Preparation process of spinosad for destroying cell anti-oxidation defense system

Abstract

The invention discloses a preparation process of spinosad for destroying a cell anti-oxidation defense system, and belongs to the technical field of spinosad. The preparation process comprises the following steps: preparing an emulsifier-metal nanoparticle microgel solid; the preparation method comprises the following steps: adding an emulsifier into a Cu-Fe NPs solution to prepare a suspension, extruding through a needle, applying a voltage to a needle tip, when the applied voltage exceeds a critical value, enabling liquid at the needle tip to overcome surface tension to form a micron-sized droplet-shaped charged jet flow, and crosslinking the droplets to obtain the emulsifier-metal nanoparticle microgel; drying the emulsifier-metal nanoparticle microgel, calcining the dried emulsifier-metal nanoparticle microgel, and grinding the calcined emulsifier-metal nanoparticle microgel to obtain an emulsifier-metal nanoparticle microgel solid; modified methyl oleate; adjusting the pH value; performing centrifugation; performing extraction; the isomerized modified methyl oleate obtained through emulsifying agent-metal nanoparticle microgel solid modification can effectively inhibit the activity of SOD enzyme and CAT enzyme, so that an antioxidant defense system is destroyed, and the insecticidal effect of spinosad is improved.

Description

Technical Field

[0001] The invention relates to the technical field of spinosyns, and in particular to a process for preparing spinosyns that destroys a cell antioxidant defense system. Background Art

[0002] Spinosad, also known as spinosad, is a macrolide, pollution-free and highly effective biological insecticide extracted from the fermentation broth of Polyspora spinosa. It has the safety of biological pesticides and the rapid effectiveness of chemical synthetic pesticides. It won the U.S. "President's Green Chemicals Challenge Award" for its low toxicity, low residue, safety against insect natural enemies, and rapid natural decomposition.

[0003] For example, Chinese patent CN113416225B discloses a spinosad extraction method, comprising the following steps: extraction, stripping, alkali crystallization extraction, decolorization, secondary stripping, secondary alkali crystallization, alcohol crystallization and drying; the adopted process eliminates the steps of plate and frame filtration, mushroom residue drying, mushroom residue leaching and the like in the traditional process, which not only simplifies the equipment, but also shortens the production cycle, while avoiding new pollution to the environment during the mushroom residue leaching process; at the same time, the optimal solid-liquid ratio of the spinosad product amount to methyl oleate, the solid-liquid ratio of the spinosad product amount to citric acid or tartaric acid solution, and the solid-liquid ratio of the spinosad product amount to ethanol are given, while reducing the usage of methyl oleate, citric acid or tartaric acid and ethanol, the yield of spinosad is improved, the spinosad extraction yield of each step is >92%, and the total extraction yield of spinosad is ≥88%, laying a foundation for realizing large-scale industrial production.

[0004] However, in the above patent, methyl oleate is used as a solvent, and methyl oleate will affect the dissolved oxygen in the fermentation broth, thereby affecting the activity of spinosad.

[0005] In addition, the methyl oleate molecule contains unsaturated bonds such as carbon-carbon double bonds and carbon-oxygen double bonds. The presence of these unsaturated bonds makes the methyl oleate molecule highly reactive, and it is easy to react with oxidants such as oxygen to produce harmful substances such as aldehydes, ketones, carboxylic acids, etc., thereby affecting the quality of the extracted spinosad.

[0006] Based on this, the present invention designs a process for preparing spinosad that destroys the cell antioxidant defense system to solve the above problems. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, the present invention provides a process for preparing spinosad that destroys the cell antioxidant defense system.

[0008] To achieve the above objectives, the present invention is implemented by the following technical methods:

[0009] A process for preparing spinosad that destroys the cell antioxidant defense system comprises the following steps:

[0010] S1: Preparation of emulsifier-metal nanoparticle microgel solid;

[0011] S1.1: Add an emulsifier to a Cu-Fe NPs solution to prepare a suspension, extrude it through a needle, and apply a voltage at the needle tip. When the applied voltage exceeds a critical value, the liquid at the needle tip overcomes the surface tension to form a micrometer-sized droplet-shaped charged jet, and these droplets are cross-linked to obtain an emulsifier-metal nanoparticle microgel;

[0012] S1.2: drying the emulsifier-metal nanoparticle microgel, calcining it, and grinding it to obtain an emulsifier-metal nanoparticle microgel solid;

[0013] S2. Modified methyl oleate;

[0014] S2.1: Add methyl oleate, water and emulsifier-metal nanoparticle microgel solid into the reactor and mix well;

[0015] S2.2: introducing nitrogen to expel the air in the reactor and stirring the reaction;

[0016] S2.3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate;

[0017] S3: adjusting the pH value of the spinosad fermentation broth to 9-11;

[0018] S4: centrifuging the fermented liquid after adjusting the pH value, discarding the supernatant, and collecting the sediment at the bottom of the centrifuge tube;

[0019] S5: washing the obtained precipitate with pure water, adding methanol, fully mixing the methanol and the precipitate, and leaching to obtain a methanol extract;

[0020] S6: centrifuging the methanol extract, discarding the solid precipitate, and collecting the extract supernatant;

[0021] S7: concentrating the supernatant under reduced pressure, adding tartaric acid solution, adjusting the pH value to 9, and obtaining spinosad crystal precipitate;

[0022] S8: Wash the polysyns crystalline precipitate with pure water and dry it to obtain the polysyns crystalline product.

[0023] Furthermore, in S1.1, the mass ratio of the emulsifier to the Cu-Fe NPs solution is 13-24:34-56.

[0024] Furthermore, the emulsifier is selected from one or more of emulsifiers A-858, A-115, OEO-110, and OEO-103.

[0025] Furthermore, S1.2 is specifically as follows: drying the emulsifier-metal nanoparticle microgel, calcining at 580-650° C. for 2-3 h, and grinding to 180-200 mesh to obtain an emulsifier-metal nanoparticle microgel solid.

[0026] Furthermore, S2.1 specifically comprises: adding 25-36 parts of methyl oleate, 100-150 parts of water and 5-18 parts of emulsifier-metal nanoparticle microgel solid into a reaction kettle and mixing them evenly.

[0027] Furthermore, S2.2 is specifically as follows: introducing nitrogen to exhaust the air in the reactor, adjusting the air pressure to 0.6-0.8 MPa, stirring at 50-60°C and 300 r / min for 40-50 min.

[0028] Furthermore, S4 specifically comprises: centrifuging the pH-adjusted fermentation broth at a speed of 1000-2000 g for 10-12 min, discarding the supernatant, and collecting the precipitate at the bottom of the centrifuge tube.

[0029] Furthermore, S5 specifically comprises: washing the obtained precipitate with pure water for 1-3 times, adding methanol, mixing thoroughly, and extracting for 2-3 hours to obtain a methanol extract.

[0030] Furthermore, in S5, the volume ratio of the methanol to the precipitate is 1.5-2:1.

[0031] Furthermore, S6: centrifuge the methanol extract at a speed of 1000-2000g for 10-12min, discard the solid precipitate, and collect the extract supernatant.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention successfully prepared isomerized modified methyl oleate with unique properties by introducing an emulsifier-metal nanoparticle microgel solid for innovative modification. This innovative achievement has shown significant advantages in biological activity regulation, especially its ability to effectively inhibit the activity of key antioxidant enzymes - SOD (superoxide dismutase) and CAT (catalase). The modified methyl oleate can significantly reduce the activity of SOD enzyme-catalyzed O 2- (superoxide anion free radical) dismutation, thereby blocking the O 2- is normally converted into H2O2 and O2. This effect promotes O 2-It directly participates in the reduction reaction of nitroblue tetrazolium to generate a colored formazan product. This process not only provides a visual means for the evaluation of antioxidant enzyme activity, but also indirectly reveals the profound effect of modified oleic acid methyl ester on the intracellular redox balance.

[0034] 2. The present invention can also effectively weaken the ability of CAT enzyme to catalyze the decomposition of H2O2 through modified methyl oleate, which is an important part of the cell's antioxidant defense system. When CAT enzyme activity is inhibited, the accumulation of H2O2 in the cell will increase significantly, thereby destroying the redox homeostasis in the cell and causing an imbalance in the antioxidant defense system. This imbalanced state will further cause abnormalities in various physiological activities inside the cell, including but not limited to cell metabolism, signal transduction, and cell cycle regulation, etc., providing more favorable conditions for external factors (such as spinosad) to invade and play a role.

[0035] 3. The present invention uses an innovative emulsifier-metal nanoparticle microgel solid modification technology to isomerize methyl oleate. This unique modification method significantly optimizes the performance of methyl oleate. More importantly, when the modified methyl oleate is in contact with the fermentation broth, it can maintain a stable dissolved oxygen level in the fermentation broth, effectively avoiding the adverse effects on the activity of spinosad caused by changes in dissolved oxygen, and ensuring that the biological activity of spinosad is fully retained.

[0036] 4. The present invention also significantly reduces the reactivity of methyl oleate molecules. This change reduces the possibility of unnecessary chemical reactions between methyl oleate and oxygen or other oxidants. Thus, the generation of potential harmful substances such as aldehydes, ketones, and carboxylic acids by the reaction is effectively avoided. The generation of these substances often poses a threat to the quality of the extracted spinosad, affecting its purity and application effect. Therefore, the modification technology of the present invention not only ensures the high activity of spinosad, but also significantly improves the quality and safety of its final product. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical methods and advantages of the embodiments of the present invention clearer, the technical methods in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are 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.

[0038] Embodiment 1

[0039] This embodiment provides a method for preparing a spinosad fermentation broth; the method comprises the following steps:

[0040] (1) Inoculating the actinomycete Saccharopolyspora spinosa S078-8 into a shake flask containing a seed culture solution, culturing at 30° C. for 2 days, and then inoculating the seed solution into a seed tank. During the inoculation, the pH of the medium in the tank was 7;

[0041] (2) Filling the seed tank with culture medium, sterilizing it, and then inoculating it with activated seed solution from a shake flask;

[0042] (3) The fermentation tank is filled with culture medium, sterilized, cooled to 30° C., and seed solution is added at 10% of the feed volume;

[0043] (4) Seed tank culture: at a temperature of 30°C and a tank pressure of 0.5 kg / cm 2 , aeration rate 1:0.8:0.9 (V / V), stirring rate 230 rpm, culture for 2 days;

[0044] (5) Fermentation tank culture: at a temperature of 30°C and a tank pressure of 0.6 kg / cm 2 , aeration rate of 1:0.7:1.5 (V / V), stirring rate of 400rpm, culture for 10 days; when 70% of the mycelium is lysed under microscope, the fermentation is terminated and the tank is released to obtain the spinosad fermentation liquid;

[0045] The seed culture medium formula is: glucose 15g / L, soybean powder 50g / L, yeast powder 8g / L, magnesium sulfate 2.8g / L, dapoxetine 0.06%, pH before sterilization is 8, pH after sterilization is 7;

[0046] The fermentation tank culture medium formula is: 130g / L glucose, 2.0g / L yeast powder, 8.0g / L bean cake powder, 9g / L corn powder, 80ml / L methyl oleate, 5.0g / L calcium carbonate, 30g / L cottonseed powder; 0.07% folate, pH before sterilization is 8.0, and pH after sterilization is 7.30.

[0047] The preparation method of the spinosad fermentation broth is taken from Chinese patent CN101629203B.

[0048] Embodiment 2

[0049] This embodiment provides a method for preparing a spinosad fermentation broth; the method comprises the following steps:

[0050] S1. Activation of bacterial strains: inoculating Saccharopolyspora spinosa into a seed culture solution for activation culture to obtain activated bacterial strains;

[0051] S2, expansion culture: the activated strain obtained in step S1 is inoculated into the proliferation culture solution, and the proliferation culture is carried out to obtain the proliferated strain;

[0052] The proliferation culture medium comprises: 40 g / L corn starch, 25 g / L soybean powder, 12 g / L seaweed powder, 0.2 g / L magnesium sulfate and 0.8 g / L ammonium molybdate; inactivated at 123°C for 30 min;

[0053] The enlarged culture described in step S2 is carried out in a culture tank, and the conditions for the enlarged culture are: the stirring speed is 130 rpm, the tank pressure is 0.6 kg / cm 2 , the dissolved oxygen content of the proliferation culture medium was 55%, and the proliferation culture was carried out at 32°C for 85 hours;

[0054] S3, fermentation to produce spinosad:

[0055] The proliferated strain obtained in step S2 is inoculated into a fermentation culture solution for fermentation to obtain a fermentation solution containing spinosad;

[0056] The fermentation culture medium comprises: 8 g / L glucose, 4 g / L dextrin, 18 g / L soybean powder, 12 g / L seaweed powder, 0.2 g / L magnesium sulfate and 0.7 g / L ammonium molybdate; inactivated at 128°C for 30 min;

[0057] The fermentation culture in step S3 is carried out in a culture tank under the following conditions: stirring speed of 140 rpm, tank pressure of 0.24 kg / cm 2 The dissolved oxygen content of the fermentation culture liquid was 38%, and the spinosad fermentation liquid was obtained after fermentation culture at 28°C for 90 hours.

[0058] The preparation method of the spinosad fermentation broth is taken from Chinese patent CN109628529B.

[0059] Embodiment 3

[0060] This embodiment provides a method for preparing Cu-Fe NPs (bimetallic nanoparticles); comprising the following steps:

[0061] Accurately weigh 25.27g CuSO4·5H2O, 27.38g FeCl3·6H2O, and 11.08g catechol into a beaker, dissolve them in deionized water, and prepare a solution with a concentration of 1 mol / L;

[0062] Take 500mL of CuSO4 and 500mL of FeCl3 solution and mix them in a conical flask. Add the mixed solution of CuSO4 and FeCl3 dropwise into 3000mL of catechol solution (the pH is adjusted to 11 with 0.1mol / L NaOH). The addition and ultrasound are carried out simultaneously. The ultrasonic reaction is carried out for 5h to obtain a Cu-Fe NPs solution.

[0063] The above method is excerpted from [1] Liu Songlin, Zhang Zhipeng, Han Xue, et al. Preparation and antibacterial activity of Cu-Fe bimetallic nanoparticles [J]. Biological Disaster Science, 2024, 47(01): 125-133.

[0064] Embodiment 4

[0065] This embodiment provides a process for preparing spinosad that destroys the cellular antioxidant defense system, comprising the following steps:

[0066] Step 1: isomerization of methyl oleate;

[0067] (1) preparing emulsifier-metal nanoparticle microgel solid;

[0068] S1: 24 g of emulsifier OEO-110 (oleic acid polyoxyethylene ether) was added to 56 g of the Cu-Fe NPs solution prepared in Example 3 to prepare a suspension, which was extruded through a 35G needle at a flow rate of 0.4 mL / h. A voltage of 40 kV was applied at the needle tip at a receiving distance of 120 mm. When the applied voltage exceeded the critical value, the liquid at the needle tip overcame the surface tension to form a micron-sized droplet-shaped charged jet. These droplets were added to glucose for cross-linking, thereby obtaining a 4 μm emulsifier-metal nanoparticle microgel;

[0069] S2: drying the emulsifier-metal nanoparticle microgel, calcining it at 650°C for 3h, and grinding it into 200 mesh to obtain an emulsifier-metal nanoparticle microgel solid;

[0070] (2) modified methyl oleate;

[0071] S1: Add 36 g of methyl oleate, 150 g of water and 18 g of emulsifier-metal nanoparticle microgel solid into a reactor and mix well;

[0072] S2: nitrogen was introduced to expel the air in the reactor, the pressure was adjusted to 0.8 MPa, and the reactor was stirred at 60°C and 300 r / min for 50 min;

[0073] S3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate;

[0074] Step 2: adjusting the pH value of the spinosad fermentation broth prepared in Example 2 to 11;

[0075] Step 3: Centrifuge the pH-adjusted fermentation broth at 2000 g for 12 min, discard the supernatant, and collect the sediment at the bottom of the centrifuge tube;

[0076] Step 4: washing the obtained precipitate with pure water for 3 times, adding methanol, wherein the volume ratio of methanol to the precipitate is 2:1; mixing well, and leaching for 3 hours to obtain a methanol extract;

[0077] Step 5: centrifuge the methanol extract at 2000 g for 12 min, discard the solid precipitate, and collect the supernatant;

[0078] Step 6: The supernatant liquid is concentrated to 1 / 10 of the volume of the original fermentation liquid by reduced pressure, and a 0.2 mol / L tartaric acid solution having the same volume as the fermentation liquid is added, and the pH value is adjusted to 9 to obtain a spinosad crystal precipitate;

[0079] Step 7: Wash the polysyn crystalline precipitate with pure water and dry it to obtain the polysyn crystalline product.

[0080] Embodiment 5

[0081] This embodiment provides a process for preparing spinosad that destroys the cellular antioxidant defense system, comprising the following steps:

[0082] Step 1: isomerization of methyl oleate;

[0083] (1) preparing emulsifier-metal nanoparticle microgel solid;

[0084] S1: 13 g of emulsifier A-858 (polyoxyethylene sorbitan monooleate, also known as Tween 80) was added to 34 g of the Cu-Fe NPs solution prepared in Example 3 to prepare a suspension, and the suspension was extruded through a 30G needle at a flow rate of 0.2 mL / h. A voltage of 35 kV was applied at the needle tip at a receiving distance of 100 mm. When the applied voltage exceeded the critical value, the liquid at the needle tip overcame the surface tension to form a micron-sized droplet-shaped charged jet. These droplets were added to glucose for cross-linking, thereby obtaining a 2 μm emulsifier-metal nanoparticle microgel;

[0085] S2: drying the emulsifier-metal nanoparticle microgel, calcining it at 580°C for 2h, and grinding it to 180 mesh to obtain an emulsifier-metal nanoparticle microgel solid;

[0086] (2) modified methyl oleate;

[0087] S1: Add 25 g of methyl oleate, 100 g of water and 5 g of emulsifier-metal nanoparticle microgel solid into a reactor and mix well;

[0088] S2: nitrogen was introduced to expel the air in the reactor, the pressure was adjusted to 0.6 MPa, and the reactor was stirred at 50°C and 300 r / min for 40 min;

[0089] S3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate;

[0090] Step 2: adjusting the pH value of the spinosad fermentation broth prepared in Example 1 to 9;

[0091] Step 3: Centrifuge the pH-adjusted fermentation broth at 1000 g for 10 min, discard the supernatant, and collect the precipitate at the bottom of the centrifuge tube;

[0092] Step 4: washing the obtained precipitate with pure water once, adding methanol, wherein the volume ratio of methanol to precipitate is 1.5:1; mixing well, and leaching for 2 hours to obtain a methanol extract;

[0093] Step 5: centrifuge the methanol extract at 1000 g for 10 min, discard the solid precipitate, and collect the supernatant;

[0094] Step 6: The supernatant liquid is concentrated to 1 / 10 of the volume of the original fermentation liquid by reduced pressure, and a 0.2 mol / L tartaric acid solution having the same volume as the fermentation liquid is added, and the pH value is adjusted to 9 to obtain a spinosad crystal precipitate;

[0095] Step 7: Wash the polysyn crystalline precipitate with pure water and dry it to obtain the polysyn crystalline product.

[0096] Embodiment 6

[0097] This embodiment provides a process for preparing spinosad that destroys the cellular antioxidant defense system, comprising the following steps:

[0098] Step 1: isomerization of methyl oleate;

[0099] (1) preparing emulsifier-metal nanoparticle microgel solid;

[0100] S1: 18 g of emulsifier OEO-103 (polyoxyethylene stearate) was added to 52 g of the Cu-Fe NPs solution prepared in Example 3 to prepare a suspension, which was extruded through a 32G needle at a flow rate of 0.3 mL / h. A voltage of 38 kV was applied at the needle tip at a receiving distance of 106 mm. When the applied voltage exceeded the critical value, the liquid at the needle tip overcame the surface tension to form a micron-sized droplet-shaped charged jet. These droplets were added to glucose for cross-linking, thereby obtaining a 3 μm emulsifier-metal nanoparticle microgel;

[0101] S2: drying the emulsifier-metal nanoparticle microgel, calcining it at 620°C for 2.5h, and grinding it to 180 mesh to obtain an emulsifier-metal nanoparticle microgel solid;

[0102] (2) modified methyl oleate;

[0103] S1: Add 31 g of methyl oleate, 120 g of water and 11 g of emulsifier-metal nanoparticle microgel solid into a reactor and mix well;

[0104] S2: nitrogen was introduced to expel the air in the reactor, the pressure was adjusted to 0.7 MPa, and the reactor was stirred at 57°C and 300 r / min for 48 min;

[0105] S3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate;

[0106] Step 2: adjusting the pH value of the spinosad fermentation broth prepared in Example 2 to 10;

[0107] Step 3: Centrifuge the pH-adjusted fermentation broth at 1500 g for 11 min, discard the supernatant, and collect the sediment at the bottom of the centrifuge tube;

[0108] Step 4: washing the obtained precipitate with pure water twice, adding methanol, wherein the volume ratio of methanol to precipitate is 1.8:1; mixing well, and leaching for 2.5 hours to obtain a methanol extract;

[0109] Step 5: centrifuge the methanol extract at 1800 g for 11 min, discard the solid precipitate, and collect the supernatant;

[0110] Step 6: The supernatant liquid is concentrated to 1 / 10 of the volume of the original fermentation liquid by reduced pressure, and a 0.2 mol / L tartaric acid solution having the same volume as the fermentation liquid is added, and the pH value is adjusted to 9 to obtain a spinosad crystal precipitate;

[0111] Step 7: Wash the polysyn crystalline precipitate with pure water and dry it to obtain the polysyn crystalline product.

[0112] Embodiment 7

[0113] This embodiment provides a process for preparing spinosad that destroys the cellular antioxidant defense system, comprising the following steps:

[0114] Step 1: isomerization of methyl oleate;

[0115] (1) preparing emulsifier-metal nanoparticle microgel solid;

[0116] S1: 19g of emulsifier A-858 (polyoxyethylene sorbitan monooleate, also known as Tween 80) was added to 41g of the Cu-Fe NPs solution prepared in Example 3 to prepare a suspension, and extruded through a 31G needle at a flow rate of 0.4mL / h. At a receiving distance of 111mm, a voltage of 36kV was applied at the needle tip. When the applied voltage exceeded the critical value, the liquid at the needle tip overcame the surface tension to form a micron-sized droplet-shaped charged jet. These droplets were added with glucose for cross-linking, and 2μm emulsifier-metal nanoparticle microgel was obtained;

[0117] S2: drying the emulsifier-metal nanoparticle microgel, calcining it at 640°C for 3h, and grinding it to 180 mesh to obtain an emulsifier-metal nanoparticle microgel solid;

[0118] (2) modified methyl oleate;

[0119] S1: Add 31 g of methyl oleate, 130 g of water and 7 g of emulsifier-metal nanoparticle microgel solid into a reactor and mix well;

[0120] S2: nitrogen was introduced to expel the air in the reactor, the pressure was adjusted to 0.8 MPa, and the reactor was stirred at 56°C and 300 r / min for 47 min;

[0121] S3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate;

[0122] Step 2: adjusting the pH value of the spinosad fermentation broth prepared in Example 1 to 9;

[0123] Step 3: Centrifuge the pH-adjusted fermentation broth at 1400 g for 12 min, discard the supernatant, and collect the precipitate at the bottom of the centrifuge tube;

[0124] Step 4: washing the obtained precipitate with pure water for 3 times, adding methanol, wherein the volume ratio of methanol to precipitate is 1.6:1; mixing well, and leaching for 2 hours to obtain a methanol extract;

[0125] Step 5: centrifuge the methanol extract at a speed of 1900 g for 12 minutes, discard the solid precipitate, and collect the extract supernatant;

[0126] Step 6: The supernatant liquid is concentrated to 1 / 10 of the volume of the original fermentation liquid by reduced pressure, and a 0.2 mol / L tartaric acid solution having the same volume as the fermentation liquid is added, and the pH value is adjusted to 9 to obtain a spinosad crystal precipitate;

[0127] Step 7: Wash the polysyn crystalline precipitate with pure water and dry it to obtain the polysyn crystalline product.

[0128] Embodiment 8

[0129] This embodiment provides a process for preparing spinosad that destroys the cellular antioxidant defense system, comprising the following steps:

[0130] Step 1: isomerization of methyl oleate;

[0131] (1) preparing emulsifier-metal nanoparticle microgel solid;

[0132] S1: 14 g of emulsifier A-115 (oleic acid polyoxyethylene ester) was added to 45 g of the Cu-Fe NPs solution prepared in Example 3 to prepare a suspension, and the suspension was extruded through a 34G needle at a flow rate of 0.3 mL / h. A voltage of 38 kV was applied at the needle tip at a receiving distance of 115 mm. When the applied voltage exceeded the critical value, the liquid at the needle tip overcame the surface tension to form a micron-sized droplet-shaped charged jet. These droplets were added to glucose for cross-linking, and 4 μm emulsifier-metal nanoparticle microgel was obtained;

[0133] S2: drying the emulsifier-metal nanoparticle microgel, calcining it at 610°C for 3h, and grinding it to 180 mesh to obtain an emulsifier-metal nanoparticle microgel solid;

[0134] (2) modified methyl oleate;

[0135] S1: Add 32 g of methyl oleate, 140 g of water and 13 g of emulsifier-metal nanoparticle microgel solid into a reactor and mix well;

[0136] S2: nitrogen was introduced to expel the air in the reactor, the pressure was adjusted to 0.6 MPa, and the reactor was stirred at 52°C and 300 r / min for 42 min;

[0137] S3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate;

[0138] Step 2: adjusting the pH value of the spinosad fermentation broth prepared in Example 2 to 10;

[0139] Step 3: Centrifuge the pH-adjusted fermentation broth at 1300 g for 10 min, discard the supernatant, and collect the precipitate at the bottom of the centrifuge tube;

[0140] Step 4: washing the obtained precipitate with pure water for 3 times, adding methanol, wherein the volume ratio of methanol to the precipitate is 2:1; mixing well, and leaching for 3 hours to obtain a methanol extract;

[0141] Step 5: centrifuge the methanol extract at 1800 g for 12 min, discard the solid precipitate, and collect the supernatant;

[0142] Step 6: The supernatant liquid is concentrated to 1 / 10 of the volume of the original fermentation liquid by reduced pressure, and a 0.2 mol / L tartaric acid solution having the same volume as the fermentation liquid is added, and the pH value is adjusted to 9 to obtain a spinosad crystal precipitate;

[0143] Step 7: Wash the polysyn crystalline precipitate with pure water and dry it to obtain the polysyn crystalline product.

[0144] Embodiment 9

[0145] This embodiment provides a process for preparing spinosad that destroys the cellular antioxidant defense system, comprising the following steps:

[0146] Step 1: isomerization of methyl oleate;

[0147] (1) preparing emulsifier-metal nanoparticle microgel solid;

[0148] S1: 22 g of emulsifier OEO-110 (oleic acid polyoxyethylene ether) was added to 54 g of the Cu-Fe NPs solution prepared in Example 3 to prepare a suspension, and the suspension was extruded through a 31G needle at a flow rate of 0.4 mL / h. A voltage of 38 kV was applied at the needle tip at a receiving distance of 113 mm. When the applied voltage exceeded the critical value, the liquid at the needle tip overcame the surface tension to form a micron-sized droplet-shaped charged jet. These droplets were added to glucose for cross-linking, thereby obtaining a 2 μm emulsifier-metal nanoparticle microgel;

[0149] S2: drying the emulsifier-metal nanoparticle microgel, calcining it at 590°C for 2h, and grinding it into 200 mesh to obtain an emulsifier-metal nanoparticle microgel solid;

[0150] (2) modified methyl oleate;

[0151] S1: Add 28g of methyl oleate, 140g of water and 6g of emulsifier-metal nanoparticle microgel solid into a reactor and mix well;

[0152] S2: nitrogen was introduced to expel the air in the reactor, the pressure was adjusted to 0.6 MPa, and the reactor was stirred at 52°C and 300 r / min for 46 min;

[0153] S3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate;

[0154] Step 2: adjusting the pH value of the spinosad fermentation broth prepared in Example 2 to 9-11;

[0155] Step 3: Centrifuge the pH-adjusted fermentation broth at 1300 g for 12 min, discard the supernatant, and collect the sediment at the bottom of the centrifuge tube;

[0156] Step 4: washing the obtained precipitate with pure water for 3 times, adding methanol, wherein the volume ratio of methanol to precipitate is 1.8:1; mixing well, and leaching for 3 hours to obtain a methanol extract;

[0157] Step 5: centrifuge the methanol extract at a speed of 1700g for 12 minutes, discard the solid precipitate, and collect the extract supernatant;

[0158] Step 6: The supernatant liquid is concentrated to 1 / 10 of the volume of the original fermentation liquid by reduced pressure, and a 0.2 mol / L tartaric acid solution having the same volume as the fermentation liquid is added, and the pH value is adjusted to 9 to obtain a spinosad crystal precipitate;

[0159] Step 7: Wash the polysyn crystalline precipitate with pure water and dry it to obtain the polysyn crystalline product.

[0160] Comparative Example 1

[0161] The difference between this comparative example and Example 7 is: the ratio of emulsifier to Cu-Fe NPs solution;

[0162] This comparative example provides a process for preparing spinosad that destroys the cell antioxidant defense system, comprising the following steps:

[0163] Step 1: isomerization of methyl oleate;

[0164] (1) preparing emulsifier-metal nanoparticle microgel solid;

[0165] S1: 9 g of emulsifier A-858 (polyoxyethylene sorbitan monooleate, also known as Tween 80) was added to 65 g of the Cu-Fe NPs solution prepared in Example 3 to prepare a suspension, which was extruded through a 31G needle at a flow rate of 0.4 mL / h. A voltage of 36 kV was applied at the needle tip at a receiving distance of 111 mm. When the applied voltage exceeded the critical value, the liquid at the needle tip overcame the surface tension to form a micron-sized droplet-shaped charged jet. These droplets were added with glucose for cross-linking, thereby obtaining a 2 μm emulsifier-metal nanoparticle microgel;

[0166] S2: drying the emulsifier-metal nanoparticle microgel, calcining it at 640°C for 3h, and grinding it to 180 mesh to obtain an emulsifier-metal nanoparticle microgel solid;

[0167] (2) modified methyl oleate;

[0168] S1: Add 31 g of methyl oleate, 130 g of water and 7 g of emulsifier-metal nanoparticle microgel solid into a reactor and mix well;

[0169] S2: nitrogen was introduced to expel the air in the reactor, the pressure was adjusted to 0.8 MPa, and the reactor was stirred at 56°C and 300 r / min for 47 min;

[0170] S3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate;

[0171] Step 2: adjusting the pH value of the spinosad fermentation broth prepared in Example 1 to 9;

[0172] Step 3: Centrifuge the pH-adjusted fermentation broth at 1400 g for 12 min, discard the supernatant, and collect the precipitate at the bottom of the centrifuge tube;

[0173] Step 4: washing the obtained precipitate with pure water for 3 times, adding methanol, wherein the volume ratio of methanol to precipitate is 1.6:1; mixing well, and leaching for 2 hours to obtain a methanol extract;

[0174] Step 5: centrifuge the methanol extract at a speed of 1900 g for 12 minutes, discard the solid precipitate, and collect the extract supernatant;

[0175] Step 6: The supernatant liquid is concentrated to 1 / 10 of the volume of the original fermentation liquid by reduced pressure, and a 0.2 mol / L tartaric acid solution having the same volume as the fermentation liquid is added, and the pH value is adjusted to 9 to obtain a spinosad crystal precipitate;

[0176] Step 7: Wash the polysyn crystalline precipitate with pure water and dry it to obtain the polysyn crystalline product.

[0177] Comparative Example 2

[0178] The difference between this comparative example and Example 7 is that no calcination is performed in step 1;

[0179] This embodiment provides a process for preparing spinosad that destroys the cellular antioxidant defense system, comprising the following steps:

[0180] Step 1: isomerization of methyl oleate;

[0181] (1) preparing emulsifier-metal nanoparticle microgel solid;

[0182] S1: 19g of emulsifier A-858 (polyoxyethylene sorbitan monooleate, also known as Tween 80) was added to 41g of the Cu-Fe NPs solution prepared in Example 3 to prepare a suspension, and extruded through a 31G needle at a flow rate of 0.4mL / h. At a receiving distance of 111mm, a voltage of 36kV was applied at the needle tip. When the applied voltage exceeded the critical value, the liquid at the needle tip overcame the surface tension to form a micron-sized droplet-shaped charged jet. These droplets were added with glucose for cross-linking, and 2μm emulsifier-metal nanoparticle microgel was obtained;

[0183] (2) modified methyl oleate;

[0184] S1: Add 31 g of methyl oleate, 130 g of water and 7 g of emulsifier-metal nanoparticle microgel into a reactor and mix well;

[0185] S2: nitrogen was introduced to expel the air in the reactor, the pressure was adjusted to 0.8 MPa, and the reactor was stirred at 56°C and 300 r / min for 47 min;

[0186] S3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate;

[0187] Step 2: adjusting the pH value of the spinosad fermentation broth prepared in Example 1 to 9;

[0188] Step 3: Centrifuge the pH-adjusted fermentation broth at 1400 g for 12 min, discard the supernatant, and collect the precipitate at the bottom of the centrifuge tube;

[0189] Step 4: washing the obtained precipitate with pure water for 3 times, adding methanol, wherein the volume ratio of methanol to precipitate is 1.6:1; mixing well, and leaching for 2 hours to obtain a methanol extract;

[0190] Step 5: centrifuge the methanol extract at a speed of 1900 g for 12 minutes, discard the solid precipitate, and collect the extract supernatant;

[0191] Step 6: The supernatant liquid is concentrated to 1 / 10 of the volume of the original fermentation liquid by reduced pressure, and a 0.2 mol / L tartaric acid solution having the same volume as the fermentation liquid is added, and the pH value is adjusted to 9 to obtain a spinosad crystal precipitate;

[0192] Step 7: Wash the polysyn crystalline precipitate with pure water and dry it to obtain the polysyn crystalline product.

[0193] Experimental Example 1

[0194] Data detection was performed on the spinosad prepared in Examples 4 to 8 and Comparative Examples 1 and 2;

[0195] The concentration of spinosad was determined by liquid chromatography as follows:

[0196] Instrument: High performance liquid chromatography;

[0197] Reagents: anhydrous ethanol or 95% ethanol, chromatographic grade acetonitrile, ammonium acetate;

[0198] Chromatographic column: C18 4.6mm×250mm, 5μm;

[0199] Detection wavelength: 250nm;

[0200] Flow rate: 1.0 mL / min;

[0201] Injection volume: 20 μL;

[0202] Column temperature: 30°C;

[0203] Mobile phase: acetonitrile: methanol: 0.05% ammonium acetate aqueous solution = 45:45:10;

[0204] Weigh 50 mg of sample, place in a 100 mL volumetric flask, add mobile phase to dissolve and dilute to scale, shake well, filter, repeat injection 2 times, and record the peak area of ​​the chromatogram;

[0205] Spinosad yield = recovered amount of spinosad (g) ÷ initial content of spinosad in fermentation broth (g) × 100%;

[0206] Purity = net content of spinosad (g) ÷ recovered amount of spinosad (g) × 100%.

[0207] Experimental Example 2

[0208] Determination of SOD enzyme activity;

[0209] Spinosad can achieve insecticidal effects by destroying the antioxidant defense system, leading to abnormalities in various physiological activities of cells, such as antioxidant consumption, lipid peroxidation, and DNA damage. The antioxidant enzymes in cells mainly include SOD, CAT, GST, etc.

[0210] O 2- It can reduce nitroblue tetrazolium (NBT) to form blue formazan, which has a maximum light absorption value at 560nm. SOD can catalyze O 2- Disproportionation occurs to generate H2O2 and oxygen, thereby inhibiting the formation of formazan;

[0211] Sf9 cells were exposed to 60 μmol / L spinosad for 24 h, cell lysis buffer was added, and the supernatant was obtained by centrifugation to obtain a cell lysis protein solution; 20 μL and 40 μL SOD detection buffer were added to a 96-well cell culture plate as blank control 1 and blank control 2, or 20 μL of each cell lysis protein solution, and then 160 μL NBT-enzyme detection working solution and 20 μL reaction start working solution were added in sequence, incubated at 37°C for 30 min, and the optical density value OD at 560 nm was measured using a microplate reader;

[0212] Formazan inhibition rate (%) = (OD blank control group 1-OD sample) / (OD blank control group 1-OD blank control group 2)*100%;

[0213] SOD enzyme activity unit = inhibition rate / (1-inhibition rate)

[0214] Experimental Example 3

[0215] Determination of CAT enzyme activity;

[0216] Under the condition of excess H2O2, part of H2O2 can be decomposed into H2O and O2 by CAT, and the remaining H2O2 reacts with the chromogenic substrate under the catalysis of peroxidase to generate a red product with a maximum light absorption value at 520nm;

[0217] Make a standard curve:

[0218] The H2O2 standard solution was diluted to 0, 0.625, 1.25, 2.5 and 3.75 mmol / L with catalase detection buffer, 4 μL of each solution was added to a 96-well cell culture plate, 200 μL of color development working solution was added, and the plates were incubated at 25°C for 15 min. The optical density at 520 nm was measured by an enzyme reader;

[0219] Sf9 cells were exposed to 60 μmol / L spinosad for 24 h, cell lysis buffer was added, and the supernatant was collected by centrifugation to obtain a cell lysis protein solution;

[0220] Take 20 μL of cell lysis protein solution, add 20 μL of catalase detection buffer, mix thoroughly, then add 10 μL of 250 mmol / L H2O2, mix thoroughly, react at 25°C for 5 min, add 450 μL of catalase reaction stop solution, mix thoroughly to terminate the reaction;

[0221] The above solution was diluted with catalase detection buffer at a ratio of 1:4, 10 μL was taken out and added to a 96-well cell culture plate, and then 200 μL of color development working solution was added, incubated at 25°C for 15 min, and the optical density value at 520 nm was measured by an enzyme reader;

[0222] Calculate the amount of remaining H2O2 based on the standard curve;

[0223] Definition of CAT activity unit: Under the conditions of 25℃ and pH 7.0, the CAT activity that catalyzes the decomposition of 10μmol H2O2 to generate H2O and O2 within 1min is defined as 1 enzyme activity unit;

[0224] CAT enzyme activity unit = amount of H2O2 consumed * sample dilution multiple / reaction time;

[0225] The results are as follows:

[0226] sample Yield purity SOD enzyme activity CAT enzyme activity Embodiment 4 82.6％ 98.5％ 0.85 U / mg 2.73 U / mg Embodiment 5 81.4％ 97.4％ 0.76 U / mg 2.84 U / mg Embodiment 6 80.7％ 98.2％ 0.92 U / mg 2.81 U / mg Embodiment 7 83.2％ 96.7％ 0.83 U / mg 2.72 U / mg Embodiment 8 82.3％ 95.8％ 0.71 U / mg 2.65 U / mg Comparative Example 1 80.9％ 92.6％ 1.52 U / mg 3.68 U / mg Comparative Example 2 81.6％ 97.3％ 1.85 U / mg 3.95 U / mg

[0227] As can be seen from the above table, the modified methyl oleate obtained by isomerization through emulsifier-metal nanoparticle microgel solid modification can effectively inhibit the activity of SOD and CAT enzymes, thereby inhibiting O 2- Disproportionation occurs, making O 2- Reduce nitroblue tetrazolium to form formazan; at the same time, weaken the ability of CAT or catalytic decomposition of H2O2, thereby destroying the antioxidant defense system, leading to abnormalities in various physiological activities of cells and enhancing the insecticidal effect of spinosad.

[0228] The isomerized modified methyl oleate obtained by emulsifier-metal nanoparticle microgel solid modification will not affect the dissolved oxygen in the fermentation broth, thereby avoiding affecting the activity of spinosad; and reduces the reactivity of the methyl oleate molecule, avoiding reaction with oxidants such as oxygen to produce harmful substances such as aldehydes, ketones, carboxylic acids, etc., thereby affecting the quality of the extracted spinosad.

[0229] In summary, the isomerized modified methyl oleate obtained by solid modification of emulsifier-metal nanoparticle microgel in the present invention can significantly enhance the insecticidal effect of spinosad on target pests by precisely regulating the activity of antioxidant enzymes in cells, destroying the antioxidant defense mechanism of cells, and inducing abnormal cell physiological activities. This discovery not only enriches the research and development ideas of biological pesticide synergists, but also provides strong technical support for improving the efficiency of crop disease and insect pest control.

[0230] The above embodiments are only used to illustrate the technical methods of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical methods described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements will not cause the essence of the corresponding technical methods to deviate from the spirit and scope of the technical methods of the embodiments of the present invention.

Claims

1. A process for preparing spinosad that destroys the cellular antioxidant defense system, characterized in that: The following steps are involved: S1: Preparation of emulsifier-metal nanoparticle microgel solid; S1.1: Add an emulsifier to a Cu-Fe NPs solution to prepare a suspension, extrude it through a needle, and apply a voltage at the needle tip. When the applied voltage exceeds a critical value, the liquid at the needle tip overcomes the surface tension to form a micrometer-sized droplet-shaped charged jet, and these droplets are cross-linked to obtain an emulsifier-metal nanoparticle microgel; S1.2: drying the emulsifier-metal nanoparticle microgel, calcining it, and grinding it to obtain an emulsifier-metal nanoparticle microgel solid; S2. Modified methyl oleate; S2.1: Add methyl oleate, water and emulsifier-metal nanoparticle microgel solid into the reactor and mix well; S2.2: introducing nitrogen to expel the air in the reactor and stirring the reaction; S2.3: filtering and removing the unreacted emulsifier-metal nanoparticle microgel solid to obtain isomerized modified methyl oleate; S3: adjusting the pH value of the spinosad fermentation broth to 9-11; S4: centrifuging the fermented liquid after adjusting the pH value, discarding the supernatant, and collecting the sediment at the bottom of the centrifuge tube; S5: washing the obtained precipitate with pure water, adding methanol, fully mixing the methanol and the precipitate, and leaching to obtain a methanol extract; S6: centrifuging the methanol extract, discarding the solid precipitate, and collecting the extract supernatant; S7: concentrating the supernatant under reduced pressure, adding tartaric acid solution, adjusting the pH value to 9, and obtaining spinosad crystal precipitate; S8: Wash the polysyns crystalline precipitate with pure water and dry it to obtain the polysyns crystalline product.

2. The process according to claim 1, characterized in that In S1.1, the mass ratio of emulsifier to Cu-Fe NPs solution was 13-24:34-56.

3. The process according to claim 2, characterized in that The emulsifier is selected from one or more of emulsifiers A-858, A-115, OEO-110, and OEO-103.

4. The process according to claim 3, characterized in that S1.2 specifically comprises: drying the emulsifier-metal nanoparticle microgel, calcining the emulsifier-metal nanoparticle microgel at 580-650°C for 2-3h, and grinding the emulsifier-metal nanoparticle microgel to 180-200 mesh to obtain an emulsifier-metal nanoparticle microgel solid.

5. The process according to claim 4, characterized in that S2.1 specifically comprises: adding 25-36 parts of methyl oleate, 100-150 parts of water and 5-18 parts of emulsifier-metal nanoparticle microgel solid into a reaction kettle and mixing them evenly.

6. The process according to claim 5, characterized in that S2.2 specifically includes: introducing nitrogen to expel the air in the reactor, adjusting the air pressure to 0.6-0.8 MPa, stirring at 50-60°C and 300 r / min for 40-50 min.

7. The process according to claim 6, characterized in that S4 specifically comprises: centrifuging the fermented liquid after adjusting the pH value at a speed of 1000-2000 g for 10-12 minutes, discarding the supernatant, and collecting the sediment at the bottom of the centrifuge tube.

8. The process according to claim 7, characterized in that S5 is specifically as follows: washing the obtained precipitate with pure water for 1-3 times, adding methanol, mixing thoroughly, and performing leaching for 2-3 hours to obtain a methanol extract.

9. The process according to claim 8, characterized in that In S5, the volume ratio of the methanol to the precipitate is 1.5-2:

1.

10. The process according to claim 9, characterized in that S6: Centrifuge the methanol extract at a speed of 1000-2000 g for 10-12 min, discard the solid precipitate, and collect the supernatant.