CN111848297A - Flavonoid insect-proof antibacterial humic acid selenium fertilizer and preparation method thereof - Google Patents
Flavonoid insect-proof antibacterial humic acid selenium fertilizer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a flavonoid insect-proof antibacterial humic acid selenium fertilizer and a preparation method thereof, and relates to the field of agricultural fertilizer application. The flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises 1 part of humate, 0.01-0.4 part of natural plant flavonoid and 0.01-0.2 part of selenium-containing compound; the preparation method comprises the following steps: 1) weighing low-rank coal-based humate, natural plant flavonoids and selenium-containing compounds according to a proportion; 2) preparing the low-rank coal-based humate into a solution with the concentration of 2% -10%; 3) adding natural plant flavonoid substances into the low-order coal-based humate solution obtained in the step 2), and stirring to obtain a mixed solution of humate and flavonoid substances; 4) adding a selenium-containing compound into the mixed solution obtained in the step 3), controlling the temperature at 30-60 ℃, and mixing and stirring to obtain the flavonoid insect-proof antibacterial humic acid selenium fertilizer. The compound fertilizer prepared by the method is environment-friendly, and has the properties of soil restoration and improvement, insect prevention and antibiosis and crop quality improvement.
Description
Technical Field
The invention belongs to the field of agricultural fertilizer application, and particularly relates to a flavonoid insect-proof antibacterial humic acid selenium fertilizer and a preparation method thereof.
Background
Humic acid exists in nature in a large amount, has a complex structure, has various active functional groups on a molecular chain, can interact with a plurality of inorganic and organic compounds, and can play roles in improving the physical and chemical properties of soil, enhancing the fertilizer efficiency and the like. The flavonoid compounds are natural products widely existing in plants, play an important role in the aspects of plant growth and development, flowering and fruiting, bacteria prevention, disease prevention and the like, and have great medicinal values. Selenium is an essential trace element for human bodies, the selenium deficiency seriously harms the health of human beings, and due to the special function of selenium and the current situation that most of China is selenium deficiency, the selenium supplement from foods becomes a necessary way for obtaining selenium from human bodies, and selenium fertilizer is an effective means for improving the selenium content of foods.
At present, a series of problems of environmental pollution, land fertility reduction, low crop quality and the like are threatening human health due to excessive and unreasonable chemical fertilizer application, so that the invention is environment-friendly, and has important significance in repairing and improving soil, preventing insects and bacteria, improving crop quality and supplementing trace elements required by human bodies through plant acquisition.
Disclosure of Invention
The invention aims to provide a flavonoid insect-proof antibacterial humic acid selenium fertilizer and a preparation method thereof, so as to overcome the harm of the traditional fertilizer to soil and environment.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a flavonoid insect-proof antibacterial humic acid selenium fertilizer which comprises the following components in parts by weight: 1 part of humate, 0.01-0.4 part of natural plant flavonoid and 0.01-0.2 part of selenium-containing compound.
Preferably, the humate is one or more of ammonium humate, potassium humate and sodium humate.
Preferably, the natural plant flavonoid is extracted from a natural plant containing flavone, and the natural plant containing flavone is one or more of ginkgo, scutellaria baicalensis, kudzuvine root and orange peel.
Further preferably, the extraction method of the natural plant flavonoids substances comprises the following steps:
mixing natural plant containing flavone with pigment and lipid removed with solvent at solid-to-liquid ratio of 1g (1-50) ml, ultrasonic-assisted extracting at 30-70 deg.C for 0.5-3 hr, vacuum filtering, and separating and purifying filtrate to obtain natural plant flavonoid.
Further preferably, the solvent is ethanol, acetone or n-butanol.
Further preferably, the specific method for removing the pigment and lipid components from the natural plant containing flavone comprises the following steps: soaking pulverized natural plant containing flavone in petroleum ether for 12 hr, ultrasonic treating for 30min, vacuum filtering, and vacuum drying the filter residue at 40-60 deg.C.
Further preferably, the filtrate is separated and purified by a macroporous resin adsorption method, and the specific method for separating and purifying comprises the following steps:
and controlling the adsorption flow rate of filtrate with 10 times of column volume to be 4 times/h of resin column volume for adsorption, washing with 20% ethanol after complete adsorption, desorbing with 90% ethanol with 3 times of resin column volume and 2 times of resin column volume/h, fixing the volume after complete desorption, and concentrating the desorbed solution under reduced pressure to obtain the natural plant flavonoid substance.
Preferably, the selenium-containing compound is one or more of selenite, selenate, selenomethionine, selenocysteine and methylselenocysteine.
The invention also discloses a preparation method of the flavonoid insect-proof antibacterial humic acid selenium fertilizer, which comprises the following steps:
1) weighing humate, natural plant flavonoids and selenium-containing compounds according to a proportion;
2) preparing humate into an aqueous solution with the mass concentration of 2% -10%;
3) slowly adding natural plant flavonoid substances into the humate aqueous solution prepared in the step 2), and stirring to obtain a mixed solution of humate and natural plant flavonoid substances;
4) adding a selenium-containing compound into the mixed solution in the step 3), controlling the temperature at 30-60 ℃, and uniformly mixing and stirring to obtain the flavonoid insect-proof antibacterial humic acid selenium fertilizer.
Compared with the prior art, the invention has the following beneficial effects:
the application mixes humate with flavonoid substances and selenium-containing compounds in natural plants to prepare the flavonoid insect-proof antibacterial humic acid selenium fertilizer. Humic acid is an organic complexing agent and a chelating agent containing a plurality of coordination groups, can interact with a plurality of inorganic and organic compounds, and plays roles in improving the physical and chemical properties of soil, enhancing the fertilizer efficiency and the like. The natural flavonoid substances can prevent insects and bacteria in advance, improve the quality of crops and promote the growth of the crops; the trace element selenium can improve the health nutrition added value of crops. The application makes full use of the antibacterial action of flavonoids and the characteristic that humic acid can improve the physical and chemical properties of soil, achieves the purposes of repairing soil, improving soil fertility, preventing insects and resisting bacteria and the like, and promotes the photosynthesis of crops, so that the yield of the crops is increased, the selenium is enriched, and the quality of the crops is improved to some extent. The raw materials selected by the invention are green and environment-friendly, have no toxicity or harm to people and livestock, accord with the sustainable development of agriculture and the development direction of green agriculture, and have long-term social benefits. By carrying out plant fertilization verification and antibacterial detection on the flavonoid insect-proof antibacterial humic acid selenium fertilizer and the common humic acid fertilizer, compared with the common humic acid fertilizer, the flavonoid insect-proof antibacterial humic acid selenium fertilizer has the advantages that the chlorophyll content, the vitamin C content and the peroxidase activity in each growth stage are increased more obviously, the chlorophyll content in each growth stage is increased by 14.04-58.5%, the vitamin C content is increased by 4.86-15.71% and the peroxidase activity is increased by 4.26-14.78%; the flavonoid insect-proof antibacterial humic acid selenium fertilizer has more obvious reduction on the content of malondialdehyde in each growth stage, and the reduction of the content of malondialdehyde in each growth stage is 10.40-37.65%.
The application provides a preparation method of the flavonoid insect-proof antibacterial humic acid selenium fertilizer, which is simple and easy to operate, and the flavonoid insect-proof antibacterial humic acid selenium fertilizer prepared by the method is environment-friendly and has the properties of repairing and improving soil, preventing insects and resisting bacteria and improving crop quality.
Detailed Description
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
The flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following raw material components in parts by weight: the addition ratio of the sodium humate, the natural ginkgo flavonoids and the sodium selenite is 1:0.15:0.10 in parts by mass.
The preparation process of the flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following steps:
1) cleaning natural plant semen Ginkgo, drying, pulverizing, soaking in petroleum ether for 12 hr, ultrasonic treating for 30min, vacuum drying at 40 deg.C after suction filtration, and removing pigment and lipid components; carrying out ultrasonic assisted extraction on the treated plants at 30 ℃ for 3h by using acetone according to a solid-liquid ratio of 1g to 10ml, carrying out suction filtration to obtain an extraction filtrate, controlling the adsorption flow rate of the filtrate with 10 times of the column volume to be 4 times/h of the volume of the resin column, adsorbing by using macroporous adsorption resin, washing by using 20% absolute ethyl alcohol after complete adsorption, desorbing by using 90% ethyl alcohol with 3 times of the volume of the resin column and 2 times of the volume/h of the resin column at the flow rate, fixing the volume after complete desorption, and concentrating the desorption solution under reduced pressure to obtain a natural ginkgo flavonoid substance;
2) weighing low-order coal-based sodium humate, natural ginkgo flavonoids and sodium selenite according to a proportion;
3) preparing sodium humate into an aqueous solution with the concentration of 2%;
4) slowly adding the natural ginkgo flavonoids into the sodium humate solution obtained in the step 3), and fully stirring to obtain a mixed solution of the sodium humate and the natural ginkgo flavonoids;
5) adding sodium selenite into the mixed solution obtained in the step 4), controlling the temperature at 30 ℃, and uniformly mixing and stirring to obtain the flavonoid insect-proof antibacterial humic acid selenium fertilizer.
Example 2
The flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following raw material components in parts by weight: the addition ratio of the sodium humate, the natural scutellaria flavonoid and the sodium selenate is 1:0.25:0.12 in parts by mass.
The preparation process of the flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following steps:
1) cleaning natural plant Scutellariae radix, drying, pulverizing, soaking in petroleum ether for 12 hr, ultrasonic treating for 30min, vacuum drying at 60 deg.C after suction filtration to remove pigment and lipid components; carrying out ultrasonic-assisted extraction on the treated plants at 40 ℃ for 2.5h by using ethanol according to a solid-liquid ratio of 1g to 20ml, carrying out suction filtration to obtain an extraction filtrate, controlling the adsorption flow rate of the filtrate with 10 times of the column volume to be 4 times/h of the column volume of the resin, adsorbing the filtrate with macroporous adsorption resin, washing the filtrate with 20% absolute ethyl alcohol after complete adsorption, desorbing the filtrate with 90% ethanol with 3 times of the column volume of the resin and 2 times of the column volume/h of the resin, fixing the volume after complete desorption, and carrying out reduced pressure concentration on a desorption solution to obtain a natural scutellaria flavonoid substance;
2) weighing sodium humate, natural scutellaria flavonoids and sodium selenate according to the mass ratio;
3) preparing sodium humate into a 4% aqueous solution;
4) slowly adding the natural scutellaria flavonoids into the sodium humate aqueous solution obtained in the step 3), and fully stirring to obtain a mixed solution of the sodium humate and the natural scutellaria flavonoids;
5) adding sodium selenate into the mixed solution in the step 4), controlling the temperature at 60 ℃, and uniformly mixing and stirring to obtain the flavonoid insect-proof antibacterial humic acid selenium fertilizer.
Example 3
The flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following raw material components in parts by weight:
the addition ratio of the potassium humate, the natural pueraria flavonoid and the selenomethionine is 1:0.2:0.15 in parts by mass.
The preparation process of the flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following steps:
1) cleaning natural plant radix Puerariae, drying, pulverizing, soaking in petroleum ether for 12 hr, ultrasonic treating for 30min, vacuum drying at 45 deg.C after suction filtration, and removing pigment and lipid components; carrying out ultrasonic-assisted extraction on the treated plants at 60 ℃ for 2.5h by using n-butanol according to a solid-to-liquid ratio of 1g to 30ml, carrying out suction filtration to obtain an extraction filtrate, controlling the adsorption flow rate of the filtrate with 10 times of the column volume to be 4 times/h of the column volume of the resin, adsorbing the filtrate with macroporous adsorption resin, washing the filtrate with 20% absolute ethyl alcohol after complete adsorption, desorbing the filtrate with 90% ethanol with 3 times of the column volume of the resin and 2 times of the column volume/h of the resin, fixing the volume after complete desorption, and carrying out reduced pressure concentration on a desorption solution to obtain a natural pueraria flavonoid substance;
2) weighing potassium humate, natural pueraria flavonoid and selenomethionine according to a proportion;
3) preparing potassium humate into an aqueous solution with the concentration of 6%;
4) slowly adding the natural pueraria flavonoids into the potassium humate solution obtained in the step 3), and fully stirring to obtain a mixed solution of the potassium humate and the natural pueraria flavonoids;
5) adding selenomethionine into the mixed solution obtained in the step 4), controlling the temperature at 50 ℃, and uniformly mixing and stirring to obtain the flavonoid insect-proof antibacterial humic acid selenium fertilizer.
Example 4
The flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following raw material components in parts by weight: the addition ratio of the ammonium humate, the natural orange peel flavonoid and the selenocysteine is 1:0.3:0.08 in parts by mass.
The preparation process of the flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following steps:
1) cleaning natural plant pericarpium Citri Tangerinae, drying, pulverizing, soaking in petroleum ether for 12 hr, ultrasonic treating for 30min, vacuum drying at 50 deg.C after suction filtration, and removing pigment and lipid components; carrying out ultrasonic-assisted extraction on the treated plants at 50 ℃ for 2h by using ethanol according to a solid-liquid ratio of 1g to 40ml, carrying out suction filtration to obtain an extraction filtrate, controlling the adsorption flow rate of the filtrate with 10 times of the column volume to be 4 times/h of the volume of the resin column, adsorbing by using macroporous adsorption resin, washing by using 20% ethanol after complete adsorption, desorbing by using 90% ethanol with 3 times of the volume of the resin column and 2 times of the volume/h of the resin column, fixing the volume after complete desorption, and carrying out reduced pressure concentration on a desorption solution to obtain a natural orange peel flavonoid substance;
2) weighing ammonium humate, natural orange peel flavonoids and selenocysteine according to a proportion;
3) preparing ammonium humate into a water solution with the concentration of 5.5%;
4) slowly adding the flavonoid substances of the natural orange peel into the ammonium humate solution obtained in the step 3), and fully stirring to obtain a mixed solution of the ammonium humate and the flavonoid substances of the natural orange peel;
5) adding selenocysteine into the mixed solution in the step 4), controlling the temperature at 40 ℃, and uniformly mixing and stirring to prepare the flavonoid insect-proof antibacterial humic acid selenium fertilizer.
Example 5
The flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following raw material components in parts by weight: the addition ratio of the potassium humate, the natural orange peel flavonoid and the sodium selenite is 1:0.35:0.12 in parts by mass.
The preparation process of the flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following steps:
1) cleaning natural plant pericarpium Citri Tangerinae, drying, pulverizing, soaking in petroleum ether for 12 hr, ultrasonic treating for 30min, vacuum drying at 55 deg.C after suction filtration, and removing pigment and lipid components; carrying out ultrasonic-assisted extraction on the treated plants at 70 ℃ for 1.5h by using ethanol according to a solid-liquid ratio of 1g to 50ml, carrying out suction filtration to obtain an extraction filtrate, controlling the adsorption flow rate of the filtrate with 10 times of the column volume to be 4 times/h of the column volume of the resin, adsorbing the filtrate with macroporous adsorption resin, washing the filtrate with 20% absolute ethyl alcohol after complete adsorption, desorbing the filtrate with 90% ethanol with 3 times of the column volume of the resin and 2 times of the column volume/h of the resin, fixing the volume after complete desorption, and carrying out reduced pressure concentration on a desorption solution to obtain a natural orange peel flavonoid substance;
2) weighing potassium humate, natural orange peel flavonoids and sodium selenite according to a proportion;
3) preparing potassium humate into a 10% aqueous solution;
4) slowly adding the natural orange peel flavonoids into the potassium humate solution obtained in the step 3), and fully stirring to obtain a mixed solution of potassium humate and the natural orange peel flavonoids;
5) adding sodium selenite into the mixed solution obtained in the step 4), controlling the temperature at 45 ℃, and uniformly mixing and stirring to obtain the flavonoid insect-proof antibacterial humic acid selenium fertilizer.
Example 6
The flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following raw material components in parts by weight: the sodium humate, the natural ginkgo flavonoid and the methyl selenocysteine are added in a ratio of 1:0.4:0.2 in parts by mass.
The preparation process of the flavonoid insect-proof antibacterial humic acid selenium fertilizer comprises the following steps:
1) cleaning natural plant semen Ginkgo, drying, pulverizing, soaking in petroleum ether for 12 hr, ultrasonic treating for 30min, vacuum drying at 40 deg.C after suction filtration, and removing pigment and lipid components; carrying out ultrasonic-assisted extraction on the treated plants at 45 ℃ for 0.5h by using acetone according to a solid-liquid ratio of 1g to 25ml, carrying out suction filtration to obtain an extraction filtrate, controlling the adsorption flow rate of the filtrate with 10 times of the column volume to be 4 times/h of the column volume of the resin, adsorbing the filtrate with macroporous adsorption resin, washing the filtrate with 20% ethanol after complete adsorption, desorbing the filtrate with 90% ethanol with 3 times of the column volume of the resin and 2 times of the column volume/h of the resin, fixing the volume after complete desorption, and concentrating the desorption solution under reduced pressure to obtain a natural ginkgo flavonoid substance;
2) weighing sodium humate, natural ginkgo flavonoids and methyl selenocysteine according to a proportion;
3) preparing sodium humate into an aqueous solution with the concentration of 6%;
4) slowly adding the natural ginkgo biloba and scutellaria flavonoid substances into the sodium humate solution obtained in the step 3), and fully stirring to obtain a mixed solution of the sodium humate and the natural ginkgo biloba and scutellaria flavonoid substances;
5) adding methyl selenocysteine into the mixed solution in the step 4), controlling the temperature at 50 ℃, and uniformly mixing and stirring to obtain the flavonoid insect-proof antibacterial humic acid selenium fertilizer.
Potassium humate was prepared as a 10% aqueous solution as a comparative experiment (ordinary humic acid fertilizer).
The low-rank coal-based humate used in the examples is mineral humic acid extracted from low-rank coal (the lower the combustion value, the higher the humic acid content of the coal).
Plant fertilization verification and antibacterial detection are performed on the flavonoid insect-proof antibacterial humic acid selenium fertilizer and the common humic acid fertilizer prepared in the embodiments 1-6, and test results are as follows:
(1) influence of flavonoid insect-proof antibacterial humic acid selenium fertilizer on chlorophyll content of green vegetables
Chlorophyll is a substance essential for photosynthesis in plants. The chlorophyll content testing and calculating method in the application comprises the following steps: weighing 0.2g of green vegetable leaves, placing the green vegetable leaves in a mortar, and adding a little calcium carbonate. Adding 3mL of acetone, grinding uniformly, adding 2mL of acetone, grinding until the leaves turn white, standing for 3-5 minutes, and filtering the homogenate into a 25mL brown volumetric flask through a funnel. The mortar residue was washed with 5mL of acetone and transferred to a funnel for filtration. The leaves on the filter paper were discarded, and the chlorophyll remaining on the filter paper was rinsed in a brown volumetric flask with acetone, and the volume was adjusted to 25mL with acetone, and shaken up. Pure acetone solution is used as a control group, then the absorbance of the control group is respectively measured under the wavelengths of 665nm and 645nm, and the total content of chlorophyll is calculated.
Wherein: v is the total volume (mL) of the extracting solution; w is the fresh weight (g) of the plant sample.
The influence of the flavonoid insect-proof antibacterial humic acid selenium fertilizer and the common humic acid fertilizer prepared in the examples 1 to 6 on the chlorophyll content of the green vegetables is shown in table 1.
TABLE 1 Effect of the different examples on the chlorophyll content of green vegetables
As can be seen from Table 1, compared with the blank examples, the chlorophyll content of the examples 1-6 and the common humic acid fertilizer is increased in each growth stage, while the increase of the examples 1-6 is more obvious, and the increase of each stage is between 14.04% and 58.5%.
(2) Influence of flavonoid insect-proof antibacterial humic acid selenium fertilizer on vitamin C content of green vegetables
Vitamin C is an indispensable organic compound for the human body and also an essential element for life. The vitamin C content testing and calculating method in the application comprises the following steps:
the determination of the vitamin C content adopts the titration determination of the oxidized 2, 6-dichlorophenol indophenol under the acidic condition, and the titration end point is the moment when the solution is changed from colorless to pale red. Firstly, titrating a certain volume of standard Vc solution (0.1mg/mL) with 2, 6-dichlorophenol indophenol until the solution is pink and no color disappears within 15s, then taking 2% oxalic acid with the same amount as the solution as a blank control, titrating according to the method, finally weighing 1g of green vegetables, shredding, adding 5mL of 2% oxalic acid solution, grinding into slurry, transferring the slurry into a centrifuge tube, simultaneously cleaning a mortar with a little 2% oxalic acid solution, combining the slurry with liquid, centrifuging at a rotating speed of 8000r/min for 5min, transferring supernatant into a volumetric flask for constant volume, quickly titrating a certain amount of liquid with 2, 6-dichlorophenol indophenol until the liquid is pink, and determining VcAnd (4) content.
In the formula:
v is the amount (mL) of 2, 6-dichlorophenol indophenol used for titrating the sample;
V1the total volume (mL) of the sample after constant volume;
V2is the volume (mL) of the sample to be measured;
l is the content of Vc oxidized per 1mL of 2, 6-dichlorophenol indophenol solution (mg/mL) which was tested using this test method.
The influence of the flavonoid insect-proof antibacterial humic acid selenium fertilizer and the common humic acid fertilizer prepared in the embodiments 1-6 on the vitamin C content of the green vegetables is shown in table 2.
TABLE 2 Effect of the different examples on the vitamin C content of green vegetables
As can be seen from Table 2, compared with the blank examples, the vitamin C content is increased in each growth stage in examples 1-6 and the common humic acid fertilizer, and the increase of the vitamin C content in examples 1-6 is more obvious, and the increase of the vitamin C content in each stage in examples 1-6 is between 4.86% and 15.71%.
(3) Influence of flavonoid insect-proof antibacterial humic acid selenium fertilizer on malondialdehyde content of green vegetables
Malondialdehyde is one of the most important products of membrane lipid peroxidation, and its production can exacerbate membrane damage. The method for testing and calculating the content of the malonaldehyde in the application comprises the following steps:
weighing 10g of trichloroacetic acid (TCA), and fixing the volume to a 100mL volumetric flask to prepare a 10% (TCA) solution. Weighing 0.125g of thiobarbituric acid (TBA), and making a constant volume into a 25mL volumetric flask to prepare a 0.6% (TBA) solution. Weighing 0.5g of green vegetable leaves, putting the green vegetable leaves into a mortar, adding 3mL of 10% TCA, grinding and homogenizing, adding 2mL of 10% TCA, continuing grinding, centrifuging at the rotating speed of 4000r/min for 15min (4 ℃), and obtaining the supernatant which is the MDA extracting solution. Weighing 2mL of the extractive solution (volume V of the extractive solution for determination)s) Adding 2ml of 0.6 percent TBA () into a test tube with a plug, uniformly mixing, reacting in a boiling water bath for 20min, immediately transferring into ice water, rapidly cooling, centrifuging under the condition of 4000r/min, detecting the absorbance values of obtained supernatant at 532nm, 600nm and 450nm, and calculating the MDA content. The control group was 2mL of water plus 2mL of 0.6% TBA.
Wherein: v is the volume of the reaction solution (5 ml); vsFor the determination of the volume of the extract (2 ml); vtThe total volume of the extractive solution (5 ml); w: fresh weight of plant sample (0.5 g).
The influence of the flavonoid insect-proof antibacterial humic acid selenium fertilizer and the common humic acid fertilizer prepared in the embodiments 1 to 6 on the content of malondialdehyde in green vegetables is shown in table 3.
TABLE 3 Effect of the different examples on the malondialdehyde content of the vegetables
As can be seen from Table 3, compared with the blank examples, the malonaldehyde content in the examples 1-6 and the common humic acid fertilizer is reduced in each growth stage, but the reduction range of the examples 1-6 is more obvious, and the reduction range of the examples 1-6 in each stage is between 10.40% and 37.65%.
(4) Influence of flavonoid insect-proof antibacterial humic acid selenium fertilizer on activity of vegetable peroxidase
Peroxidase is an important oxidoreductase in plants, is related to photosynthesis, respiration, auxin oxidation and the like, and can ensure the permeability of cell membranes so that the plants can be prevented from being damaged by active oxygen and grow vigorously. The methods for testing and calculating peroxidase activity (POD) in the present application are:
the 100mmol/L phosphate buffer solution with pH of 6.0 is prepared by blending sodium dihydrogen phosphate and disodium hydrogen phosphate. 50mL of the above solution was placed in a beaker, 28. mu.L of guaiac wood powder was added, 19. mu.L of 30% hydrogen peroxide solution was added, and the mixture was mixed well and stored in a refrigerator as a reaction mixture for further use. Weighing 0.25g of well-grown green vegetable leaves, putting the green vegetable leaves into a mortar, adding 5mL of phosphate buffer solution with the pH value of 6.0, grinding the mixture into homogenate, centrifuging the homogenate for 15min (4 ℃) at the rotating speed of 7000r/min, transferring the supernatant into a 25mL volumetric flask, fixing the volume to 25mL by using the phosphate buffer solution, and storing the supernatant at low temperature for later use. 3mL of the reaction mixture was added to 1mL of the above extract, and the mixture was placed in a cuvette, and a stopwatch was immediately started to record the reaction time, and the absorbance at 470nm was measured and read every one minute to calculate the POD activity. The control group consisted of 3mL reaction mixture and 1mL phosphate buffer.
Wherein: delta A470Is the difference in absorbance; Δ t is the time (min) corresponding to the absorbance difference; v1: total volume of extract (25 ml); v2Volume of extract solution for measurement (1 ml): w: fresh weight of plant sample (0.25 g).
The influence of the flavonoid insect-proof antibacterial humic acid selenium fertilizer and the common humic acid fertilizer prepared in the examples 1 to 6 on the activity of the peroxidase of the green vegetables is shown in table 4.
TABLE 4 Effect of the different examples on peroxidase Activity
As can be seen from Table 4, compared with the blank examples, the peroxidase activities of examples 1 to 6 and the common humic acid fertilizer are increased in each growth stage, the increase of examples 1 to 6 is more obvious, and the increase of examples 1 to 6 in each growth stage is between 4.26% and 14.78%.
(5) Test data of antibacterial experiment of flavonoid insect-proof antibacterial humic acid selenium fertilizer
The method for testing and calculating the antibacterial property comprises the following steps:
circular Xinhua filter paper with the diameter of 6mm is subjected to dry heat sterilization (160 ℃ for 2 hours) by adopting a filter paper sheet method for later use. The sterilized paper sheets are put into flavonoid insect-proof antibacterial humic acid selenium fertilizer or common humic acid fertilizer to be fully soaked, taken out and spread in a sterile plate, and naturally volatilized to be used.
Inoculating the slant preserving strain to a beef extract peptone agar culture medium, placing the medium in a constant temperature incubator at 37 ℃, and culturing for 24 h. Taking activated strain, and preparing into 10 with sterile normal saline5cfu/mL of bacterial suspension.
0.2mL of the prepared bacterial suspension is taken, dripped into the surface of a solid culture medium, uniformly coated on the surface of the solid culture medium by using a sterile coater, and a filter paper sheet soaked with the pigment solution and volatilized from the flavonoid insect-proof antibacterial humic acid selenium fertilizer or the common humic acid fertilizer is clamped by using a sterile forceps and is put into a flat dish. A blank was prepared by evaporating the solution with distilled water. 3 replicates were made for each sample. And (3) putting the flat plate into a bacteria incubator, culturing at 37 ℃ for 18h, taking out, observing, measuring the diameter of the inhibition zone, and taking an average value.
The antibacterial properties of the flavonoid insect-proofing antibacterial humic acid selenium fertilizer and the common humic acid fertilizer prepared in the embodiments 1 to 6 are shown in table 5.
TABLE 5 antibacterial property test of various examples
The experimental data show that compared with the blank case and the common humic acid fertilizer, the examples 1-6 have remarkable antibacterial capability on staphylococcus aureus and escherichia coli, and the natural plant flavonoid, the selenium-containing compound and the humic acid are synergistic, so that the growth of crops and the improvement of the quality of the plants are facilitated.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (9)
1. The flavonoid insect-proof antibacterial humic acid selenium fertilizer is characterized by comprising the following components in parts by mass: 1 part of humate, 0.01-0.4 part of natural plant flavonoid and 0.01-0.2 part of selenium-containing compound.
2. An insect-proofing and antibacterial flavonoid humic acid selenium fertilizer according to claim 1, wherein the humate is one or more of ammonium humate, potassium humate and sodium humate.
3. The flavonoid insect-proof antibacterial humic acid selenium fertilizer as claimed in claim 1, wherein the natural plant flavonoid is extracted from natural plants containing flavone, and the natural plants containing flavone are one or more of ginkgo biloba, scutellaria baicalensis, pueraria lobata and orange peel.
4. The flavonoid insect-proof antibacterial humic acid selenium fertilizer as claimed in claim 3, wherein the extraction method of the natural plant flavonoids comprises the following steps:
mixing natural plant containing flavone with pigment and lipid removed with solvent at solid-to-liquid ratio of 1g (1-50) ml, ultrasonic-assisted extracting at 30-70 deg.C for 0.5-3 hr, vacuum filtering, and separating and purifying filtrate to obtain natural plant flavonoid.
5. The flavonoid insect-proof antibacterial humic acid selenium fertilizer as claimed in claim 4, wherein the solvent is ethanol, acetone or n-butanol.
6. The flavonoid insect-proof antibacterial humic acid selenium fertilizer as claimed in claim 4, wherein the specific method for removing pigment and lipid components from the natural plant containing flavone comprises the following steps: soaking pulverized natural plant containing flavone in petroleum ether for 12 hr, ultrasonic treating for 30min, vacuum filtering, and vacuum drying the filter residue at 40-60 deg.C.
7. The flavonoid insect-proof antibacterial humic acid selenium fertilizer as claimed in claim 4, wherein the filtrate is separated and purified by macroporous resin adsorption method, and the specific method for separation and purification is as follows:
and controlling the adsorption flow rate of filtrate with 10 times of column volume to be 4 times/h of resin column volume for adsorption, washing with 20% ethanol after complete adsorption, desorbing with 90% ethanol with 3 times of resin column volume and 2 times of resin column volume/h, fixing the volume after complete desorption, and concentrating the desorbed solution under reduced pressure to obtain the natural plant flavonoid substance.
8. The flavonoid insect-resistant antibacterial humic acid selenium fertilizer as claimed in claim 1, wherein the selenium-containing compound is one or more of selenite, selenate, selenomethionine, selenocysteine and methyl selenocysteine.
9. The preparation method of the flavonoid insect-proof antibacterial humic acid selenium fertilizer as claimed in any one of claims 1 to 8, which is characterized by comprising the following steps:
1) weighing humate, natural plant flavonoids and selenium-containing compounds according to a proportion;
2) preparing humate into an aqueous solution with the mass concentration of 2% -10%;
3) slowly adding natural plant flavonoid substances into the humate aqueous solution prepared in the step 2), and stirring to obtain a mixed solution of humate and natural plant flavonoid substances;
4) adding a selenium-containing compound into the mixed solution in the step 3), controlling the temperature at 30-60 ℃, and uniformly mixing and stirring to obtain the flavonoid insect-proof antibacterial humic acid selenium fertilizer.
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