Amino acid fertilizer for promoting crop growth

Abstract

The invention belongs to the field of fertilizer preparation, and particularly relates to an amino acid fertilizer for promoting crop growth. The amino acid fertilizer for promoting crop growth is prepared from the following raw materials in parts by mass: 700 to 850 parts of polypide micromolecular protein slurry, 45 to 55 parts of monopotassium phosphate, 100 to 120 parts of urea, 20 to 40 parts of trace elements and 10 to 20 parts of biological fermentation extract, the trace elements comprise a magnesium element, an iron element, a boron element, a zinc element and a copper element. The amino acid fertilizer is green, free of pollution, low in production cost and capable of remarkably promoting crop growth and improving the quality of agricultural products.

Description

Technical Field

[0001] The invention belongs to the field of fertilizer preparation, and in particular relates to an amino acid fertilizer for promoting crop growth. Background Art

[0002] In recent years, global protein shortage has become an increasingly urgent global problem. The world's fishmeal production has also dropped sharply due to offshore pollution, overfishing, and extended fishing seasons. Seeking new sources of protein is a research field that is currently in urgent need of development. Insect protein is a protein raw material processed from insect eggs, larvae, pupae, and adults. It has the advantages of rich protein content and balanced amino acid ratio. It has the potential to become a new feed protein resource and can alleviate the current situation of insufficient protein raw materials. Therefore, more and more research is devoted to the development of insect protein production technology.

[0003] Patent CN 118020864A discloses a protein feed that replaces fish meal and a preparation method thereof, the preparation method comprising the following steps: (1) crushing black soldier fly larvae, adding nitrogen source, carbon source, urea and calcium hydrogen phosphate thereto, and mixing uniformly to obtain a mixed raw material; (2) preparing yeast fermentation broth, adding a composite enzyme preparation to the fermentation broth, and stirring uniformly to obtain a mixed fermentation broth; (3) inoculating the mixed fermentation broth into the mixed raw material for solid fermentation, and drying after the fermentation to obtain a protein feed that replaces fish meal, the protein feed has similar nutritional components to fish meal, the protein digestibility can completely replace imported fish meal, and can promote animal growth. Patent CN 116725124A discloses an insect protein powder containing grain insects, silkworm pupae, and bee pupae and a preparation process, the insect protein powder containing grain insects, silkworm pupae, and bee pupae comprises grain insects, silkworm pupae, bee pupae and auxiliary materials in a ratio of 3:3:3:1, the insect protein powder does not need to be extracted, which reduces the waste of insect protein extraction and improves the utilization rate of insect protein.

[0004] The white-spotted flower beetle is an insect of the family Scarabaeidae in the order Coleoptera. It is widely distributed in China and neighboring countries. It has two growth stages: larvae and adults. In the adult stage, it is an important agricultural pest. However, its larvae do not harm crops and can convert agricultural waste into high-quality insect protein and fat. However, there are currently few studies on the use of white-spotted flower beetles as raw materials to make protein powder. In addition, as China has attached increasing importance to environmental protection in recent years and agriculture has entered a green development stage, higher requirements have been placed on the environmental protection of fertilizers and pesticides used in agriculture. It is necessary to build a national green fertilizer system as soon as possible, including green raw materials, green manufacturing, green products, green circulation and green application.

[0005] Therefore, developing an insect protein made from white-spotted flower beetles as raw materials and using it in environmentally friendly fertilizers is of great significance to the green and high-quality development of agriculture. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides an amino acid fertilizer for promoting crop growth. The white-spotted flower beetle is converted into a small molecule protein slurry by enzymatic hydrolysis and fermentation, and a bio-fermentation extract with a high content of amino oligosaccharides and rhamnolipids is obtained by fermentation. The protein slurry, the bio-fermentation extract and other basic fertilizers are compounded to obtain an amino acid fertilizer which is green and environmentally friendly, can significantly promote crop growth and improve the quality of agricultural products.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides an amino acid fertilizer for promoting crop growth, which comprises the following raw materials in parts by weight:

[0009] 700-850 parts of insect body small molecule protein slurry, 45-55 parts of potassium dihydrogen phosphate, 100-120 parts of urea, 20-40 parts of trace elements, and 10-20 parts of biological fermentation extract;

[0010] The trace elements include magnesium, iron, boron, zinc and copper.

[0011] In some embodiments, the preparation steps of the biofermentation extract are:

[0012] The seed liquid of Pseudomonas proteus is inoculated into a specific fermentation medium, fermented and cultured for 3-5 days at 20-30° C. in an aerobic environment, centrifuged, and the obtained supernatant is dried to obtain a biological fermentation extract.

[0013] In some embodiments, the Pseudomonas aeruginosa is Pseudomonas aeruginosa CICC 22994.

[0014] In some embodiments, the concentration of the seed solution of Pseudomonas proteus is (1-9)×10 7 CFU / mL.

[0015] Preferably, the concentration of the seed solution of Pseudomonas aeruginosa is (3-7)×10 7 CFU / mL.

[0016] In some embodiments, the components of the specific fermentation medium include: 5-10 g peptone, 3-5 g beef extract powder, 5-10 g sodium chloride, 1-2 g dipotassium hydrogen phosphate, 5-10 mg manganese sulfate monohydrate, and 1 L deionized water.

[0017] Preferably, the components of the specific fermentation medium include: 6-9 g peptone, 3-5 g beef extract powder, 7-9 g sodium chloride, 1-2 g dipotassium hydrogen phosphate, 6-8 mg manganese sulfate monohydrate, and 1 L deionized water.

[0018] In some embodiments, the specific fermentation medium further comprises 5-10 wt % chitosan, 1-2.5 wt % glycerol, 0.01-0.1 wt % arabinose and 0.1-1 wt % methanol.

[0019] Preferably, the specific fermentation medium further comprises 6-9 wt% chitosan, 1.5-2 wt% glycerol, 0.05-0.08 wt% arabinose and 0.5-1 wt% methanol.

[0020] Further preferably, the chitosan is water-soluble chitosan, and its deacetylation degree is ≥90%.

[0021] In some embodiments, the pH of the specific fermentation medium is 7-8.

[0022] In some embodiments, the inoculation amount of the seed solution of Pseudomonas proteus is 5-10 wt % of the specific fermentation medium.

[0023] Preferably, the inoculation amount of the seed solution of Pseudomonas proteus is 6-9wt% of the specific fermentation medium.

[0024] In some embodiments, the preparation steps of the insect body small molecule protein slurry are:

[0025] (1) taking dried and crushed white star flower beetle bodies and banana peels, mixing them evenly, and sterilizing them to obtain composite white star flower beetle powder;

[0026] (2) adding 3-10 times by weight of pure water to the composite white star flower beetle powder of step (1), then adding alkaline protease, stirring evenly, adjusting the pH to 8-10, enzymolyzing at 25-55° C. for 2-8 hours, inactivating the enzyme, and obtaining a white star flower beetle enzymolysis solution;

[0027] (3) The pH of the enzymatic hydrolyzate of the white-spotted flower beetle in step (2) is adjusted to 5-7, and then the mixed bacterial seed solution is inoculated therein, and shake-cultured at 25-40° C. for 3-7 days to obtain a worm body small molecule protein slurry.

[0028] In some embodiments, the mass ratio of the white-spotted flower beetle body to the banana peel is (30-45):1.

[0029] Preferably, the mass ratio of the white-spotted flower beetle body to the banana peel is 40:1.

[0030] In some embodiments, the amount of alkaline protease used in step (2) is 0.5-1.5wt% of the composite white star flower beetle powder, and the enzyme activity of the alkaline protease used is ≥200000 U / g.

[0031] Preferably, the amount of alkaline protease used in step (2) is 1 wt % of the composite white star flower beetle powder.

[0032] In some embodiments, the amount of the mixed bacteria seed solution is 1-5wt% of the enzymatic hydrolyzate of the white star flower beetle; the mixed bacteria seed solution includes a mass ratio of (1-2): 1:1 domesticated Aspergillus oryzae seed solution and domesticated Bacillus plantarum seed solution; the concentration of the Aspergillus oryzae seed solution and the Bacillus plantarum seed solution is (1-10)×10 9 CFU / mL.

[0033] Preferably, the amount of the mixed bacteria seed liquid is 3wt% of the enzymatic hydrolyzate of the white star flower beetle; the mixed bacteria seed liquid comprises a mass ratio of 1.5:1 domesticated Aspergillus oryzae seed liquid and domesticated Bacillus plantarum seed liquid; the concentration of the Aspergillus oryzae seed liquid and the Bacillus plantarum seed liquid is (3-7)×10 8 CFU / mL.

[0034] Further preferably, the Aspergillus oryzae is Aspergillus oryzae CICC 40214 and the Bacillus plantarum is Bacillus plantarum CICC 10345.

[0035] In some embodiments, the acclimation step is:

[0036] S1: picking activated Aspergillus oryzae and Bacillus plantarum respectively and inoculating them into a sterilized liquid culture medium with a pH of 5-7 and containing 1-5wt% of composite white star flower beetle powder, and culturing them at 25-40° C. with shaking for 5-10 days to obtain a primary acclimation liquid of Aspergillus oryzae and a primary acclimation liquid of Bacillus plantarum;

[0037] S2: The Aspergillus oryzae primary acclimation liquid and the plant lactobacillus plantarum primary acclimation liquid of step S1 are inoculated into a sterilized liquid culture medium having a pH of 5-7 and containing 5-10 wt % of the composite white star flower beetle powder, and cultured at 25-40° C. with shaking for 5-10 days to obtain the Aspergillus oryzae secondary acclimation liquid and the plant lactobacillus plantarum secondary acclimation liquid;

[0038] S3: The Aspergillus oryzae secondary acclimation liquid and the plant lactobacillus plantarum secondary acclimation liquid of step S2 are respectively inoculated into a sterilized liquid culture medium having a pH of 5-7 and containing 10-20wt% of the composite white star flower beetle powder, and cultured at 25-40° C. with shaking for 5-10 days to obtain the Aspergillus oryzae tertiary acclimation liquid and the plant lactobacillus plantarum tertiary acclimation liquid;

[0039] S4: Spread the three-stage acclimation liquid of Aspergillus oryzae and the three-stage acclimation liquid of Bacillus plantarum in step S3 on a plate culture medium, and culture at 25-40° C. for 1-5 days to obtain acclimated Aspergillus oryzae and acclimated Bacillus plantarum.

[0040] In some embodiments, the crude protein content of the insect body small molecule protein slurry is ≥16.24%; the total content of aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine is ≥16.29%.

[0041] Aminoligosaccharides are bactericides with disease resistance, and are effective against diseases caused by a variety of fungi, bacteria, and viruses. The present invention selects Pseudomonas proteus CICC 22994 as a fermentation strain, and carries out biological fermentation of chitosan in a specific fermentation medium to degrade chitosan into aminooligosaccharides. The nutrients, arabinose, and methanol present in the specific fermentation medium can improve the activity of Pseudomonas proteus to a certain extent, and can also promote the expression of functional genes in Pseudomonas proteus, so that Pseudomonas proteus secretes functional enzymes to degrade chitosan in the system, and obtain aminooligosaccharides with high yield, which not only has a good bactericidal effect, but also can stimulate the expression of functional genes in plants, produce chitinase, glucanase, phytoalexin, PR protein, etc. with disease resistance, can promote root growth, enhance the stress resistance of crops, and promote the growth and development of crops.

[0042] The applicant unexpectedly discovered that during the fermentation process, the glycerol component present in the specific fermentation medium can induce Pseudomonas proteus CICC 22994 to secrete more rhamnolipids, which can work synergistically with amino oligosaccharides to further improve the antibacterial properties of the biological fermentation extract. At present, when amino acid fertilizers are sprayed on the surface of crops, the effective ingredients of the fertilizers often do not stay on the surface of the crops because the hydrophobicity of the crop leaves is high, resulting in low utilization and effects of the amino acid fertilizers. Rhamnolipids are a kind of biological surfactants. When they are used in amino acid fertilizers and sprayed on the surface of crops, rhamnolipids can reduce the surface tension of the leaves, improve wettability, and make the active ingredients in the fertilizers, especially the various peptides in the insect body small molecule protein slurry better adhere to the leaf surface, and then penetrate into the inside of the crop, and better play the role of amino acid fertilizers. Rhamnolipids also have better salt tolerance than chemically synthesized surfactants, so that the amino acid fertilizer of the present invention is also suitable for saline-alkali land.

[0043] In addition, the present invention utilizes an enzymatic method combined with a fermentation method to convert the white-star flower beetle into a small-molecule protein slurry. The applicant has found that strictly controlling the process parameters during the enzymatic hydrolysis and fermentation process is beneficial to increasing the content of amino acids and crude protein in the insect body's small-molecule protein slurry. First, the present invention selects a certain amount of alkaline protease to enzymatically hydrolyze the crushed white-star flower beetle body and banana peel, and regulates the enzymatic hydrolysis process by controlling the enzyme concentration, enzymatic hydrolysis time, enzymatic hydrolysis temperature, environmental pH, and substrate concentration, so that the macromolecules that are difficult to degrade in the white-star flower beetle body and banana peel are degraded into small molecules, which is beneficial to the subsequent absorption of small molecules by Aspergillus oryzae and Bacillus plantarum, and converts them into amino acids and polypeptides, thereby improving the utilization rate of raw materials. Secondly, the invention domesticates Aspergillus oryzae and Bacillus plantarum first, and increases the composite white star flower beetle powder during the domestication period to improve the adaptability and selectivity of Aspergillus oryzae and Bacillus plantarum to the composite white star flower beetle powder. The banana peel in the composite white star flower beetle powder is rich in carbohydrates, cellulose, minerals, etc., and the mass ratio of the banana peel to the white star flower beetle powder is controlled, so that the banana peel can provide carbon sources and nutrient elements for Aspergillus oryzae and Bacillus plantarum, promote their biological utilization of the white star flower beetle powder, and avoid competitive inhibition of microorganisms due to excessive nutrition, thereby reducing the conversion rate of the white star flower beetle powder, and further reducing the content of crude protein and amino acids in the insect body small molecule protein slurry.

[0044] The applicant also found that the rhamnolipid contained in the biological fermentation extract can significantly improve the stability and biological activity of small molecule proteins and polypeptides in the insect body small molecule protein slurry. Rhamnolipid can form a complex with small molecule proteins or polypeptides to improve their solubility and absorption rate in plants, thereby enhancing their biological effects. At the same time, the antimicrobial properties of rhamnolipids also help to improve the stability of small molecule proteins and polypeptides during storage and transportation. The combined use of amino oligosaccharides, rhamnolipids and insect body small molecule protein slurry will also bring better pest control effects and crop growth promotion effects.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. The present invention discloses an amino acid fertilizer for promoting crop growth. The raw materials of the fertilizer include protein slurry for promoting crop growth and biological fermentation extract for inhibiting the growth of pathogens. The fertilizer is green and environmentally friendly, can promote the growth of crops and improve the quality of fruits.

[0047] 2. The present invention selects Pseudomonas proteus CICC 22994 as the fermentation strain, performs bio-fermentation on chitosan in a specific fermentation medium, degrades chitosan into amino-oligosaccharides with high antibacterial properties, and secretes a large amount of rhamnolipids during the fermentation process, which can further improve the antibacterial properties of the bio-fermentation extract.

[0048] 3. The present invention formulates a specific fermentation medium, which can improve the biological activity of Pseudomonas aeruginosa CICC 22994, degrade chitosan of a certain concentration, and the active ingredients therein can induce Pseudomonas aeruginosa CICC22994 to secrete a large amount of rhamnolipid.

[0049] 4. The present invention uses white-spotted flower beetle bodies and banana peels as raw materials, and ferments them using an enzymatic method combined with a microbial fermentation method to obtain a small molecule protein slurry with high protein and amino acids. The production process of the small molecule protein slurry is mild, environmentally friendly and pollution-free, and has a high cost-effectiveness.

[0050] 5. The present invention domesticates the microorganisms Aspergillus oryzae and Bacillus plantarum, improves the adaptability and selectivity of Aspergillus oryzae and Bacillus plantarum to the composite white star flower beetle powder, has a good bioavailability of the white star flower beetle powder, and further increases the content of crude protein and amino acids in the insect body small molecule protein slurry. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0052] In order to better illustrate the specific implementation mode of the present invention, further examples are given below.

[0053] The mass fractions described in the following embodiments may be grams, kilograms, tons or other mass units.

[0054] The white-star flower beetle insect body used in the invention is the white-star flower beetle larvae; the tryptone, PDA slant culture medium and LB plate culture medium used are all purchased from Guangdong Huankai Microbiological Technology Co., Ltd.; the alkaline protease used has an enzyme activity of 200000 U / g and is purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the Aspergillus oryzae used is Aspergillus oryzae CICC 40214, and the plant lactobacillus is plant lactobacillus CICC 10345; the chitosan used is chitosan oligosaccharide (Mn<10000) purchased from Nantong Lushen Bioengineering Co., Ltd.; and the Pseudomonas proteus used is Pseudomonas proteus CICC 22994.

[0055] Preparation Example 1

[0056] Domestication of Aspergillus oryzae:

[0057] In a sterile environment, the preserved Aspergillus oryzae mother strain was inoculated into a 0.5 cm×0.5 cm bacterial block on a PDA slant medium, and cultured at 37° C. until the hyphae were full, thereby obtaining activated Aspergillus oryzae;

[0058] S1: Pick the activated Aspergillus oryzae and inoculate it into a sterilized 500 mL inorganic salt liquid culture medium with a pH of 6 and containing 3 wt% of composite white star flower beetle powder, and culture it at 37° C. and 150 r / min shaking for 7 days to obtain an Aspergillus oryzae primary acclimation solution;

[0059] S2: inoculate 1 mL of the primary acclimation liquid of Aspergillus oryzae obtained in step S1 into 500 mL of a sterilized inorganic salt liquid culture medium having a pH of 6 and containing 8 wt % of composite white star flower beetle powder, and culture the medium at 37° C. with shaking for 7 days to obtain a secondary acclimation liquid of Aspergillus oryzae;

[0060] S3: inoculate 1 mL of the secondary acclimation liquid of Aspergillus oryzae obtained in step S2 into 500 mL of a sterilized inorganic salt liquid culture medium having a pH of 6 and containing 15 wt % of composite white star flower beetle powder, and culture the medium at 37° C. with shaking for 7 days to obtain a tertiary acclimation liquid of Aspergillus oryzae;

[0061] S4: Take 100 μL of the three-time acclimation solution of Aspergillus oryzae in step S3 and spread it on a PDA plate culture medium, and culture it at 37° C. for 3 days to obtain acclimated Aspergillus oryzae.

[0062] Preparation Example 2

[0063] Acclimation of Lactobacillus plantarum:

[0064] Under a sterile environment, 100 μL of bacterial solution was drawn from a glycerol tube of the preserved plant lactobacillus strain, and evenly spread on an LB plate medium by a plate streak method, and inverted and cultured at 37° C. until colonies grew out, thereby obtaining activated plant lactobacillus;

[0065] S1: picking the activated plant lactobacillus and inoculating it into a sterilized inorganic salt liquid culture medium with a pH of 6.5 and containing 3 wt% of composite white star flower beetle powder, and culturing it at 37° C. with shaking for 7 days to obtain a primary acclimation solution of plant lactobacillus;

[0066] S2: inoculate 1 mL of the plant lactobacillus primary acclimation solution of step S1 into a sterilized inorganic salt liquid culture medium having a pH of 6.5 and containing 8 wt % of composite white star flower beetle powder, and culture at 37° C. with shaking for 7 days to obtain a plant lactobacillus secondary acclimation solution;

[0067] S3: 1 mL of the secondary acclimation solution of Bacillus plantarum obtained in step S2 is inoculated into a sterilized inorganic salt liquid culture medium having a pH of 6.5 and containing 15 wt % of composite white star flower beetle powder, and cultured at 37° C. with shaking for 7 days to obtain a tertiary acclimation solution of Bacillus plantarum;

[0068] S4: Take 100 μL of the three-time acclimation solution of plant lactobacillus in step S3, spread it on the LB plate culture medium, and culture it at 37° C. for 1 day to obtain the acclimated plant lactobacillus.

[0069] Preparation Example 3

[0070] Preparation of acclimated Aspergillus oryzae seed solution:

[0071] In a sterile environment, the domesticated Aspergillus oryzae in Preparation Example 1 was inoculated into 500 mL of potato culture medium, shaken and cultured at 37°C for 3 days, and then centrifuged at 5000 r / min to obtain the bacterial cells, which were diluted with sterile water to 6 × 10 8 CFU / mL, and the domesticated Aspergillus oryzae seed liquid was obtained.

[0072] Preparation Example 4

[0073] Preparation of the acclimated Lactobacillus plantarum seed solution:

[0074] In a sterile environment, the plant lactobacillus acclimated in Preparation Example 2 was inoculated into 500 mL of liquid culture medium, shaken and cultured at 37 ° C for 1 day, and then centrifuged at 5000 r / min to obtain the bacteria, which were diluted with sterile water to 6 × 10 8 CFU / mL, and the acclimated Lactobacillus plantarum seed solution was obtained.

[0075] The preparation steps of the inorganic salt liquid culture medium used in Preparation Examples 1 and 2 are as follows: dissolve 2 g of (NH4)2SO4, 2 g of Na2HPO4·12H2O, 0.5 g of MgSO4·7H2O, 0.5 g of KH2PO4, 0.5 mL of trace element solution and 0.5 mL of vitamin solution in 500 mL of deionized water, adjust the pH to 7, sterilize at 120°C for 20 min, and naturally cool to room temperature to obtain an inorganic salt liquid culture medium;

[0076] Trace element solution formula: CaCl2 25 mg, MnCl4·4H2O 5 mg, deionized water 1 L;

[0077] Vitamin liquid formula: riboflavin 200 mg, para-aminobenzoic acid 200 mg, thiamine hydrochloride 400 mg, niacin 400 mg, pyridoxine hydrochloride 400 mg, calcium pantothenate 400 mg, folic acid 2 mg, biotin 2 mg, inositol 2000 mg, deionized water 1L.

[0078] The steps for preparing the potato culture medium used in Preparation Example 3 are as follows: peel the potatoes, cut them into pieces of about 1 cm 3100 g of potato pieces were put into 500 mL of deionized water and boiled for 30 min, filtered with double-layer gauze, 10 g of glucose was added to the filtrate, and deionized water was added to 500 mL, the pH was adjusted to 7 with 1 mol / L sodium hydroxide solution, sterilized at 120 ° C for 20 min, and naturally cooled to room temperature to obtain potato culture medium;

[0079] The preparation steps of the liquid culture medium used in Preparation Example 4 are as follows: 5 g of yeast powder, 10 g of tryptone and 10 g of NaCl are dissolved in 1 L of deionized water, sterilized at 121° C. for 20 min, and naturally cooled to room temperature to obtain a liquid culture medium.

[0080] Preparation Example 5

[0081] The preparation steps of insect body small molecule protein slurry are as follows:

[0082] (1) 400 g of dried and crushed white star flower beetle bodies and 10 g of banana peels were mixed evenly, and sterilized at 120° C. for 20 min to obtain a composite white star flower beetle powder;

[0083] (2) adding 2050 g of pure water to the composite white star flower beetle powder of step (1), and then adding 4.1 g of alkaline protease, stirring evenly, adjusting the pH to 9, performing enzymolysis at 45° C. for 5 h, and deactivating the enzyme at 100° C. for 15 min to obtain a white star flower beetle enzymolysis solution;

[0084] (3) The pH of the enzymatic hydrolyzate of the white-spotted flower beetle in step (2) is adjusted to 6, and then 3 wt % of the mixed bacterial seed solution is inoculated therein, and shake-cultured at 37° C. for 3 days to obtain a small molecule protein slurry of the insect body.

[0085] The mixed bacterial seed solution used was composed of the Aspergillus oryzae seed solution domesticated in Preparation Example 3 and the Bacillus plantarum seed solution domesticated in Preparation Example 4 in a mass ratio of 1.5:1.

[0086] Preparation Example 6

[0087] Preparation of Pseudomonas aeruginosa seed solution:

[0088] (1) Under a sterile environment, 100 μL of bacterial solution was drawn from the glycerol tube of the preserved Pseudomonas proteus strain, and evenly spread on the LB plate culture medium by the plate streak method, and inverted and cultured at 37°C until colonies grew, thereby obtaining activated Pseudomonas proteus;

[0089] (2) In a sterile environment, the activated Pseudomonas aeruginosa was inoculated into 500 mL of LB liquid culture medium, cultured at 37°C for 14 h, and then centrifuged at 5000 r / min to obtain the bacterial cells, which were diluted with sterile water to 5 × 107 CFU / mL, and obtain the Pseudomonas aeruginosa seed solution.

[0090] Preparation Example 7

[0091] The preparation steps of the bio-fermentation extract are as follows:

[0092] Take 0.8 kg of the Pseudomonas aeruginosa seed liquid obtained in Preparation Example 6 and inoculate it into 10 kg of a specific fermentation medium. Ferment and culture it at 25°C in an aerobic environment for 4 days. Centrifuge it at 5000 r / min at 4°C for 10 min. Dry the supernatant at 50°C to obtain a biological fermentation extract.

[0093] Preparation of the specific fermentation medium used: 8 g of peptone, 4 g of beef extract powder, 7 g of sodium chloride, 1.5 g of dipotassium hydrogen phosphate, 7 mg of manganese sulfate monohydrate, 80 g of chitosan, 15 g of glycerol, 0.5 g of arabinose and 5 g of methanol were dissolved in 1 L of deionized water, and the pH was adjusted to 7.5 to obtain a specific fermentation medium.

[0094] Preparation Example 8

[0095] The preparation steps of the biofermentation extract were the same as those in Preparation Example 7, except that the amount of glycerol added to the specific fermentation medium used was 30 g.

[0096] Preparation Example 9

[0097] The preparation steps of the biofermentation extract were the same as those in Preparation Example 7, except that the amount of arabinose added to the specific fermentation medium used was 1.5 g.

[0098] Preparation Example 10

[0099] The preparation steps of the biofermentation extract were the same as those in Preparation Example 7, except that the amount of methanol added to the specific fermentation medium used was 10 g.

[0100] Preparation Example 11

[0101] The preparation steps of the biofermentation extract are the same as those of Preparation Example 7, except that no glycerol is added to the specific fermentation medium used.

[0102] Preparation Example 12

[0103] The preparation steps of the biofermentation extract are the same as those of Preparation Example 7, except that arabinose is not added to the specific fermentation medium used.

[0104] Preparation Example 13

[0105] The preparation steps of the biofermentation extract are the same as those of Preparation Example 7, except that no methanol is added to the specific fermentation medium used.

[0106] Preparation Example 14

[0107] The preparation steps of the biofermentation extract were the same as those in Preparation Example 7, except that the inoculation amount of the Pseudomonas proteus seed solution was 1.2 kg.

[0108] Preparation Example 15

[0109] The preparation steps of the biofermentation extract are the same as those of Preparation Example 7, except that glycerol, arabinose and methanol are not added to the specific fermentation medium used.

[0110] Example 1

[0111] An amino acid fertilizer for promoting crop growth, comprising the following raw materials in parts by weight:

[0112] 700 parts of insect body small molecule protein slurry, 45 parts of potassium dihydrogen phosphate, 100 parts of urea, 20 parts of trace elements and 10 parts of biological fermentation extract.

[0113] The insect body small molecule protein slurry used is obtained from Preparation Example 5; the biological fermentation extract used is obtained from Preparation Example 7.

[0114] Example 2

[0115] An amino acid fertilizer for promoting crop growth, comprising the following raw materials in parts by weight:

[0116] 850 parts of insect body small molecule protein slurry, 55 parts of potassium dihydrogen phosphate, 120 parts of urea, 40 parts of trace elements and 20 parts of biological fermentation extract.

[0117] The insect body small molecule protein slurry used is obtained from Preparation Example 5; the biological fermentation extract used is obtained from Preparation Example 7.

[0118] Example 3

[0119] An amino acid fertilizer for promoting crop growth, comprising the following raw materials in parts by weight:

[0120] 800 parts of insect body small molecule protein slurry, 50 parts of potassium dihydrogen phosphate, 110 parts of urea, 30 parts of trace elements and 15 parts of biological fermentation extract.

[0121] The insect body small molecule protein slurry used is obtained from Preparation Example 5; the biological fermentation extract used is obtained from Preparation Example 7.

[0122] Example 4

[0123] An amino acid fertilizer for promoting crop growth, comprising the following raw materials in parts by weight:

[0124] 750 parts of insect body small molecule protein slurry, 48 parts of potassium dihydrogen phosphate, 105 parts of urea, 25 parts of trace elements and 18 parts of biological fermentation extract.

[0125] The insect body small molecule protein slurry used is obtained from Preparation Example 5; the biological fermentation extract used is obtained from Preparation Example 7.

[0126] Example 5

[0127] An amino acid fertilizer for promoting crop growth, comprising the following raw materials in parts by weight:

[0128] 780 parts of insect body small molecule protein slurry, 52 parts of potassium dihydrogen phosphate, 115 parts of urea, 35 parts of trace elements and 13 parts of biological fermentation extract.

[0129] The insect body small molecule protein slurry used is obtained from Preparation Example 5; the biological fermentation extract used is obtained from Preparation Example 7.

[0130] Comparative Example 1

[0131] An amino acid fertilizer for promoting crop growth, comprising the following raw materials in parts by weight:

[0132] 780 parts of insect body small molecule protein slurry, 52 parts of potassium dihydrogen phosphate, 115 parts of urea, and 35 parts of trace elements.

[0133] The insect body small molecule protein slurry used is obtained from Preparation Example 5.

[0134] The trace elements described in Examples 1-5 and Comparative Example 1 are obtained by uniformly mixing ferric chloride, boron magnesium fertilizer, zinc sulfate and copper sulfate in a mass ratio of 1:1:1:1.

[0135] Performance Testing

[0136] 1. Performance test of insect body small molecule protein slurry

[0137] The insect body small molecule protein slurry prepared in Preparation Example 5 was sent to Guangdong Microbiological Analysis and Testing Center for crude protein content testing. The crude protein content testing method was based on GB / T 6432-2018 (7.2). The analysis results showed that the crude protein content of the insect body small molecule protein slurry in Preparation Example 5 was 16.24%.

[0138] The insect body small molecule protein slurry prepared in Preparation Example 5 was then tested for amino acid content at the Guangdong Provincial Microbiological Analysis and Testing Center. The amino acid content test method referred to GB / T 18246-2019. The specific test results of the amino acid content test are shown in Table 1.

[0139] Table 1

[0140]

[0141] As can be seen from Table 1, the contents of various amino acids in the insect body small molecule protein slurry obtained in Preparation Example 5 are relatively high.

[0142] 2. Performance test of biological fermentation extract

[0143] The following tests were performed on the bio-fermentation extracts obtained in Preparation Examples 7-15:

[0144] 2.1 Test of the content of amino oligosaccharides:

[0145] Refer to Deng Xiaojiao, Yan Cong, Xue Jianqiang, et al. "A high performance liquid chromatography method for determining the content of amino-oligosaccharides in pesticides", DOI:10.16201 / j.cnki.cn10-1660 / tq.2023.11.07. The content of amino-oligosaccharides in biological fermentation extracts was determined by the method.

[0146] 2.2 Rhamnolipid content test:

[0147] Determination of rhamnolipid content by sulfuric acid-orcinol method: Use 53wt% concentrated sulfuric acid to dissolve orcinol to make the final concentration of orcinol solution 0.19%, then use ultrapure water to prepare rhamnose solutions of different concentrations of 50, 100, 200, 300, 400, and 500 mg / L, and react with orcinol solution according to a volume ratio of 1:9. After heating at 80°C for 30 minutes, let stand at room temperature for 15 minutes, and then measure the absorbance at 421nm. Then, use rhamnose concentration as the horizontal axis and OD421 absorbance value as the vertical axis to make a rhamnolipid standard curve.

[0148] Then, 0.1 g of the biofermentation extract obtained in Preparation Examples 7-15 was taken respectively, and a sample solution of 100 mg / L was prepared with ultrapure water. After reacting with the orcinol solution according to the above method, the absorbance value at 421 nm was measured, and it was substituted into the rhamnolipid standard curve to calculate the rhamnolipid content in the biofermentation extract.

[0149] Specific test data are shown in Table 2.

[0150] Table 2

[0151] serial number Aminooligosaccharide content (%) Rhamnolipid content (%) Preparation Example 7 75.3 8.5 Preparation Example 8 70.2 5.3 Preparation Example 9 68.5 4.7 Preparation Example 10 66.9 5.0 Preparation Example 11 60.4 3.3 Preparation Example 12 58.8 3.9 Preparation Example 14 72.5 7.2 Preparation Example 15 50.6 2.6

[0152] As shown in Table 3, the biofermentation extract obtained in Preparation Example 7 contains more amino oligosaccharides and rhamnolipids. Compared with Preparation Example 7, the addition amount of one of glycerol, arabinose and methanol was changed in Preparation Examples 8-10, respectively, the induction effect on Pseudomonas proteus was weakened, and the secretion of amino oligosaccharides and rhamnolipids was reduced; one of glycerol, arabinose and methanol was missing in Preparation Examples 11-13, and glycerol, arabinose and methanol were not added in Preparation Example 15, which would lead to a significant reduction in the secretion of amino oligosaccharides and rhamnolipids by Pseudomonas proteus; in Preparation Example 14, the inoculation amount of Pseudomonas proteus was increased, the nutrients in the specific fermentation medium were limited, competitive inhibition was produced, the degradation effect of Pseudomonas proteus on chitosan was weakened, and the contents of amino oligosaccharides and rhamnolipids were reduced.

[0153] As can be seen from Table 3, the biofermentation extract obtained in Preparation Example 7 is applied to amino acid fertilizer.

[0154] 3. Performance test of amino acid fertilizer

[0155] The amino acid fertilizer obtained by mixing the raw materials in Example 3 and Comparative Example 1 was used to carry out a crop growth promotion experiment.

[0156] 3.1 Materials and methods

[0157] (1) Experimental location: farmland near the chemical industry park in Tiemenguan Economic and Technological Development Zone;

[0158] (2) Experimental crops: Tomato (Pink D-80), seedling raising on May 10, 2023, planting on July 1, 2023, 4,000 plants / mu, harvesting from August 10, 2023 to September 30, 2023;

[0159] (3) Experimental design: The experiment has four treatments, each with three replicates, arranged in random blocks. Each plot is 50 square meters in size and rectangular with protection rows. The experimental treatment design is as follows:

[0160] Treatment 1: basic fertilization + foliar spraying of the aminoacid fertilizer of Example 3;

[0161] Treatment 2: basic fertilization + foliar spraying of the control fertilizer prepared in Comparative Example 1;

[0162] Treatment 3 (blank control): basic fertilization + foliar spraying of an equal amount of water;

[0163] Basic fertilization includes: 2500kg / mu high-quality decomposed organic fertilizer (our company's product), 30kg / mu superphosphate, 20kg / mu potassium sulfate, and 10kg / mu urea;

[0164] (4) Application method: The application method of amino acid fertilizer in treatments 1 and 2 is: spray a 600-fold diluted solution of amino acid fertilizer on the tomato leaves once on the 1st and 15th of each month starting from July 15, 2023, with each application amount of 0.8 L / mu; in treatment 3, spray with clean water, and the number and amount are the same as those of other treatments.

[0165] 3.2 Test Results

[0166] The biological characteristics of tomatoes in each treatment were analyzed by measuring the following data:

[0167] (1) Leaf thickness and chlorophyll content: On July 30, 2023, 10 plants were randomly selected from each treatment, and 6 leaves in the middle of each plant were selected to measure the leaf thickness and chlorophyll content to obtain the average value. The leaf thickness was measured using a leaf thickness meter, and the chlorophyll content was measured using a chlorophyll meter.

[0168] (2) Fruit number and single fruit weight: Ten plants were randomly selected from each treatment, and the number and weight of fruits during the harvest period were counted. The total number of fruits per plant and the average fruit weight were calculated.

[0169] The specific measurement results are shown in Table 4.

[0170] Table 4

[0171]

[0172] It can be seen from Table 4 that compared with Treatments 2 and 3, the amino acid fertilizer used in Treatment 1 can significantly increase the thickness and chlorophyll content of the leaves and improve the quality of tomato fruits. This is because the amino acid fertilizer in Example 3 contains not only various nutrients for crop growth, but also amino oligosaccharides and rhamnolipids, which can kill some harmful bacteria, improve the absorption of amino acid fertilizers, and promote the growth and fruiting of crops.

[0173] The above is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present application. Although the present application is disclosed as above in the preferred embodiment, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution.

Claims

1. An amino acid fertilizer for promoting crop growth, It is characterized in that According to the weight percentage, it includes the following raw materials: 700-850 parts of insect body small molecule protein slurry, 45-55 parts of potassium dihydrogen phosphate, 100-120 parts of urea, 20-40 parts of trace elements, and 10-20 parts of biological fermentation extract; The trace elements include magnesium, iron, boron, zinc and copper.

2. The amino acid fertilizer according to claim 1, It is characterized in that The preparation steps of the biological fermentation extract are: The seed liquid of Pseudomonas proteus is inoculated into a specific fermentation medium, fermented and cultured for 3-5 days at 20-30° C. in an aerobic environment, centrifuged, and the obtained supernatant is dried to obtain a biological fermentation extract.

3. The amino acid fertilizer according to claim 2, It is characterized in that The Pseudomonas aeruginosa is Pseudomonas aeruginosa CICC 22994.

4. The amino acid fertilizer according to claim 3, It is characterized in that The concentration of the seed solution of Pseudomonas proteus is (1-9)×10 7 CFU / mL.

5. The amino acid fertilizer according to claim 2, It is characterized in that The components of the specific fermentation medium include: 5-10g of peptone, 3-5g of beef extract powder, 5-10g of sodium chloride, 1-2g of dipotassium hydrogen phosphate, 5-10mg of manganese sulfate monohydrate, and 1L of deionized water.

6. The amino acid fertilizer according to claim 5, It is characterized in that The specific fermentation medium further comprises 5-10 wt % chitosan, 1-2.5 wt % glycerol, 0.01-0.1 wt % arabinose and 0.1-1 wt % methanol.

7. The amino acid fertilizer according to claim 6, It is characterized in that The pH of the specific fermentation medium is 7-8.

8. The amino acid fertilizer according to claim 7, It is characterized in that The inoculation amount of the seed solution of Pseudomonas proteus is 5-10 wt % of the specific fermentation medium.

9. The amino acid fertilizer according to claim 1, It is characterized in that The preparation steps of the insect body small molecule protein slurry are as follows: (1) taking dried and crushed white star flower beetle bodies and banana peels, mixing them evenly, and sterilizing them to obtain composite white star flower beetle powder; (2) adding 3-10 times by weight of pure water to the composite white star flower beetle powder of step (1), then adding alkaline protease, stirring evenly, adjusting the pH to 8-10, enzymolyzing at 25-55° C. for 2-8 hours, inactivating the enzyme, and obtaining a white star flower beetle enzymolysis solution; (3) The pH of the enzymatic hydrolyzate of the white-spotted flower beetle in step (2) is adjusted to 5-7, and then the mixed bacterial seed solution is inoculated therein, and shake-cultured at 25-40° C. for 3-7 days to obtain a worm body small molecule protein slurry.

10. The amino acid fertilizer according to claim 9, It is characterized in that The crude protein content of the insect body small molecule protein slurry is ≥16.24%; the total content of aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine is ≥16.29%.