Disclosure of Invention
In order to solve the problems mentioned in the background art, the invention provides a pesticide composition based on marine bioactive substances and a preparation method thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The pesticide composition based on marine bioactive substances comprises, by weight, 20-25% of sulfonated brown algae polyphenol microcapsules, 8-12% of sodium alginate, 0.1-1% of phosphatidylcholine, 2-3% of alkylphenol ethoxylates, 1-3% of fatty alcohol ethoxylates, 0.5-2% of azone, 2-5% of propylene glycol, 0.2-1% of organosilicon defoamer and the balance of water.
Further, the sulfonated brown algae polyphenol microcapsule is prepared by the following steps:
S1, crushing Sargassum horneri, sieving, adding ethanol and phytic acid, performing ultrasonic treatment, centrifuging at 8000rpm for 10-15min, collecting supernatant, and concentrating by rotary evaporation to 1/10 of the original volume to obtain brown algae polyphenol extract;
s2, mixing the brown algae polyphenol extract and 3 '-adenosine phosphate-5' -phosphorus acyl sulfuric acid, controlling the temperature to stir and react at constant temperature in a phosphate buffer reaction environment with the pH value of 7.5, centrifuging in a water bath at the speed of 8000rpm for 10-15min after the reaction is finished, and collecting supernatant to obtain a sulfonated brown algae polyphenol solution;
s3, adding the chitooligosaccharide solution into the primary emulsion, dropwise adding the sulfonated brown algae polyphenol solution, adding sodium tripolyphosphate, controlling the temperature, magnetically stirring, centrifuging at 8000rpm for 10-15min, collecting precipitate, washing, vacuum drying for 48h, and sieving with 200-400 meshes to obtain the sulfonated brown algae polyphenol microcapsule.
Further, in the step S1, the mass ratio of the Sargassum horneri to the ethanol to the phytic acid is (1-1.05): (16-19): (0.005-0.006), the power of ultrasonic treatment is 300-400W, the power is 50-55kHz, the temperature during ultrasonic treatment is 50-55 ℃, and the time during ultrasonic treatment is 20-30min.
Further, in the step S2, the mass ratio of the brown algae polyphenol extract to the 3 '-adenosine-5' -phosphoryl sulfuric acid is (7-8) (0.95-1.05), the control temperature is 60-65 ℃, the stirring speed is 60-100rpm, the reaction time is 8-10 hours, the water bath temperature is 90-95 ℃, and the water bath time is 10-20 minutes.
Further, in the step S3, the mass ratio of the chitooligosaccharide solution, the primary emulsion, the sulfonated brown algae polyphenol solution and the sodium tripolyphosphate is 1 (20-22) (0.4-0.5) (0.2-0.3), the concentration of the chitooligosaccharide solution is 1.0-1.3% (w/v), the solvent is 1% acetic acid solution with pH value=5.0, the temperature is controlled to be 40 ℃, the magnetic stirring speed is 300-400rpm, and the stirring time is 2-3 hours.
Further, the primary emulsion in step S3 is prepared by the steps of:
a1, adding corn oil and vitamin E acetate into a container, heating in a water bath until the corn oil and the vitamin E acetate are completely melted, adding Tween 80 and polyglycerol polyricinoleate, and magnetically stirring until the corn oil and the vitamin E acetate are uniform and transparent to obtain an oil phase;
A2, adding sodium hyaluronate into deionized water, performing ultrasonic treatment, adding glycerol, magnetically stirring until the solution is clear, and controlling the pH value to be 5.5-6.0 to obtain a water phase;
And A3, slowly pouring the water phase into the oil phase, mixing, homogenizing and refining, transferring the emulsion into a water bath, magnetically stirring, naturally cooling to below 30 ℃, and regulating the pH value to 5.5-6.0 by using citric acid or triethanolamine to obtain the primary emulsion.
Further, in the step A1, the mass ratio of the corn oil to the vitamin E acetate to the Tween 80 to the polyglycerol polyricinoleate is (182-186), 1-1.1, 6-7, 0.09-0.11, the temperature of the water bath is 60-65 ℃, and the magnetic stirring speed is 500-600rpm.
Further, in the step A2, the mass ratio of deionized water, sodium hyaluronate and glycerin is (128-130) (1.3-1.5) (0.09-0.1), the temperature during ultrasonic treatment is 40-45 ℃, the power of ultrasonic treatment is 200-300W, the power is 40-45kHz, the time during ultrasonic treatment is 10-20min, and the magnetic stirring speed is 300-400rpm.
Further, the temperature of mixing in the step A3 is 70-75 ℃, the homogenizing speed is 5000-6000rpm, the homogenizing time is 3-5min, the temperature of the water bath is 45-47 ℃, and the magnetic stirring speed is 100-200rpm.
According to another aspect of the present invention, there is provided a method for preparing the above pesticidal composition, comprising the steps of:
Adding water into a reaction kettle according to weight percentage, heating to 50-55 ℃, adding sodium alginate, stirring at a speed of 100-200rpm until the sodium alginate is completely dissolved, adding sulfonated brown algae polyphenol microcapsules, phosphatidylcholine, alkylphenol ethoxylates and fatty alcohol ethoxylates, stirring at a speed of 300-400rpm for 10-20min, adding azone and organosilicon defoamer, and shearing and emulsifying at 3000-4000rpm for 15-25min to obtain the pesticide composition based on marine bioactive substances.
The invention has the beneficial effects that:
1. in the technical scheme of the invention, the 3 '-adenosine phosphate-5' -phosphosulfate (PAPS) sulfonated modified brown algae polyphenol strengthens the interaction between the brown algae polyphenol and the pathogenic bacteria cell membrane through charge density regulation and active site exposure. Electrostatic repulsion of the sulfonic acid groups with the membrane phospholipid heads disrupts membrane integrity, while insertion of hydrophobic aromatic rings into the membrane bilayer initiates perforation. In addition, the degradation products of the chitooligosaccharide interfere the formation of the biological film of pathogenic bacteria and form a multi-target antibacterial synergistic effect with sulfonated brown algae polyphenol.
2. In the technical scheme of the invention, the core-shell heterostructure of the sulfonated brown algae polyphenol microcapsule constructs a compact interface layer through the electrostatic action of the chitosan oligosaccharide and the sodium tripolyphosphate and the bonding of the sulfonic group and the hydroxyl group, so as to form a physical barrier. Not only blocking the external environment such as ultraviolet rays, oxidative free radicals and the like, damaging the inner core polyphenol, but also obviously delaying the oxidative degradation of the brown algae polyphenol through the amino free radical scavenging capability of the chitooligosaccharide and the metal chelation, and improving the stability and the utilization efficiency of the active ingredients, thereby improving the durability and the effect of the pesticide.
3. The pesticide composition prepared by the invention has good environmental compatibility, and the microcapsule shell layer is composed of the chitooligosaccharide and the sodium tripolyphosphate, so that the microcapsule shell layer can be degraded into non-toxic products by soil microorganisms in natural environment, thereby avoiding accumulation and pollution of chemical pesticides in the environment and being beneficial to reducing the influence on the environment.
4. In the technical scheme of the invention, the sulfonated brown algae polyphenol microcapsule has a certain slow release effect. Under the alkaline environment of plant leaf surface, the shell ion crosslinking network dissociates to release active component, while under the neutral or weak acid storage condition, the crosslinking structure is stable to realize long-acting slow release. Meanwhile, chitinase secreted by plant pathogenic bacteria can specifically hydrolyze beta-1, 4 glycosidic bonds of chitosan oligosaccharide, so that sulfonated brown algae polyphenol precisely acts on a pathogenic bacteria enrichment area, the control efficiency is improved, and the application frequency is reduced.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specified, the raw materials used in the present invention are all derived from conventional products purchased in the market.
Preparation example 1
The primary emulsion is prepared by the following steps:
A1, adding 182g of corn oil and 1g of vitamin E acetate into a container, heating in a water bath at 60 ℃ until the mixture is completely melted, adding 6g of Tween 80 and 0.09g of polyglycerol polyricinoleate, and magnetically stirring at a speed of 500rpm until the mixture is uniform and transparent to obtain an oil phase;
A2, adding 1.3g of sodium hyaluronate into 128g of deionized water, carrying out ultrasonic treatment at 40 ℃ for 10min, wherein the ultrasonic treatment power is 200W, the ultrasonic treatment power is 40kHz, adding 0.09g of glycerol, magnetically stirring at a speed of 300-400rpm until the solution is clear, and controlling the pH value to be 5.5 to obtain a water phase;
and A3, slowly pouring the water phase into the oil phase, mixing at 70 ℃, homogenizing and refining for 3min at a speed of 5000rpm, transferring the emulsion into a water bath at 45 ℃, magnetically stirring at a speed of 100rpm, naturally cooling to 25 ℃, and regulating the pH value to 5.5 by using citric acid to obtain the primary emulsion.
The sulfonated brown algae polyphenol microcapsule is prepared by the following steps:
S1, crushing and sieving 10g of Sargassum horneri, adding 160g of ethanol and 0.05g of phytic acid, performing ultrasonic treatment at 50 ℃ for 20min, wherein the power of ultrasonic treatment is 300W, the power is 50kHz, centrifuging at 8000rpm for 10min, collecting supernatant, and performing rotary evaporation and concentration to 1/10 of the original volume to obtain brown algae polyphenol extract;
S2, mixing 70g of brown algae polyphenol extract and 9.5g of 3 '-adenosine phosphate-5' -phosphorylsulfuric acid, stirring at a constant temperature of 60 ℃ for reaction for 8 hours at a speed of 60rpm in a phosphate buffer reaction environment with a pH value of 7.5, carrying out water bath at 90 ℃ for 10 minutes after the reaction is finished, and centrifuging at a speed of 8000rpm for 10 minutes to collect supernatant to obtain a sulfonated brown algae polyphenol solution;
S3, dissolving 0.1g of chitosan oligosaccharide powder in 9.9g of 1% acetic acid solution with pH=5.0 to prepare chitosan oligosaccharide solution, adding 10g of chitosan oligosaccharide solution into 200g of the prepared primary emulsion, dropwise adding 4g of sulfonated brown algae polyphenol solution, adding 2g of sodium tripolyphosphate, controlling the temperature to be 40 ℃, magnetically stirring at a speed of 300rpm for 2 hours, centrifuging at a speed of 8000rpm for 10 minutes to collect precipitate, washing, vacuum drying for 48 hours, sieving with 200 meshes to obtain sulfonated brown algae polyphenol microcapsules.
Preparation example 2
The primary emulsion is prepared by the following steps:
A1, adding 184g of corn oil and 1.05g of vitamin E acetate into a container, heating in a water bath at 62 ℃ until the corn oil and the vitamin E acetate are completely melted, adding 6.5g of Tween 80 and 0.1g of polyglycerol polyricinoleate, and magnetically stirring at a speed of 550rpm until the corn oil and the vitamin E acetate are uniform and transparent to obtain an oil phase;
a2, adding 1.4g of sodium hyaluronate into 129g of deionized water, carrying out ultrasonic treatment at 42 ℃ for 15min, wherein the ultrasonic treatment power is 250W, the ultrasonic treatment power is 42kHz, adding 0.095g of glycerol, magnetically stirring at a speed of 350rpm until the solution is clear, and controlling the pH value to be 5.7 to obtain a water phase;
and A3, slowly pouring the water phase into the oil phase, mixing at 72 ℃, homogenizing and refining for 4min at the speed of 5500rpm, transferring the emulsion into a 46 ℃ water bath, magnetically stirring at the speed of 150rpm, naturally cooling to 20 ℃, and regulating the pH value to 5.8 by using citric acid to obtain the primary emulsion.
The sulfonated brown algae polyphenol microcapsule is prepared by the following steps:
s1, crushing 10.2g of Sargassum horneri, sieving, adding 180g of ethanol and 0.055g of phytic acid, performing ultrasonic treatment at 52 ℃ for 25min, performing ultrasonic treatment with power of 350W and power of 52kHz, centrifuging at 8000rpm for 12min, collecting supernatant, and performing rotary evaporation concentration to 1/10 of the original volume to obtain brown algae polyphenol extract;
s2, mixing 74g of brown algae polyphenol extract and 10g of 3 '-adenosine phosphate-5' -phosphorylsulfuric acid, stirring at a constant temperature of 62 ℃ at a speed of 80rpm for reaction for 9 hours in a phosphate buffer reaction environment with a pH value of 7.5, carrying out water bath at 92 ℃ for 15 minutes after the reaction is finished, and centrifuging at a speed of 8000rpm for 12 minutes to collect supernatant to obtain a sulfonated brown algae polyphenol solution;
S3, dissolving 0.12g of chitosan oligosaccharide powder in 9.88g of 1% acetic acid solution with pH=5.0 to prepare chitosan oligosaccharide solution, adding 10g of chitosan oligosaccharide solution into 210g of the prepared primary emulsion, dropwise adding 4.5g of sulfonated brown algae polyphenol solution, adding 2.3g of sodium tripolyphosphate, controlling the temperature to be 40 ℃, magnetically stirring at a speed of 350rpm for 2.5h, centrifuging at a speed of 8000rpm for 12min to collect precipitate, washing, vacuum drying for 48h, and sieving through 300 meshes to obtain sulfonated brown algae polyphenol microcapsules.
Preparation example 3
The primary emulsion is prepared by the following steps:
A1, adding 186g of corn oil and 1.1g of vitamin E acetate into a container, heating in a water bath at 65 ℃ until the corn oil and the vitamin E acetate are completely melted, adding 7g of Tween 80 and 0.11g of polyglycerol polyricinoleate, and magnetically stirring at a speed of 600rpm until the corn oil and the vitamin E acetate are uniform and transparent to obtain an oil phase;
A2, adding 1.5g of sodium hyaluronate into 130g of deionized water, carrying out ultrasonic treatment at 45 ℃ for 20min, wherein the ultrasonic treatment power is 300W, the ultrasonic treatment power is 45kHz, adding 0.1g of glycerol, magnetically stirring at 400rpm until the solution is clear, and controlling the pH value to be 6.0 to obtain a water phase;
And A3, slowly pouring the water phase into the oil phase, mixing at 75 ℃, homogenizing and refining for 5min at 6000rpm, transferring the emulsion into a 47 ℃ water bath, magnetically stirring at 200rpm, naturally cooling to 25 ℃, and regulating the pH value to 6.0 by using citric acid to obtain the primary emulsion.
The sulfonated brown algae polyphenol microcapsule is prepared by the following steps:
S1, crushing 10.5g of Sargassum horneri, sieving, adding 190g of ethanol and 0.06g of phytic acid, performing ultrasonic treatment at 55 ℃ for 30min, wherein the power of ultrasonic treatment is 400W, the power is 55kHz, centrifuging at 8000rpm for 15min, collecting supernatant, and performing rotary evaporation concentration to 1/10 of the original volume to obtain brown algae polyphenol extract;
S2, mixing 80g of brown algae polyphenol extract and 10.5g of 3 '-adenosine phosphate-5' -phosphorylsulfuric acid, stirring at a constant temperature of 65 ℃ for reaction for 10 hours at a speed of 100rpm in a phosphate buffer reaction environment with a pH value of 7.5, carrying out water bath at 95 ℃ for 20 minutes after the reaction is finished, and centrifuging at a speed of 8000rpm for 15 minutes to collect supernatant to obtain a sulfonated brown algae polyphenol solution;
s3, dissolving 0.13g of chitosan oligosaccharide powder in 9.87g of 1% acetic acid solution with pH=5.0 to prepare chitosan oligosaccharide solution, adding 10g of chitosan oligosaccharide solution into 220g of the prepared primary emulsion, dropwise adding 5g of sulfonated brown algae polyphenol solution, adding 3g of sodium tripolyphosphate, controlling the temperature to be 40 ℃, magnetically stirring for 3 hours at a speed of 400rpm, centrifuging for 15 minutes at a speed of 8000rpm, collecting precipitate, washing, vacuum drying for 48 hours, sieving through 400 meshes, and obtaining sulfonated brown algae polyphenol microcapsules.
Example 1
A method for preparing a pesticide composition based on marine bioactive substances, comprising the steps of:
59.5g of water is added into a reaction kettle according to the weight percentage, 8g of sodium alginate is added after the temperature is raised to 50 ℃, the mixture is stirred at the speed of 100rpm until the mixture is completely dissolved, 20g of the sulfonated brown algae polyphenol microcapsule prepared in preparation example 1, 0.5g of phosphatidylcholine, 3g of alkylphenol ethoxylate and 1g of fatty alcohol ethoxylate are added, the mixture is stirred at the speed of 300rpm for 10min, 2g of azone and 1g of organosilicon defoamer are added, and the mixture is sheared and emulsified at the speed of 3000rpm for 15min, so that the pesticide composition based on marine bioactive substances is obtained.
Example 2
A method for preparing a pesticide composition based on marine bioactive substances, comprising the steps of:
53.7g of water, 12g of sodium alginate and 25g of sulfonated brown algae polyphenol microcapsule prepared in preparation example 2, 0.3g of phosphatidylcholine, 2g of alkylphenol ethoxylate and 3g of fatty alcohol ethoxylate are added into a reaction kettle according to weight percentage, the mixture is heated to 52 ℃, then stirred at a speed of 150rpm until the mixture is completely dissolved, stirred at a speed of 350rpm for 15min, 0.5g of azone and 0.5g of organosilicon defoamer are added, and shearing and emulsifying at 3500rpm for 20min, so that the pesticide composition based on marine bioactive substances is obtained.
Example 3
A method for preparing a pesticide composition based on marine bioactive substances, comprising the steps of:
59.8g of water is added into a reaction kettle according to the weight percentage, 10g of sodium alginate is added after the temperature is raised to 50-55 ℃, stirring is carried out at the speed of 200rpm until the sodium alginate is completely dissolved, 22g of the sulfonated brown algae polyphenol microcapsule prepared in preparation example 3, 1g of phosphatidylcholine, 2g of alkylphenol ethoxylate and 2g of fatty alcohol ethoxylate are added, stirring is carried out at the speed of 400rpm for 20min, 1g of azone and 0.2g of organosilicon defoamer are added, and shearing and emulsifying are carried out at the speed of 4000rpm for 25min, thus obtaining the pesticide composition based on marine bioactive substances.
Comparative example 1
This comparative example is different from example 1 in that the sulfonated brown algae polyphenol microcapsule prepared in preparation example 1 is replaced with brown algae polyphenol, and the remaining steps are the same as those of example 1.
Comparative example 2
This comparative example is different from example 2 in that the sulfonated brown algae polyphenol microcapsule prepared in preparation example 2 is replaced with chitooligosaccharide, and the remaining steps are the same as in example 2.
Comparative example 3
This comparative example is different from example 3 in that sodium tripolyphosphate is used instead of the sulfonated brown algae polyphenol microcapsule prepared in preparation example 3, and the rest of the procedure is the same as that of example 3.
(I) Indoor toxicity measurement:
Collecting larva of Plutella xylostella of 3 years old, weighing about 0.5-1.0 mg/head, and starving for 4 hr.
The pesticide compositions prepared in examples 1 to 3 and comparative examples 1 to 3 were respectively prepared into 2% pesticide solutions to be tested according to mass fractions, distilled water containing 0.05% Triton X-100 was used as a solvent, and a blank control group of 0.05% Triton X-100 solution and a commercially available positive control group of 5% avermectin emulsifiable concentrate were additionally provided.
Fresh cabbage leaves are cut into 3X 5 cm leaf discs, the leaf discs are immersed in pesticide solutions for 10s and naturally dried, the leaf discs are placed in culture dishes with the diameter of 9 cm, filter paper is padded at the bottom to moisten the leaf discs, 20 plutella xylostella larvae are inoculated into each dish for culture, the culture condition is that the temperature is 26+/-1 ℃, the relative humidity is 70%, the light-dark ratio is 14:10h, the leaf discs cannot be crawled normally or cannot be completely moved, and 3 repetitions are set. After 24h, 48h, 72h, corrected mortality was calculated, corrected mortality (%) = (treatment mortality-control mortality)/(1-control mortality) ×100. The results are shown in Table 1:
TABLE 1 corrected mortality for examples 1-3 and comparative examples 1-3 and control group
(II) earthworm avoidance behavior test:
Mixing the above materials according to the proportion of 70% quartz sand, 20% kaolin and 10% peat, controlling pH value to 6.0+ -0.5, and organic matter content to 2%, and preparing artificial soil. The pesticidal compositions of examples 1 to 3 and comparative examples 1 to 3 were uniformly mixed into soil at a ratio of 1% (w/w), and left to stand for 24 hours after being thoroughly mixed. 6 groups of 20cm×10cm×5cm transparent plastic boxes were prepared, which were divided into a contaminated zone, an intermediate isolation zone and a control zone, the intermediate isolation zone separating the contaminated zone and the control zone by means of an isolation plate. The middle isolation zone was 2cm wide for initial placement of earthworms, and the contaminated zone contained 200g of soil containing 1% of the pesticide composition prepared in each of the examples and comparative examples, and the control zone contained 200g of uncontaminated artificial soil. The environmental conditions are set to be 20+/-1 ℃ and 70% humidity, and are light-proof and dark conditions.
Selected adult healthy Eisenia foetida, weighing 300-500mg, was pre-conditioned in artificial soil for 7 days, during which time cow dung was fed. Earthworms were randomly divided into 6 groups of 10 replicates (n=4) corresponding to examples 1-3 and comparative examples 1-3. A blank group without the pesticide composition was also set.
The earthworms were placed in the middle isolation region, the isolation plates were removed to move freely, and after 48 hours, the numbers of earthworms in the contaminated region and the control region were counted, and the avoidance rate (%) = (number of earthworms in the control region—number of earthworms in the contaminated region)/total number of earthworms was multiplied by 100 was calculated. The results are shown in Table 2:
TABLE 2 distribution of Lumbricus in examples 1-3 and comparative examples 1-3 and blank
As can be seen from Table 1, the 24-hour corrected mortality rate of examples 1-3 is 35-39%, and the rate is raised to 95-97.5% after 72 hours, which is close to the positive control (5% avermectin emulsifiable concentrate, and the 72-hour mortality rate is 99.1%), which shows that the sulfonated brown algae polyphenol microcapsule pesticide has certain quick-acting property and lasting property. The mortality rate of 72 hours of comparative example 1 was 65%, indicating that non-microencapsulated brown algae polyphenols may cause efficacy decay due to rapid degradation or photolysis. The 72h mortality rates of comparative example 2 and comparative example 3 were 50.2% and 42.3%, respectively, indicating that the active ingredient release may not be controlled, resulting in reduced efficacy.
As shown in Table 2, the avoidance rate of examples 1-3 is 15-22%, which indicates that the sulfonated brown algae polyphenol microcapsules have no remarkable repellency or toxicity to earthworms and are environment-friendly. The avoidance rate of comparative example 1 and comparative example 3 was 85-90%, indicating that free brown algae polyphenols or sodium tripolyphosphate may stimulate the mucus layer or nerve receptor on the earthworm body surface, and induce strong avoidance behavior. The avoidance rate of comparative example 2 was 40% and could interfere with earthworm osmotic pressure regulation due to the cationic character of the chitooligosaccharide.
In conclusion, the pesticide composition prepared by the invention prolongs the duration and improves the bioavailability through slow release and targeted release. Simultaneously, the exposure of non-target organisms is reduced, and the ecological toxicity is reduced.
In the description of the present specification, reference to the terms "preparation", "embodiment", "embodiments", etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or preparation is included in at least one embodiment or preparation of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same examples or preparations. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or preparations.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.