ+ form of γ-poly glutamate, a ΝΗ4 + form of γ-poly glutamate, a Mg++ form Γ-poly glutamate or gamma-poly glutamate in the form of Ca++. 107342-990209.doc