Complex enzyme preparation for improving absorption and utilization rate of fertilizer and preparation method of complex enzyme preparation

Abstract

The invention discloses a compound enzyme preparation for improving the absorption and utilization rate of a fertilizer and a preparation method thereof, and belongs to the technical field of enzyme preparations. The compound enzyme preparation is prepared from the following components in parts by weight: 100 parts of purified water, 6 to 8 parts of modified cellulase, 10 to 12 parts of immobilized protease, 10 to 12 parts of phosphatase, 10 to 14 parts of urease, 8 to 10 parts of nitrate reductase, 10 to 12 parts of pectinase, 8 to 10 parts of chitinase, 6 to 8 parts of xylanase, 10 to 12 parts of beta-glucosidase, 10 to 12 parts of superoxide dismutase and 4.5 to 6 parts of a microbial agent, the modified cellulase is obtained by chemically modifying cellulase; the immobilized protease is prepared by the following steps: adsorbing activated and coupled modified polylactic acid and a protease solution, crosslinking with glutaraldehyde, filtering, washing and drying. The compound enzyme preparation improves the fertilizer absorption and utilization rate of plants.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme preparations, and in particular relates to a composite enzyme preparation for improving fertilizer absorption and utilization rate and a preparation method thereof. Background Art

[0002] In the agricultural production process, fertilizer, as an important source of nutrients required by plants, has a decisive influence on the growth and yield of crops. However, the traditional method of applying chemical fertilizers has problems such as low utilization rate, serious nutrient loss, and environmental pollution, which has become one of the key factors restricting the sustainable development of agriculture. Therefore, the development of a composite enzyme preparation that can improve the absorption and utilization rate of fertilizers is of great significance for improving agricultural production efficiency, reducing the use of chemical fertilizers, reducing environmental pollution, and promoting green development of agriculture.

[0003] Excessive use of fertilizers not only leads to the destruction of soil structure and the decline of fertility, but also increases the risk of eutrophication of water bodies and greenhouse gas emissions. According to statistics, my country's fertilizer use accounts for one-third of the global total, but the utilization rate is relatively low, with nitrogen utilization rate of only 30% to 35%, phosphorus utilization rate of about 10% to 20%, and potassium utilization rate of about 35% to 50%. This means that a large amount of fertilizer nutrients are not effectively used in the soil, but are lost through leaching, volatilization and fixation, which not only wastes resources but also pollutes the environment.

[0004] As a biological fertilizer, compound enzyme preparations can promote the decomposition and mineralization of organic matter in the soil through enzymatic reactions, releasing nutrients that can be directly absorbed and utilized by plants. At the same time, compound enzyme preparations can also improve soil structure, increase soil permeability and water retention capacity, and provide a better growth environment for plant roots. In addition, compound enzyme preparations also have the advantages of being non-toxic, harmless, and residue-free, and will not pollute the environment, meeting the requirements of green development of modern agriculture. Therefore, a compound enzyme preparation that can improve the absorption and utilization rate of fertilizers is developed to solve the technical problem of low absorption and utilization rate of fertilizers in the prior art. Summary of the invention

[0005] The purpose of the present invention is to provide a composite enzyme preparation for improving fertilizer absorption and utilization rate and a preparation method thereof, so as to solve the technical problem of low fertilizer absorption and utilization rate in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A composite enzyme preparation for improving fertilizer absorption and utilization rate, comprising the following components in parts by weight: 100 parts of purified water, 6-8 parts of modified cellulase, 10-12 parts of immobilized protease, 10-12 parts of phosphatase, 10-14 parts of urease, 8-10 parts of nitrate reductase, 10-12 parts of pectinase, 8-10 parts of chitinase, 6-8 parts of xylanase, 10-12 parts of β-glucosidase, 10-12 parts of superoxide dismutase and 4.5-6 parts of microbial agent; the modified cellulase The cellulase is obtained by chemically modifying the cellulase; the immobilized protease is obtained by adsorbing the activated and coupled modified polylactic acid and the protease solution, and then filtering, washing and drying after the glutaraldehyde cross-linking reaction; the microbial agent is a mixture of filamentous yeast, rhizopus, polymyxoactinomycetes, jelly-like bacillus and pentosaceae, and the mass ratio of the yeast, rhizopus, polymyxoactinomycetes, jelly-like bacillus and pentosaceae is 1:0.5-1:1-2:1:1.

[0008] Furthermore, the preparation method of the modified cellulase comprises the following steps:

[0009] (1) preparing a chemical modification solution: adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to a polyethylene glycol solution, stirring to react, and obtaining a chemical modification solution;

[0010] (2) Connecting cellulase: dissolving cellulase in phosphate buffer to obtain a cellulase solution, slowly adding the chemical modification solution into the cellulase solution, stirring the reaction, and obtaining a modified cellulase.

[0011] Furthermore, in step (1), the concentration of the polyethylene glycol solution is 5% to 20% (W / V), the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to polyethylene glycol is 0.1% to 1%, the mass ratio of N-hydroxysuccinimide to polyethylene glycol is 0.1% to 1%, the stirring temperature is 30-40°C, the stirring speed is 50-80rpm, and the reaction time is 30-60min; in step (2), the concentration of the phosphate buffer is 0.1M, the concentration of the cellulase solution is 1-10mg / mL, the stirring speed is 80-100rpm, the reaction temperature is 25-30°C, and the reaction time is 2-3h.

[0012] Furthermore, the preparation method of the immobilized protease comprises the following steps:

[0013] S1, activation: adding dimethyl sulfoxide and epichlorohydrin to a NaOH solution, mixing evenly, to obtain an activation solution; adding polylactic acid to the activation solution, stirring for reaction, to obtain an activated polylactic acid solution; filtering the activated polylactic acid solution to obtain an activated polylactic acid filter residue, washing and drying to obtain an activated polylactic acid;

[0014] S2, coupling: adding ethanol with a volume concentration of 90% and a coupling agent to the activated polylactic acid, adjusting the pH, connecting a reflux device, heating and stirring the reaction in a three-necked flask to obtain a modified polylactic acid solution; filtering the modified polylactic acid solution to obtain a modified polylactic acid filter residue, washing and drying to obtain a modified polylactic acid;

[0015] S3, adsorption: weighing protease lyophilized powder, dissolving it in phosphate buffer solution to obtain a protease solution, and then weighing modified polylactic acid and dispersing it in the protease solution for adsorption to obtain a modified polylactic acid protease solution;

[0016] S4, cross-linking: adding glutaraldehyde solution to the modified polylactic acid protease solution, performing cross-linking reaction under stirring to obtain a crude immobilized protease, filtering the crude immobilized protease to obtain an immobilized protease residue, washing and naturally drying to obtain an immobilized protease.

[0017] Furthermore, the mass concentration of the NaOH solution in S1 is 40-45wt%, the mass percentage of dimethyl sulfoxide in the NaOH solution is 1-5wt%, the mass percentage of epichlorohydrin in the NaOH solution is 1-5wt%, the volume ratio of polylactic acid to the activation solution is 1:10-20, and the activated polylactic acid residue is washed 2-3 times with deionized water; the mass ratio of polylactic acid to the coupling agent in S2 is 5-6:1, the mass ratio of the coupling agent to the volume of 90% ethanol is 1:200-250g / mL, the coupling agent is a mixture of N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane and 3-aminopropyltriethoxysilane, and the mass ratio of N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane to 3-aminopropyltriethoxysilane is 1: 1. Adjust pH=2-2.5 with dilute hydrochloric acid, heat to 100°C for 40-80min, and stir at a magnetic speed of 140-150rpm. Wash the modified polylactic acid residue with 95% ethanol solution and then with deionized water until the filtrate is neutral. The concentration of the phosphate buffer solution in S3 is 0.2mol / L, the enzyme activity of the protease solution is 10-10000U / mL, the concentration of the protease solution is 0.5-1g / mL, the mass ratio of the protease to the modified polylactic acid is 1:1, and the adsorption time is 0.5-2h. The concentration of the glutaraldehyde solution in S4 is 0.25-2.5% (W / V), the stirring speed is 30-60rpm, the cross-linking time is 1-4h, and the immobilized protease residue is washed with deionized water until the filtrate is neutral.

[0018] A method for preparing a composite enzyme preparation for improving fertilizer absorption and utilization rate comprises the following steps:

[0019] M1. Primary fermentation: weigh modified cellulase, immobilized protease, phosphatase, urease, nitrate reductase, pectinase, chitinase, xylanase, β-glucosidase and superoxide dismutase into a fermentation tank, add purified water and glucose, raise the temperature for fermentation, and obtain a primary fermentation product;

[0020] M2, secondary fermentation: adjust the temperature and continue fermentation. After the fermentation is completed, obtain the secondary fermentation product;

[0021] M3, tertiary fermentation: adjust the temperature and continue fermentation. After the fermentation is completed, the tertiary fermentation product is obtained;

[0022] M4, final fermentation: adjust the temperature, add glucose, continue fermentation, and obtain the final fermentation product after completion;

[0023] M5. Maturation: Place the final fermentation product and filter it to obtain a mature product. Add microbial agents and stir to obtain a composite enzyme preparation that improves fertilizer absorption and utilization.

[0024] Furthermore, in M1, the mass ratio of glucose to purified water is 0.8-1:20, and the temperature is raised to 40°C and maintained for 24-26 hours; in M2, the temperature is adjusted to 37°C and fermented for 72-74 hours; in M3, the temperature is adjusted to 39°C and fermented for 16 days; in M4, the mass ratio of glucose to purified water is 0.8-1:20, the temperature is adjusted to 37°C and fermented for 10 days; in M5, the temperature is 30°C and left for 30 days, and oxygen is added to the bottom for 20 minutes every 24 hours during the entire fermentation process.

[0025] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] 1. The composite enzyme preparation of the present invention can quickly release fixed nutrients in the soil through the synergistic effect of multiple enzymes, significantly improve the absorption and utilization rate of fertilizers, and at the same time, the addition of microbial agents promotes the diversity and activity of soil microorganisms, and can secrete a variety of beneficial substances, such as vitamins, amino acids and hormones, etc., which are helpful to improve soil structure and increase soil organic matter. The addition of antioxidant enzymes such as superoxide dismutase helps to improve the tolerance of plants to environmental stress (such as salt stress, drought, etc.), thereby promoting the growth and development of plants. In addition, the preparation process gradually activates and optimizes the activity of various enzymes through fermentation at different temperatures and times, which is beneficial to temperature and time control, and ensures the stability of the composite enzyme preparation during storage and use.

[0027] 2. The present invention obtains modified cellulase by chemically modifying cellulase. The cellulase chemically modified with polyethylene glycol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide has enhanced enzyme activity and stability. The modified cellulase can more effectively decompose cellulose in the soil and release carbon sources and energy sources that are easily absorbed by plants. The chemical modification enhances the stability of the cellulase in the environment, prolongs the service life of the enzyme, and reduces the loss of the enzyme. The enhanced decomposition ability of the cellulase helps to accelerate the decomposition and mineralization of soil organic matter, provide more nutrients for plants, and thus improve the absorption and utilization rate of fertilizers.

[0028] 3. The present invention immobilizes the protease on the polylactic acid carrier. The immobilized protease can not only decompose the protein in the soil and release nutrients such as ammonia nitrogen that can be absorbed by plants, but also avoid the self-degradation of the enzyme, thereby improving the stability of the enzyme in the environment, prolonging the action time, and promoting the soil nitrogen cycle, thereby improving the absorption and utilization rate of the fertilizer. The polylactic acid carrier can be completely degraded by microorganisms in the natural environment, and finally generates carbon dioxide and water, which will not cause environmental pollution. Therefore, while improving the absorption and utilization rate of the fertilizer, it also protects the environment. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Embodiment 1:

[0031] The present embodiment discloses a composite enzyme preparation for improving fertilizer absorption and utilization rate, which is composed of the following components in parts by weight: 100kg purified water, 6kg modified cellulase, 10kg immobilized protease, 10kg phosphatase, 10kg urease, 8kg nitrate reductase, 10kg pectinase, 8kg chitinase, 6kg xylanase, 10 kg β-glucosidase, 10kg superoxide dismutase, 1kg filamentous yeast, 0.5kg Rhizopus, 1kg Actinomyces polymyxa, 1kg Bacillus jelly-like and 1kg Pediococcus pentosaceus; the modified cellulase is obtained by chemically modifying the cellulase; the immobilized protease is obtained by adsorbing activated and coupled modified polylactic acid with a protease solution, cross-linking with glutaraldehyde, filtering, washing and drying.

[0032] This embodiment discloses a method for preparing a modified cellulase, comprising the following steps:

[0033] (1) preparing a chemical modification solution: adding 0.1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 g of N-hydroxysuccinimide to 100 mL of a 5% (W / V) polyethylene glycol solution, stirring at 30° C. and 50 rpm for 30 min to obtain a chemical modification solution;

[0034] (2) Connecting cellulase: Dissolve 10 mg of cellulase in 10 mL of 0.1 M phosphate buffer to obtain a cellulase solution, slowly add the chemical modification solution into the cellulase solution, stir at 80 rpm, react at 25° C., and react for 2 h to obtain modified cellulase.

[0035] This embodiment discloses a method for preparing an immobilized protease, comprising the following steps:

[0036] S1, activation: add 1-5g of dimethyl sulfoxide and 1-5g of epichlorohydrin to 40-45wt% 100mL NaOH solution, mix well, and obtain an activation solution; add 5g of polylactic acid to the activation solution, stir and react, and obtain an activated polylactic acid solution; filter the activated polylactic acid solution to obtain an activated polylactic acid residue, wash it with deionized water 2-3 times, and dry it to obtain an activated polylactic acid;

[0037] S2, coupling: add 90% ethanol, 0.425-1g N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane and 0.425-1g 3-aminopropyltriethoxysilane in a volume concentration to the activated polylactic acid, adjust the pH to 2-2.5, connect a reflux device, heat and stir the reaction in a three-necked flask, heat to 100°C, the time is 40-80min, the magnetic stirring rate is 140-150rpm, and a modified polylactic acid solution is obtained; filter the modified polylactic acid solution to obtain a modified polylactic acid filter residue, first wash it with a 95% ethanol solution, then wash it with deionized water until the filtrate is neutral, and dry it to obtain a modified polylactic acid;

[0038] S3, adsorption: weigh 5-10g of lyophilized protease powder, dissolve it in 10mL of 0.2mol / L phosphate buffer solution to obtain a protease solution, the enzyme activity of the protease solution is 10-10000U / mL, and then weigh 5-10g of modified polylactic acid and disperse it in the protease solution for adsorption for 0.5-2h to obtain a modified polylactic acid protease solution;

[0039] S4, cross-linking: add 0.25-2.5% (W / V) 10mL glutaraldehyde solution to the modified polylactic acid protease solution, stir at 30-60rpm, and cross-link for 1-4h to obtain a crude immobilized protease. Filter the crude immobilized protease to obtain an immobilized protease residue, wash with deionized water until the filtrate is neutral, and naturally dry to obtain an immobilized protease.

[0040] This embodiment discloses a method for preparing a composite enzyme preparation for improving fertilizer absorption and utilization rate, comprising the following steps:

[0041] M1. Primary fermentation: weigh modified cellulase, immobilized protease, phosphatase, urease, nitrate reductase, pectinase, chitinase, xylanase, β-glucosidase and superoxide dismutase into a fermentation tank, add purified water and 4-5 kg ​​of glucose, raise the temperature to 40°C and maintain for 24-26 hours to obtain the primary fermentation product;

[0042] M2, secondary fermentation: adjust the temperature to 37°C and ferment for 72-74 hours. After the fermentation is completed, obtain the secondary fermentation product;

[0043] M3, tertiary fermentation: adjust the temperature to 39°C and ferment for 16 days. After the fermentation is completed, the tertiary fermentation product is obtained;

[0044] M4, final fermentation: add 4-5 kg ​​of glucose, adjust the temperature to 37 ° C, ferment for 10 days, and obtain the final fermentation product after the end;

[0045] M5. Maturation: Place the final fermentation product at 30°C for 30 days. Add oxygen at the bottom for 20 minutes every 24 hours during the entire fermentation process. After filtration, a mature product is obtained. Microbial agents are added and stirred to obtain a composite enzyme preparation that improves the absorption and utilization rate of fertilizers.

[0046] Embodiment 2:

[0047] The present embodiment discloses a composite enzyme preparation for improving the absorption and utilization rate of fertilizers, which is composed of the following components in parts by weight: 100kg purified water, 6-8kg modified cellulase, 10-12kg immobilized protease, 10-12kg phosphatase, 10-14kg urease, 8-10kg nitrate reductase, 10-12kg pectinase, 8-10kg chitinase, 6-8kg xylanase, 10-12kg β-glucosidase, 10-12kg superoxide dismutase, 1kg filamentous yeast, 0.5-1kg Rhizopus, 1-2kg Actinomycetes polymyxa, 1kg Bacillus jelly-like and 1kg Pediococcus pentosaceus; 3.5-6kg microbial agent; the modified cellulase is obtained by chemically modifying the cellulase; the immobilized protease is obtained by adsorbing activated and coupled modified polylactic acid with a protease solution, cross-linking with glutaraldehyde, filtering, washing and drying.

[0048] This embodiment discloses a method for preparing a modified cellulase, comprising the following steps:

[0049] (1) preparing a chemical modification solution: adding 0.1-1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to 100 mL of a 5% to 20% (W / V) polyethylene glycol solution, stirring at 30-40° C. and 50-80 rpm for 30-60 min to obtain a chemical modification solution;

[0050] (2) Connecting cellulase: Dissolve 10-100 mg of cellulase in 10 mL of 0.1 M phosphate buffer to obtain a cellulase solution, slowly add the chemical modification solution into the cellulase solution at a stirring speed of 80-100 rpm and a reaction temperature of 25-30° C. for 2-3 h to obtain modified cellulase.

[0051] This embodiment discloses a method for preparing an immobilized protease, comprising the following steps:

[0052] S1, activation: add 1-5g of dimethyl sulfoxide and 1-5g of epichlorohydrin to 40-45wt% 100mL NaOH solution, mix well, and obtain an activation solution; add 5-10g of polylactic acid to the activation solution, stir and react, and obtain an activated polylactic acid solution; filter the activated polylactic acid solution to obtain an activated polylactic acid residue, wash it with deionized water 2-3 times, and dry it to obtain an activated polylactic acid;

[0053] S2, coupling: add 90% ethanol, 0.425-1g N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane and 0.425-1g 3-aminopropyltriethoxysilane in a volume concentration to the activated polylactic acid, adjust the pH to 2-2.5, connect a reflux device, heat and stir the reaction in a three-necked flask, heat to 100°C, the time is 40-80min, the magnetic stirring rate is 140-150rpm, and a modified polylactic acid solution is obtained; filter the modified polylactic acid solution to obtain a modified polylactic acid filter residue, first wash it with a 95% ethanol solution, then wash it with deionized water until the filtrate is neutral, and dry it to obtain a modified polylactic acid;

[0054] S3, adsorption: weigh 5-10g of lyophilized protease powder, dissolve it in 10mL of 0.2mol / L phosphate buffer solution to obtain a protease solution, the enzyme activity of the protease solution is 10-10000U / mL, and then weigh 5-10g of modified polylactic acid and disperse it in the protease solution for adsorption for 0.5-2h to obtain a modified polylactic acid protease solution;

[0055] S4, cross-linking: add 0.25-2.5% (W / V) 10mL glutaraldehyde solution to the modified polylactic acid protease solution, stir at 30-60rpm, and cross-link for 1-4h to obtain a crude immobilized protease. Filter the crude immobilized protease to obtain an immobilized protease residue, wash with deionized water until the filtrate is neutral, and naturally dry to obtain an immobilized protease.

[0056] This embodiment discloses a method for preparing a composite enzyme preparation for improving fertilizer absorption and utilization rate, comprising the following steps:

[0057] M1. Primary fermentation: weigh modified cellulase, immobilized protease, phosphatase, urease, nitrate reductase, pectinase, chitinase, xylanase, β-glucosidase and superoxide dismutase into a fermentation tank, add purified water and 4-5 kg ​​of glucose, raise the temperature to 40°C and maintain for 24-26 hours to obtain the primary fermentation product;

[0058] M2, secondary fermentation: adjust the temperature to 37°C and ferment for 72-74 hours. After the fermentation is completed, obtain the secondary fermentation product;

[0059] M3, tertiary fermentation: adjust the temperature to 39°C and ferment for 16 days. After the fermentation is completed, the tertiary fermentation product is obtained;

[0060] M4, final fermentation: add 4-5 kg ​​of glucose, adjust the temperature to 37 ° C, ferment for 10 days, and obtain the final fermentation product after the end;

[0061] M5. Maturation: Place the final fermentation product at 30°C for 30 days. Add oxygen at the bottom for 20 minutes every 24 hours during the entire fermentation process. After filtration, a mature product is obtained. Microbial agents are added and stirred to obtain a composite enzyme preparation that improves the absorption and utilization rate of fertilizers.

[0062] Embodiment 3:

[0063] The present embodiment discloses a composite enzyme preparation for improving the absorption and utilization rate of fertilizers, which is composed of the following components in parts by weight: 100kg purified water, 6-8kg modified cellulase, 10-12kg immobilized protease, 10-12kg phosphatase, 10-14kg urease, 8-10kg nitrate reductase, 10-12kg pectinase, 8-10kg chitinase, 6-8kg xylanase, 10-12kg β-glucosidase, 10-12kg superoxide dismutase, 1kg filamentous yeast, 0.5-1kg Rhizopus, 1-2kg Actinomycetes polymyxa, 1kg Bacillus jelly-like and 1kg Pediococcus pentosaceus; 3.5-6kg microbial agent; the modified cellulase is obtained by chemically modifying the cellulase; the immobilized protease is obtained by adsorbing activated and coupled modified polylactic acid with a protease solution, cross-linking with glutaraldehyde, filtering, washing and drying.

[0064] This embodiment discloses a method for preparing a modified cellulase, comprising the following steps:

[0065] (1) preparing a chemical modification solution: adding 0.1-1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to 100 mL of a 5% to 20% (W / V) polyethylene glycol solution, stirring at 30-40° C. and 50-80 rpm for 30-60 min to obtain a chemical modification solution;

[0066] (2) Connecting cellulase: Dissolve 10-100 mg of cellulase in 10 mL of 0.1 M phosphate buffer to obtain a cellulase solution, slowly add the chemical modification solution into the cellulase solution at a stirring speed of 80-100 rpm and a reaction temperature of 25-30° C. for 2-3 h to obtain modified cellulase.

[0067] This embodiment discloses a method for preparing an immobilized protease, comprising the following steps:

[0068] S1, activation: add 1-5g of dimethyl sulfoxide and 1-5g of epichlorohydrin to 40-45wt% 100mL NaOH solution, mix well, and obtain an activation solution; add 5-10g of polylactic acid to the activation solution, stir and react, and obtain an activated polylactic acid solution; filter the activated polylactic acid solution to obtain an activated polylactic acid residue, wash it with deionized water 2-3 times, and dry it to obtain an activated polylactic acid;

[0069] S2, coupling: add 90% ethanol, 0.425-1g N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane and 0.425-1g 3-aminopropyltriethoxysilane in a volume concentration to the activated polylactic acid, adjust the pH to 2-2.5, connect a reflux device, heat and stir the reaction in a three-necked flask, heat to 100°C, the time is 40-80min, the magnetic stirring rate is 140-150rpm, and a modified polylactic acid solution is obtained; filter the modified polylactic acid solution to obtain a modified polylactic acid filter residue, first wash it with a 95% ethanol solution, then wash it with deionized water until the filtrate is neutral, and dry it to obtain a modified polylactic acid;

[0070] S3, adsorption: weigh 5-10g of lyophilized protease powder, dissolve it in 10mL of 0.2mol / L phosphate buffer solution to obtain a protease solution, the enzyme activity of the protease solution is 10-10000U / mL, and then weigh 5-10g of modified polylactic acid and disperse it in the protease solution for adsorption for 0.5-2h to obtain a modified polylactic acid protease solution;

[0071] S4, cross-linking: add 0.25-2.5% (W / V) 10mL glutaraldehyde solution to the modified polylactic acid protease solution, stir at 30-60rpm, and cross-link for 1-4h to obtain a crude immobilized protease. Filter the crude immobilized protease to obtain an immobilized protease residue, wash with deionized water until the filtrate is neutral, and naturally dry to obtain an immobilized protease.

[0072] This embodiment discloses a method for preparing a composite enzyme preparation for improving fertilizer absorption and utilization rate, comprising the following steps:

[0073] M1. Primary fermentation: weigh modified cellulase, immobilized protease, phosphatase, urease, nitrate reductase, pectinase, chitinase, xylanase, β-glucosidase and superoxide dismutase into a fermentation tank, add purified water and 4-5 kg ​​of glucose, raise the temperature to 40°C and maintain for 24-26 hours to obtain the primary fermentation product;

[0074] M2, secondary fermentation: adjust the temperature to 37°C and ferment for 72-74 hours. After the fermentation is completed, obtain the secondary fermentation product;

[0075] M3, tertiary fermentation: adjust the temperature to 39°C and ferment for 16 days. After the fermentation is completed, the tertiary fermentation product is obtained;

[0076] M4, final fermentation: add 4-5 kg ​​of glucose, adjust the temperature to 37 ° C, ferment for 10 days, and obtain the final fermentation product after the end;

[0077] M5. Maturation: Place the final fermentation product at 30°C for 30 days. Add oxygen at the bottom for 20 minutes every 24 hours during the entire fermentation process. After filtration, a mature product is obtained. Microbial agents are added and stirred to obtain a composite enzyme preparation that improves the absorption and utilization rate of fertilizers.

[0078] Comparative Example 1:

[0079] Comparative Example 1 Compared with Example 3, in the preparation process of the composite enzyme preparation in Comparative Example 1, the modified cellulase was replaced by cellulase, and other conditions remained unchanged.

[0080] Comparative Example 2:

[0081] Comparative Example 2 Compared with Example 3, in the preparation process of the composite enzyme preparation in Comparative Example 2, the immobilized protease was replaced with protease, and other conditions remained unchanged.

[0082] Comparative Example 3:

[0083] Comparative Example 3 Compared with Example 3, in the preparation process of the composite enzyme preparation in Comparative Example 3, no microbial agent was added, and other conditions remained unchanged.

[0084] Experimental example

[0085] The composite enzyme preparations prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to the following experimental determinations:

[0086] 1. Determination of agronomic indicators of broccoli

[0087] The composite enzyme preparations prepared in Examples 1-3 and Comparative Examples 1-3 were diluted 200 times to obtain a composite enzyme preparation liquid, and the composite enzyme preparation liquid was mixed with 50% composite fertilizer liquid to obtain a nutrient solution, which was sprayed on the leaves with an electric sprayer during the planting period, seedling period and rosette period of broccoli growth, respectively, wherein the blank control group was sprayed with an equal amount of water as the composite enzyme preparation liquid on the leaves during the same period, and the agronomic indicators of broccoli were counted on the 35th day after planting, wherein SPAD is the relative content of chlorophyll, and the statistical results are shown in Table 1:

[0088] Table 1

[0089] Group Plant height / cm Crown width / cm SPAD Single fruit weight / kg Shimizu 8.3 24.3 26.12 0.56 Example 1 18.1 58.6 48.55 1.54 Example 2 18.4 58.7 48.72 1.62 Example 3 18.7 59.1 49.13 1.69 Comparative Example 1 13.0 41.2 30.10 1.13 Comparative Example 2 12.5 40.5 28.94 1.08 Comparative Example 3 13.4 41.5 30.47 1.15

[0090] According to the statistical results in Table 1, the composite enzyme preparations prepared in Examples 1-3 of the present invention improve the fertilizer absorption and utilization rate of broccoli. By comparing Comparative Examples 1-3 with Examples 1-3, it can be seen that adding modified cellulase, immobilized protease and microbial agents can effectively improve the fertilizer absorption and utilization rate of plants.

[0091] 2. Determination of tomato agronomic indicators

[0092] The composite enzyme preparations prepared in Examples 1-3 and Comparative Examples 1-3 were diluted 200 times to obtain composite enzyme preparation liquid, and the composite enzyme preparation liquid was mixed with 50% composite fertilizer liquid to obtain a nutrient solution, and the composite enzyme was sprayed after seedling recovery, before flowering, early fruit setting, late fruit setting, and fruit swelling during the growth period of tomatoes, respectively. The blank control group was sprayed with an equal amount of water as the composite enzyme preparation liquid on the leaves at the same time. The agronomic indicators of tomatoes were counted on the 130th day after planting, and the statistical results are shown in Table 2:

[0093] Table 2

[0094] Group Single fruit weight / g Horizontal diameter / cm Longitudinal diameter / cm Fruit shape index Brix Shimizu 146.70 4.19 3.76 0.57 4.88 Example 1 211.69 6.91 5.63 0.82 6.73 Example 2 212.04 7.23 5.84 0.85 6.89 Example 3 212.31 7.42 5.96 0.86 7.05 Comparative Example 1 178.45 5.90 4.16 0.64 5.36 Comparative Example 2 176.33 5.81 4.02 0.60 5.20 Comparative Example 3 180.46 6.06 4.25 0.66 5.43

[0095] According to the statistical results in Table 1, the composite enzyme preparations prepared in Examples 1-3 of the present invention improve the fertilizer absorption and utilization rate of tomatoes. By comparing Comparative Examples 1-3 with Examples 1-3, it can be seen that adding modified cellulase, immobilized protease and microbial agents can effectively improve the fertilizer absorption and utilization rate of plants.

[0096] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0097] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A composite enzyme preparation for improving fertilizer absorption and utilization rate, characterized in that: The invention comprises the following components in parts by weight: 100 parts of purified water, 6-8 parts of modified cellulase, 10-12 parts of immobilized protease, 10-12 parts of phosphatase, 10-14 parts of urease, 8-10 parts of nitrate reductase, 10-12 parts of pectinase, 8-10 parts of chitinase, 6-8 parts of xylanase, 10-12 parts of beta-glucosidase, 10-12 parts of superoxide dismutase and 4.5-6 parts of microbial agent; the modified cellulase is obtained by chemically modifying the cellulase; and the immobilized protease is obtained by adsorbing activated and coupled modified polylactic acid and protease solution, performing glutaraldehyde cross-linking reaction, filtering, washing and drying.

2. A composite enzyme preparation for improving fertilizer absorption and utilization rate according to claim 1, characterized in that: The microbial agent is a mixture of filamentous yeast, rhizopus, polymyxoactinomyces, jelly-like bacillus and Pediococcus pentosaceus, and the mass ratio of yeast, rhizopus, polymyxoactinomyces, jelly-like bacillus and Pediococcus pentosaceus is 1:0.5-1:1-2:1:1; the preparation method of modified cellulase comprises the following steps: (1) preparing a chemical modification solution: adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to a polyethylene glycol solution, stirring to react, and obtaining a chemical modification solution; (2) Connecting cellulase: dissolving cellulase in phosphate buffer to obtain a cellulase solution, slowly adding the chemical modification solution into the cellulase solution, stirring the reaction, and obtaining a modified cellulase.

3. A composite enzyme preparation for improving fertilizer absorption and utilization rate according to claim 2, characterized in that: The concentration of the polyethylene glycol solution in step (1) is 5% to 20% (W / V), the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to polyethylene glycol is 0.1% to 1%, the mass ratio of N-hydroxysuccinimide to polyethylene glycol is 0.1% to 1%, the stirring temperature is 30-40° C., the stirring speed is 50-80 rpm, and the reaction time is 30-60 min.

4. A composite enzyme preparation for improving fertilizer absorption and utilization rate according to claim 2, characterized in that: In step (2), the concentration of the phosphate buffer is 0.1 M, the concentration of the cellulase solution is 1-10 mg / mL, the stirring speed is 80-100 rpm, the reaction temperature is 25-30° C., and the reaction time is 2-3 h.

5. The composite enzyme preparation for improving fertilizer absorption and utilization rate according to claim 1, characterized in that: The preparation method of immobilized protease comprises the following steps: S1, activation: adding dimethyl sulfoxide and epichlorohydrin to a NaOH solution, mixing evenly, to obtain an activation solution; adding polylactic acid to the activation solution, stirring for reaction, to obtain an activated polylactic acid solution; The activated polylactic acid solution is filtered to obtain an activated polylactic acid residue, and the activated polylactic acid is obtained after washing and drying; S2, coupling: adding ethanol with a volume concentration of 90% and a coupling agent to the activated polylactic acid, adjusting the pH, connecting a reflux device, heating and stirring in a three-necked flask to react, and obtaining a modified polylactic acid solution; The modified polylactic acid solution is filtered to obtain a modified polylactic acid residue, which is then washed and dried to obtain the modified polylactic acid; S3, adsorption: weighing protease lyophilized powder, dissolving it in phosphate buffer solution to obtain a protease solution, and then weighing modified polylactic acid and dispersing it in the protease solution for adsorption to obtain a modified polylactic acid protease solution; S4, cross-linking: adding glutaraldehyde solution to the modified polylactic acid protease solution, performing cross-linking reaction under stirring to obtain a crude immobilized protease, filtering the crude immobilized protease to obtain an immobilized protease residue, washing and naturally drying to obtain an immobilized protease.

6. The composite enzyme preparation for improving fertilizer absorption and utilization rate according to claim 5, characterized in that: The mass concentration of the NaOH solution in S1 is 40-45wt%, the mass percentage of dimethyl sulfoxide in the NaOH solution is 1-5wt%, the mass percentage of epichlorohydrin in the NaOH solution is 1-5wt%, the volume ratio of polylactic acid to the activation solution is 1:10-20, and the activated polylactic acid filter residue is washed 2-3 times with deionized water.

7. The composite enzyme preparation for improving fertilizer absorption and utilization rate according to claim 5, characterized in that: The mass ratio of polylactic acid to coupling agent in S2 is 5-6:1, the mass ratio of coupling agent to volume of 90% ethanol is 1:200-250 g / mL, the coupling agent is a mixture of N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane and 3-aminopropyltriethoxysilane, the mass ratio of N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane and 3-aminopropyltriethoxysilane is 1:1, the pH is adjusted to 2-2.5 with dilute hydrochloric acid, heated to 100°C for 40-80 minutes, the magnetic stirring rate is 140-150 rpm, and the modified polylactic acid filter residue is first washed with 95% ethanol solution and then washed with deionized water until the filtrate is neutral.

8. The composite enzyme preparation for improving fertilizer absorption and utilization rate according to claim 5, characterized in that: The concentration of the phosphate buffer solution in S3 is 0.2 mol / L, the enzyme activity of the protease solution is 10-10000 U / mL, the concentration of the protease solution is 0.5-1 g / mL, the mass ratio of the protease to the modified polylactic acid is 1:1, and the adsorption time is 0.5-2 h; the concentration of the glutaraldehyde solution in S4 is 0.25-2.5% (W / V), the stirring speed is 30-60 rpm, the cross-linking time is 1-4 h, and the immobilized protease residue is washed with deionized water until the filtrate is neutral.

9. A method for preparing a composite enzyme preparation for improving fertilizer absorption and utilization rate, characterized in that: The following steps are involved: M1. Primary fermentation: weigh modified cellulase, immobilized protease, phosphatase, urease, nitrate reductase, pectinase, chitinase, xylanase, β-glucosidase and superoxide dismutase into a fermentation tank, add purified water and glucose, raise the temperature for fermentation, and obtain a primary fermentation product; M2, secondary fermentation: adjust the temperature and continue fermentation. After the fermentation is completed, obtain the secondary fermentation product; M3, tertiary fermentation: adjust the temperature and continue fermentation. After the fermentation is completed, the tertiary fermentation product is obtained; M4, final fermentation: adjust the temperature, add glucose, continue fermentation, and obtain the final fermentation product after completion; M5. Maturation: Place the final fermentation product and filter it to obtain a mature product. Add microbial agents and stir to obtain a composite enzyme preparation that improves fertilizer absorption and utilization.

10. The method for preparing a composite enzyme preparation for improving fertilizer absorption and utilization rate according to claim 9, characterized in that: The mass ratio of glucose to purified water in M1 is 0.8-1:20, and the temperature is raised to 40°C and maintained for 24-26 hours; the temperature in M2 is adjusted to 37°C and fermented for 72-74 hours; the temperature in M3 is adjusted to 39°C and fermented for 16 days; the mass ratio of glucose to purified water in M4 is 0.8-1:20, the temperature is adjusted to 37°C and fermented for 10 days; the temperature in M5 is 30°C for 30 days, and oxygen is added to the bottom for 20 minutes every 24 hours during the entire fermentation process.