A germination promoter for Stipa breviflora seeds and its application

Abstract

The invention discloses a stipa brevis seed germination promoter and application, and belongs to the technical field of seed germination promoter preparation. The stipa brevis seed germination promoter is composed of the following components in parts by weight: 42-55 parts of cow dung, 28-42 parts of wheat bran, 30-45 parts of rice milk, 5-9 parts of PBS buffer, 2-3 parts of composite hydrogel, 6-10 parts of immobilized enzyme, wherein the composite hydrogel is prepared from sodium chloride, cellulose nanofiber, sodium periodate, γ-polyglutamic acid and ethylene glycol diglycidol, and the immobilized enzyme is prepared from tribenzaldehyde, acetic acid buffer, p-phenylenediamine and composite enzyme. The promoter prepared by the method can effectively improve the germination rate and plant height of stipa brevis.

Description

Technical Field

[0001] The invention belongs to the technical field of seed germination promoter preparation, and particularly relates to a Stipa breviflora seed germination promoter and application thereof. Background Art

[0002] Stipa breviflora, as a superior forage grass widely distributed in Northwest and Southwest China, grows mostly in rocky hillsides, dry hillsides or river valley terraces at an altitude of 700-4700 meters. It has strong adaptability and stress resistance. However, under natural conditions, the germination of Stipa breviflora seeds is often restricted by a variety of environmental factors, such as insufficient water, temperature fluctuations, poor soil, and the dormancy characteristics of the seeds themselves. These factors together limit the population renewal and ecological restoration capabilities of Stipa breviflora. Therefore, the development of an accelerator that can effectively promote the germination of Stipa breviflora seeds is of great significance for increasing forage yields, improving grassland ecology, and promoting the sustainable development of animal husbandry.

[0003] Traditional methods for promoting the germination of forage seeds mainly include physical treatment, chemical treatment and biological treatment. However, these methods often have problems such as unstable effects, high costs or potential risks to the environment in practical applications. In response to these problems, the research and development of Stipa breviflora seed germination promoter came into being.

[0004] Patent CN115956581A discloses a graphene-based Codonopsis pilosula seed germination promoter, and a preparation method and application thereof. The preparation steps are: using water as a solvent, adding graphene oxide and ultrasonically treating it, standing to obtain an upper layer solution, and then drying to obtain the ultrasonically treated graphene oxide; dissolving the ultrasonically treated graphene oxide in water, stirring evenly, adding aminated nanosilver, and reacting at 50-60°C to obtain a graphene-based Codonopsis pilosula seed germination promoter, which can effectively promote the germination and growth of Codonopsis pilosula seeds, but the seed germination promoter prepared by this method still has room for improvement in the germination rate of seeds. Summary of the invention

[0005] The object of the present invention is to provide a Stipa breviflora seed germination promoter and application thereof, so as to solve the technical problem of poor germination rate of Stipa breviflora seeds in the prior art.

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

[0007] The invention provides a Stipa breviflora seed germination promoter, which is composed of the following components in parts by weight: 42-55 parts of cow dung, 28-42 parts of wheat bran, 30-45 parts of rice milk, 5-9 parts of PBS buffer, 2-3 parts of composite hydrogel, and 6-10 parts of immobilized enzyme, wherein the composite hydrogel is prepared from sodium chloride, cellulose nanofiber, sodium periodate, gamma-polyglutamic acid and ethylene glycol diglycidol, and the immobilized enzyme is prepared from mesitylene tribenzaldehyde, acetic acid buffer, p-phenylenediamine and the composite enzyme.

[0008] Preferably, the method for preparing the composite hydrogel comprises the following steps:

[0009] Q1: Add sodium chloride to deionized water, stir to dissolve, then add cellulose nanofibers, add sodium periodate after ultrasonic dispersion, stir to dissolve, adjust pH, protect from light, filter, wash, and freeze-dry to obtain modified cellulose nanofibers;

[0010] Q2: The modified cellulose nanofibers are cut and added to deionized water, and a suspension is obtained after high-speed stirring. Then, γ-polyglutamic acid is added and stirred to dissolve. Then, ethylene glycol diglycidol is added, and after adjusting the pH, the mixture is allowed to stand and react to obtain a composite hydrogel.

[0011] In the above process, sodium periodate, as a strong oxidant, can react with the hydroxyl groups on the surface of cellulose nanofibers to generate aldehyde groups, thereby obtaining modified cellulose nanofibers with excellent reactivity. Subsequently, the epoxy units in ethylene glycol diglycidol can react with the hydroxyl groups on the surface of the modified cellulose nanofibers and the carboxylate ions of γ-polyglutamic acid to undergo cross-linking reactions, thereby obtaining a composite hydrogel.

[0012] Preferably, in Q1, the dosage ratio of sodium chloride, deionized water, cellulose nanofibers and sodium periodate is (1.3-3.5) g: (200-300) mL: (1-2) g: (1-2) g, the ultrasonic dispersion time is 10-30 min, the stirring and dissolving time after adding sodium periodate is 1-2 h, the pH is adjusted to 4-4.2 with 0.1 mol / L hydrochloric acid, the light-proof treatment time is 2-4 h, the stirring speed during the light-proof process is 200-300 rpm, the treatment temperature is 40-60 ° C, the filter is filtered with a 0.45 μm filter membrane, and the filter is washed with deionized water.

[0013] Preferably, in Q2, the amount ratio of modified cellulose nanofibers, deionized water, γ-polyglutamic acid and ethylene glycol diglycidol is (0.65-2.17) g: (40-60) mL: (6-8) g: (2.8-4.2) g, the high-speed stirring speed is 20000-28000 rpm, the time is 30-45 min, the average molecular weight of γ-polyglutamic acid is 1100 kDa, the purity is 92.2%, the stirring and dissolving time is 30-45 min, the epoxy value of ethylene glycol diglycidol is greater than 0.65, the pH is adjusted to 4.8-5.2 with 6 mol / L hydrochloric acid, the standing time is 2-4 h, the reaction temperature is 50-55 ° C, the humidity is 80-90% RH, and the time is 8-10 h.

[0014] Preferably, the method for preparing the immobilized enzyme comprises the following steps:

[0015] S1: adding isotriphenylaldehyde to acetic acid buffer, and ultrasonically treating the solution to obtain solution 1; adding p-phenylenediamine to acetic acid buffer, and ultrasonically treating the solution to obtain solution 2;

[0016] S2: Add the complex enzyme to solution 1, incubate, then add solution 2 dropwise to solution 1 through a peristaltic pump, react with magnetic stirring, centrifuge, wash, and dry to obtain the immobilized enzyme.

[0017] In the above process, iso-tritylaldehyde and p-phenylenediamine are used as raw materials, acetic acid buffer with pH=4.5 is used as a catalyst and a reaction medium, and the composite enzyme is fixed to prepare an immobilized enzyme.

[0018] Preferably, in S1, the dosage ratio of triphenylaldehyde to acetic acid buffer is (0.032-0.05) g: (5-7.5) mL, the ultrasonic treatment time is 5-9 min, the dosage ratio of p-phenylenediamine to acetic acid buffer is (0.03-0.054) g: (5-8) mL, the pH of the acetic acid buffer is 4.5, and the ultrasonic treatment time is 5-7 min.

[0019] Preferably, in S2, the dosage ratio of solution 1 and complex enzyme is (6-8) mL: (0.02-0.05) g, the complex enzyme consists of phytase, α-amylase and β-glucose enzyme, the incubation time is 5-8 min, the magnetic stirring speed is 1000-1500 rpm, the reaction time is 30-45 min, the centrifugal speed is 5000-7000 rpm, the time is 10-12 min, washing is performed with methanol, the drying temperature is 30-32° C., and the time is 6-8 h.

[0020] Preferably, the method for preparing the Stipa breviflora seed germination promoter comprises the following steps:

[0021] Step 1: Mix cow dung, wheat bran and rice milk evenly to obtain a basic mixture;

[0022] Step 2: Add PBS buffer to the basic mixture, adjust the pH to the enzyme activity range, then continue to add the composite hydrogel, stir evenly, add the immobilized enzyme, mix evenly, and ferment to obtain the Stipa breviflora seed germination promoter.

[0023] The application of the Stipa breviflora seed germination promoter is to apply the obtained Stipa breviflora seed germination promoter to the germination and growth of Stipa breviflora seeds.

[0024] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0025] Sodium periodate used in the present invention as a strong oxidant can selectively oxidize the hydroxyl groups (mainly the hydroxyl groups at the C2 and C3 positions) on the surface of cellulose nanofibers to form aldehyde groups, and decomposes after attacking the C3-C3 ortho-hydroxyl groups of glucose units through iodine oxygen bonds to form unstable intermediates. The modification process not only enhances the chemical activity of cellulose nanofibers, but also improves their dispersibility and stability, laying a good foundation for the preparation of subsequent composite hydrogels. Ethylene glycol diglycidol cross-links with the hydroxyl groups on the surface of modified cellulose nanofibers and the carboxylate ions of γ-polyglutamic acid to form a composite hydrogel with a tight network structure. The composite hydrogel has excellent water retention and water absorption, provides necessary moisture for the germination of short-flowered needle grass seeds, and has excellent stability and mechanical properties. Subsequently, the polymer generated by isotriphenylaldehyde and p-phenylenediamine under the catalysis of acetic acid buffer at pH=4.5 is used as a carrier, and the composite enzyme is fixed by covalent bonds, ionic bonding or physical adsorption, thereby improving the stability and reusability of the enzyme, and making it evenly distributed in the composite hydrogel network, enhancing the contact opportunity and catalytic efficiency of the enzyme and the substrate. The synergistic effect of the composite hydrogel and immobilized enzyme created an excellent microenvironment for the germination of Stipa breviflora seeds, improved the germination rate and growth performance of the seeds, enhanced the seed resistance, and promoted the activity of soil microorganisms and ecological balance. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1: This example discloses a method for preparing a composite hydrogel, comprising the following steps:

[0028] Q1: Add 2.4g sodium chloride to 250mL deionized water, stir to dissolve, then add 1.5g cellulose nanofibers, add 1.5g sodium periodate after ultrasonic dispersion for 30min, stir to dissolve for 2h, adjust pH to 4 with 0.1mol / L hydrochloric acid, 300rpm, 50℃, avoid light for 4h, filter with 0.45μm filter membrane, wash with deionized water, freeze-dry to obtain modified cellulose nanofibers;

[0029] Q2: 1.42 g of modified cellulose nanofibers were cut and added to 50 mL of deionized water. After high-speed stirring at 28,000 rpm for 30 min, a suspension was obtained. Then, 7 g of γ-polyglutamic acid with a purity of 92.2% and an average molecular weight of 1100 kDa was added and stirred for 45 min to dissolve. Then, 3.5 g of ethylene glycol diglycidol with an epoxy value of >0.65 was added. After adjusting the pH to 4.8 with 6 mol / L hydrochloric acid, the mixture was allowed to stand for 4 h and reacted at 55 °C and 90% RH for 10 h to obtain a composite hydrogel.

[0030] This embodiment discloses a method for preparing an immobilized enzyme, comprising the following steps:

[0031] S1: 0.041 g of triphenylaldehyde was added to 6.25 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 1. 0.042 g of p-phenylenediamine was added to 6.5 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 2.

[0032] S2: Add 0.035 g of complex enzyme (consisting of phytase, α-amylase and β-glucosidase) to 7 mL of solution 1, incubate for 8 min, then add solution 2 dropwise into solution 1 through a peristaltic pump, react with magnetic stirring at 1500 rpm for 45 min, centrifuge at 7000 rpm for 10 min, wash with methanol, and dry at 32°C for 8 h to obtain immobilized enzyme.

[0033] The present embodiment discloses a Stipa breviflora seed germination promoter, which is composed of the following ingredients in parts by weight: 47 parts of cow dung, 35 parts of wheat bran, 37.5 parts of rice milk, 7 parts of PBS buffer, 2.5 parts of composite hydrogel, and 8 parts of immobilized enzyme.

[0034] This embodiment discloses a method for preparing a Stipa breviflora seed germination promoter, comprising the following steps:

[0035] Step 1: Mix cow dung, wheat bran and rice milk evenly to obtain a basic mixture;

[0036] Step 2: Add PBS buffer to the basic mixture, adjust the pH to the enzyme activity range, then continue to add the composite hydrogel, stir evenly, add the immobilized enzyme, mix evenly, and ferment to obtain the Stipa breviflora seed germination promoter.

[0037] Example 2: This example discloses a method for preparing a composite hydrogel, comprising the following steps:

[0038] Q1: Add 1.3g sodium chloride to 200mL deionized water, stir to dissolve, then add 1g cellulose nanofibers, ultrasonically disperse for 30min, add 2g sodium periodate, stir to dissolve for 2h, adjust pH to 4 with 0.1mol / L hydrochloric acid, 300rpm, 50℃, avoid light for 4h, filter with 0.45μm filter membrane, wash with deionized water, freeze-dry to obtain modified cellulose nanofibers;

[0039] Q2: 0.65 g of modified cellulose nanofibers were cut and added to 40 mL of deionized water. The suspension was obtained after high-speed stirring at 28,000 rpm for 30 min. Then, 6 g of γ-polyglutamic acid with a purity of 92.2% and an average molecular weight of 1100 kDa was added and stirred for 45 min to dissolve. Then, 2.8 g of ethylene glycol diglycidol with an epoxy value of >0.65 was added. The pH was adjusted to 4.8 with 6 mol / L hydrochloric acid, and the mixture was allowed to stand for 4 h. The reaction was then carried out at 55 °C and 90% RH for 10 h to obtain a composite hydrogel.

[0040] This embodiment discloses a method for preparing an immobilized enzyme, comprising the following steps:

[0041] S1: 0.032 g of iso-tritylaldehyde was added to 5 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 1. 0.054 g of p-phenylenediamine was added to 8 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 2.

[0042] S2: Add 0.02 g of complex enzyme (consisting of phytase, α-amylase and β-glucosidase) to 6 mL of solution 1, incubate for 8 min, then add solution 2 dropwise into solution 1 through a peristaltic pump, react with magnetic stirring at 1500 rpm for 45 min, centrifuge at 7000 rpm for 10 min, wash with methanol, and dry at 32°C for 8 h to obtain immobilized enzyme.

[0043] The present embodiment discloses a Stipa breviflora seed germination promoter, which is composed of the following ingredients in parts by weight: 42 parts of cow dung, 28 parts of wheat bran, 45 parts of rice milk, 5 parts of PBS buffer, 3 parts of composite hydrogel, and 10 parts of immobilized enzyme.

[0044] This embodiment discloses a method for preparing a Stipa breviflora seed germination promoter, comprising the following steps:

[0045] Step 1: Mix cow dung, wheat bran and rice milk evenly to obtain a basic mixture;

[0046] Step 2: Add PBS buffer to the basic mixture, adjust the pH to the enzyme activity range, then continue to add the composite hydrogel, stir evenly, add the immobilized enzyme, mix evenly, and ferment to obtain the Stipa breviflora seed germination promoter.

[0047] Example 3: This example discloses a method for preparing a composite hydrogel, comprising the following steps:

[0048] Q1: Add 3.5g sodium chloride to 300mL deionized water, stir to dissolve, then add 2g cellulose nanofibers, add 1g sodium periodate after ultrasonic dispersion for 30min, stir to dissolve for 2h, adjust pH to 4 with 0.1mol / L hydrochloric acid, 300rpm, 50℃, avoid light for 4h, filter with 0.45μm filter membrane, wash with deionized water, freeze-dry to obtain modified cellulose nanofibers;

[0049] Q2: 2.17 g of modified cellulose nanofibers were cut and added to 60 mL of deionized water. The suspension was obtained after high-speed stirring at 28,000 rpm for 30 min. Then, 8 g of γ-polyglutamic acid with a purity of 92.2% and an average molecular weight of 1100 kDa was added and stirred for 45 min to dissolve. Then, 4.2 g of ethylene glycol diglycidol with an epoxy value of >0.65 was added. The pH was adjusted to 4.8 with 6 mol / L hydrochloric acid, and the mixture was allowed to stand for 4 h. The reaction was then carried out at 55 °C and 90% RH for 10 h to obtain a composite hydrogel.

[0050] This embodiment discloses a method for preparing an immobilized enzyme, comprising the following steps:

[0051] S1: 0.05 g of triphenylaldehyde was added to 7.5 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 1. 0.03 g of p-phenylenediamine was added to 5 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 2.

[0052] S2: Add 0.05 g of complex enzyme (consisting of phytase, α-amylase and β-glucose) to 8 mL of solution 1, incubate for 8 min, then add solution 2 dropwise into solution 1 through a peristaltic pump, react with magnetic stirring at 1500 rpm for 45 min, centrifuge at 7000 rpm for 10 min, wash with methanol, and dry at 32 °C for 8 h to obtain immobilized enzyme.

[0053] The present embodiment discloses a Stipa breviflora seed germination promoter, which is composed of the following ingredients in parts by weight: 55 parts of cow dung, 42 parts of wheat bran, 30 parts of rice milk, 9 parts of PBS buffer, 2 parts of composite hydrogel, and 6 parts of immobilized enzyme.

[0054] This embodiment discloses a method for preparing a Stipa breviflora seed germination promoter, comprising the following steps:

[0055] Step 1: Mix cow dung, wheat bran and rice milk evenly to obtain a basic mixture;

[0056] Step 2: Add PBS buffer to the basic mixture, adjust the pH to the enzyme activity range, then continue to add the composite hydrogel, stir evenly, add the immobilized enzyme, mix evenly, and ferment to obtain the Stipa breviflora seed germination promoter.

[0057] Example 4: This example discloses a method for preparing a composite hydrogel, comprising the following steps:

[0058] Q1: Add 1.8g sodium chloride to 280mL deionized water, stir to dissolve, then add 1.2g cellulose nanofibers, add 1.7g sodium periodate after ultrasonic dispersion for 30min, stir to dissolve for 2h, adjust pH to 4 with 0.1mol / L hydrochloric acid, 300rpm, 50℃, avoid light for 4h, filter with 0.45μm filter membrane, wash with deionized water, freeze-dry to obtain modified cellulose nanofibers;

[0059] Q2: 1.03 g of modified cellulose nanofibers were cut and added to 55 mL of deionized water. After high-speed stirring at 28,000 rpm for 30 min, a suspension was obtained. Then, 6.5 g of γ-polyglutamic acid with a purity of 92.2% and an average molecular weight of 1100 kDa was added and stirred for 45 min to dissolve. Then, 3.2 g of ethylene glycol diglycidol with an epoxy value of >0.65 was added. After adjusting the pH to 4.8 with 6 mol / L hydrochloric acid, the mixture was allowed to stand for 4 h and reacted at 55 °C and 90% RH for 10 h to obtain a composite hydrogel.

[0060] This embodiment discloses a method for preparing an immobilized enzyme, comprising the following steps:

[0061] S1: 0.038 g of triphenylaldehyde was added to 5.5 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 1. 0.035 g of p-phenylenediamine was added to 6 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 2.

[0062] S2: Add 0.03 g of complex enzyme (consisting of phytase, α-amylase and β-glucose) to 6.5 mL of solution 1, incubate for 8 min, then add solution 2 dropwise into solution 1 through a peristaltic pump, react with magnetic stirring at 1500 rpm for 45 min, centrifuge at 7000 rpm for 10 min, wash with methanol, and dry at 32°C for 8 h to obtain immobilized enzyme.

[0063] The present embodiment discloses a Stipa breviflora seed germination promoter, which is composed of the following ingredients in parts by weight: 45 parts of cow dung, 33 parts of wheat bran, 35 parts of rice milk, 6 parts of PBS buffer, 2.2 parts of composite hydrogel, and 7 parts of immobilized enzyme.

[0064] This embodiment discloses a method for preparing a Stipa breviflora seed germination promoter, comprising the following steps:

[0065] Step 1: Mix cow dung, wheat bran and rice milk evenly to obtain a basic mixture;

[0066] Step 2: Add PBS buffer to the basic mixture, adjust the pH to the enzyme activity range, then continue to add the composite hydrogel, stir evenly, add the immobilized enzyme, mix evenly, and ferment to obtain the Stipa breviflora seed germination promoter.

[0067] Example 5: This example discloses a method for preparing a composite hydrogel, comprising the following steps:

[0068] Q1: Add 2.9g sodium chloride to 220mL deionized water, stir to dissolve, then add 1.7g cellulose nanofibers, add 1.2g sodium periodate after ultrasonic dispersion for 30min, stir to dissolve for 2h, adjust pH to 4 with 0.1mol / L hydrochloric acid, 300rpm, 50℃, avoid light for 4h, filter with 0.45μm filter membrane, wash with deionized water, freeze-dry to obtain modified cellulose nanofibers;

[0069] Q2: 1.95 g of modified cellulose nanofibers were cut and added to 45 mL of deionized water. After high-speed stirring at 28,000 rpm for 30 min, a suspension was obtained. Then, 7.5 g of γ-polyglutamic acid with a purity of 92.2% and an average molecular weight of 1100 kDa was added and stirred for 45 min to dissolve. Then, 4.1 g of ethylene glycol diglycidol with an epoxy value of >0.65 was added. After adjusting the pH to 4.8 with 6 mol / L hydrochloric acid, the mixture was allowed to stand for 4 h and reacted at 55 °C and 90% RH for 10 h to obtain a composite hydrogel.

[0070] This embodiment discloses a method for preparing an immobilized enzyme, comprising the following steps:

[0071] S1: 0.047 g of triphenylaldehyde was added to 7.2 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 1. 0.048 g of p-phenylenediamine was added to 7 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 2.

[0072] S2: Add 0.04 g of complex enzyme (consisting of phytase, α-amylase and β-glucose) to 7.5 mL of solution 1, incubate for 8 min, then add solution 2 dropwise into solution 1 through a peristaltic pump, react with magnetic stirring at 1500 rpm for 45 min, centrifuge at 7000 rpm for 10 min, wash with methanol, and dry at 32°C for 8 h to obtain immobilized enzyme.

[0073] The present embodiment discloses a Stipa breviflora seed germination promoter, which is composed of the following ingredients in parts by weight: 50 parts of cow dung, 40 parts of wheat bran, 40 parts of rice milk, 8 parts of PBS buffer, 2.8 parts of composite hydrogel, and 9 parts of immobilized enzyme.

[0074] This embodiment discloses a method for preparing a Stipa breviflora seed germination promoter, comprising the following steps:

[0075] Step 1: Mix cow dung, wheat bran and rice milk evenly to obtain a basic mixture;

[0076] Step 2: Add PBS buffer to the basic mixture, adjust the pH to the enzyme activity range, then continue to add the composite hydrogel, stir evenly, add the immobilized enzyme, mix evenly, and ferment to obtain the Stipa breviflora seed germination promoter.

[0077] Example 6: This example discloses a method for preparing a composite hydrogel, comprising the following steps:

[0078] Q1: Add 2.7g sodium chloride to 260mL deionized water, stir to dissolve, then add 1.3g cellulose nanofibers, add 1.8g sodium periodate after ultrasonic dispersion for 30min, stir to dissolve for 2h, adjust pH to 4 with 0.1mol / L hydrochloric acid, 300rpm, 50℃ in dark for 4h, filter with 0.45μm filter membrane, wash with deionized water, freeze-dry to obtain modified cellulose nanofibers;

[0079] Q2: 1.17 g of modified cellulose nanofibers were cut and added to 52 mL of deionized water. After high-speed stirring at 28,000 rpm for 30 min, a suspension was obtained. Then, 6.4 g of γ-polyglutamic acid with a purity of 92.2% and an average molecular weight of 1100 kDa was added and stirred for 45 min to dissolve. Then, 3.1 g of ethylene glycol diglycidol with an epoxy value of >0.65 was added. After adjusting the pH to 4.8 with 6 mol / L hydrochloric acid, the mixture was allowed to stand for 4 h and reacted at 55 °C and 90% RH for 10 h to obtain a composite hydrogel.

[0080] This embodiment discloses a method for preparing an immobilized enzyme, comprising the following steps:

[0081] S1: 0.039 g of triphenylaldehyde was added to 6.5 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 1. 0.046 g of p-phenylenediamine was added to 6.8 mL of acetic acid buffer (pH = 4.5), and ultrasonic treatment was performed for 6 min to obtain solution 2.

[0082] S2: Add 0.035 g of complex enzyme (consisting of phytase, α-amylase and β-glucose) to 6.8 mL of solution 1, incubate for 8 min, then add solution 2 dropwise into solution 1 through a peristaltic pump, react with magnetic stirring at 1500 rpm for 45 min, centrifuge at 7000 rpm for 10 min, wash with methanol, and dry at 32°C for 8 h to obtain immobilized enzyme.

[0083] The present embodiment discloses a Stipa breviflora seed germination promoter, which is composed of the following ingredients in parts by weight: 53 parts of cow dung, 38 parts of wheat bran, 34 parts of rice milk, 6.5 parts of PBS buffer, 2.6 parts of composite hydrogel, and 8.5 parts of immobilized enzyme.

[0084] This embodiment discloses a method for preparing a Stipa breviflora seed germination promoter, comprising the following steps:

[0085] Step 1: Mix cow dung, wheat bran and rice milk evenly to obtain a basic mixture;

[0086] Step 2: Add PBS buffer to the basic mixture, adjust the pH to the enzyme activity range, then continue to add the composite hydrogel, stir evenly, add the immobilized enzyme, mix evenly, and ferment to obtain the Stipa breviflora seed germination promoter.

[0087] Comparative Example 1: Compared with Example 1, in the preparation process of the composite hydrogel in Comparative Example 1, no modified cellulose nanofibers are added, and other conditions remain unchanged.

[0088] Comparative Example 2: Compared with Example 1, in the preparation process of the immobilized enzyme in Comparative Example 2, no triphenylaldehyde was added, and other conditions remained unchanged.

[0089] Comparative Example 3: Compared with Example 1, in Comparative Example 3, during the preparation of the Stipa breviflora seed germination promoter, no composite hydrogel was added, and other conditions remained unchanged.

[0090] Comparative Example 4: Compared with Example 1, in Comparative Example 4, during the preparation of the Stipa breviflora seed germination promoter, no immobilized enzyme was added, and other conditions remained unchanged.

[0091] Experimental example: Select healthy, pest-free, and uniformly mature Stipa brevisflower seeds as test materials, divide the seeds into 11 groups on average, treat 10 groups with the prepared samples, and do not treat 1 group (blank group). Ensure that the temperature of the seeds is 15-25°C during the treatment process, observe and record the germination of the seeds regularly, calculate the germination rate, and measure the plant height. The test results are shown in Table 1:

[0092]

[0093] It can be seen from the test results in Table 1 that the Stipa breviflora seed germination promoter prepared in Examples 1-6 of the present invention can effectively improve the emergence rate and plant height of Stipa breviflora. By comparing Comparative Example 1 with Examples 1-6, it can be seen that the addition of modified cellulose nanofibers can effectively improve the emergence rate and plant height of Stipa breviflora; by comparing Comparative Example 2 with Examples 1-6, it can be seen that the addition of triphenylaldehyde can effectively improve the emergence rate and plant height of Stipa breviflora; by comparing Comparative Example 3 with Examples 1-6, it can be seen that the addition of composite hydrogel can effectively improve the emergence rate and plant height of Stipa breviflora; by comparing Comparative Example 4 with Examples 1-6, it can be seen that the addition of immobilized enzymes can improve the emergence rate and plant height of rice and effectively improve the emergence rate and plant height of Stipa breviflora.

[0094] 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.

[0095] 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 germination promoter for Stipa breviflora seeds, characterized in that: The invention is composed of the following ingredients in parts by weight: 42-55 parts of cow dung, 28-42 parts of wheat bran, 30-45 parts of rice milk, 5-9 parts of PBS buffer, 2-3 parts of composite hydrogel, and 6-10 parts of immobilized enzyme, wherein the composite hydrogel is prepared from sodium chloride, cellulose nanofiber, sodium periodate, γ-polyglutamic acid and ethylene glycol diglycidol, and the immobilized enzyme is prepared from mesitylene tribenzaldehyde, acetic acid buffer, p-phenylenediamine and composite enzyme. The preparation method of the composite hydrogel comprises the following steps: Q1: Sodium chloride is added to deionized water, stirred to dissolve, and then cellulose nanofibers are added. After ultrasonic dispersion, sodium periodate is added, stirred to dissolve, the pH value is adjusted, the mixture is protected from light, filtered, washed, and freeze-dried to obtain modified cellulose nanofibers. The dosage ratio of sodium chloride, deionized water, cellulose nanofibers, and sodium periodate is (1.3-3.5) g: (200-300) mL: (1-2) g: (1-2) g; Q2: The modified cellulose nanofibers were sheared and added to deionized water, and a suspension was obtained after high-speed stirring. Then, γ-polyglutamic acid was added and stirred to dissolve. Then, ethylene glycol diglycidol was added, and the pH was adjusted. The mixture was allowed to stand and react to obtain a composite hydrogel. The amount ratio of the modified cellulose nanofibers, deionized water, γ-polyglutamic acid and ethylene glycol diglycidol was (0.65-2.17) g: (40-60) mL: (6-8) g: (2.8-4.2) g. The average molecular weight of γ-polyglutamic acid was 1100 kDa. The method for preparing the immobilized enzyme comprises the following steps: S1: adding iso-tritylaldehyde to acetic acid buffer, and ultrasonically treating to obtain solution 1; adding p-phenylenediamine to acetic acid buffer, and ultrasonically treating to obtain solution 2; the amount ratio of iso-tritylaldehyde to acetic acid buffer is (0.032-0.05) g: (5-7.5) mL; the amount ratio of p-phenylenediamine to acetic acid buffer is (0.03-0.054) g: (5-8) mL; S2: Add the complex enzyme to solution 1, incubate, then add solution 2 dropwise into solution 1 through a peristaltic pump, react by magnetic stirring, centrifuge, wash, and dry to obtain immobilized enzyme. The dosage ratio of solution 1 and complex enzyme is (6-8) mL: (0.02-0.05) g. The complex enzyme consists of phytase, α-amylase and β-glucose enzyme.

2. The Stipa breviflora seed germination promoter according to claim 1, characterized in that In the Q1, the ultrasonic dispersion time is 10-30 min, the stirring and dissolving time after adding sodium periodate is 1-2 h, the pH is adjusted to 4-4.2 with 0.1 mol / L hydrochloric acid, the light-proof treatment time is 2-4 h, the stirring speed during the light-proof process is 200-300 rpm, the treatment temperature is 40-60°C, it is filtered with a 0.45 μm filter membrane, and washed with deionized water.

3. The Stipa breviflora seed germination promoter according to claim 1, characterized in that In the Q2, the high-speed stirring speed is 20000-28000rpm, the time is 30-45min, the purity is 92.2%, the stirring and dissolving time is 30-45min, the epoxy value of ethylene glycol diglycidol is greater than 0.65, the pH is adjusted to 4.8-5.2 with 6mol / L hydrochloric acid, the standing time is 2-4h, the reaction temperature is 50-55°C, the humidity is 80-90%RH, and the time is 8-10h.

4. The Stipa breviflora seed germination promoter according to claim 1, characterized in that In S1, the ultrasonic treatment time is 5-9 min, the pH of the acetic acid buffer is 4.5, and the ultrasonic treatment time is 5-7 min.

5. The Stipa breviflora seed germination promoter according to claim 1, characterized in that In the S2, the incubation time is 5-8 min, the magnetic stirring speed is 1000-1500 rpm, the reaction time is 30-45 min, the centrifugal speed is 5000-7000 rpm, the time is 10-12 min, washing is performed with methanol, the drying temperature is 30-32° C., and the time is 6-8 h.

6. The Stipa breviflora seed germination promoter according to claims 1-5, characterized in that: The preparation method of the Stipa breviflora seed germination promoter comprises the following steps: Step 1: Mix cow dung, wheat bran and rice milk evenly to obtain a basic mixture; Step 2: Add PBS buffer to the basic mixture, adjust the pH to the enzyme activity range, then continue to add the composite hydrogel, stir evenly, add the immobilized enzyme, mix evenly, and ferment to obtain the Stipa breviflora seed germination promoter.

7. Use of a Stipa breviflora seed germination promoter according to any one of claims 1 to 6, characterized in that: The obtained Stipa breviflora seed germination promoter was applied to the germination of Stipa breviflora seeds.

8. Use of a Stipa breviflora seed germination promoter according to any one of claims 1 to 6, characterized in that: The obtained Stipa breviflora seed germination promoter was applied to the growth of Stipa breviflora seeds.

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