Purification method of konjak alkaloid and application of konjak alkaloid in pest control

Abstract

The invention discloses a method for purifying konjak alkaloid, which comprises the following steps: soaking konjak powder in a phosphate buffer solution, adding cellulase, pectinase and protease for enzymolysis to prepare a konjak alkaloid crude product, putting the konjak alkaloid crude product into an extraction kettle, extracting with carbon dioxide as a supercritical fluid, and extracting with ethanol to obtain the konjak alkaloid. Extracting by taking an ethanol water solution as an entrainer; and after the extraction is completed, separating the supercritical carbon dioxide fluid from the alkaloid to obtain the konjak alkaloid extract. The invention further discloses application of the konjac alkaloid in pest control. The konjac alkaloid and chitinase are mixed to prepare a pesticide preparation to be sprayed on the surfaces of plants. The method can significantly improve the purity of konjac alkaloid, has a significant effect in pest control, is environmentally friendly, does not produce drug resistance, and provides a new thought for development of green botanical pesticides.

Description

Technical Field

[0001] The invention belongs to the technical field of plant effective component extraction and application, and particularly relates to a method for purifying konjac alkaloids and application thereof in pest control. Background Art

[0002] Konjac, as a plant with wide application prospects, has significant biological activity and physiological function in its alkaloids. Konjac alkaloids can not only degrade rapidly, without ecological pollution problems, but also have higher selectivity, are safe for humans and animals and do not produce resistance. Therefore, konjac alkaloids have broad application prospects in the fields of pesticides, antiseptic, medicine and health care. However, the existing konjac alkaloid purification method has the problems of low efficiency and low purity, therefore, developing a kind of efficient and feasible konjac alkaloid purification method has important practical significance. Meanwhile, the exploration for the application of konjac alkaloids is also one of the hot spots of current research. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0004] Another object of the present invention is to provide a method for purifying konjac alkaloids.

[0005] Another object of the present invention is to provide the application of konjac alkaloids in preventing and controlling insect pests.

[0006] To this end, the technical solution provided by the present invention is: A method for purifying konjac alkaloids comprises the steps of: Step one, get konjac raw material, be prepared into konjac powder of 10~200 mesh, place konjac powder and soak in the phosphate buffer solution that pH value is 7.0, add cellulase, pectinase and protease in the konjac powder suspension after soaking and carry out enzymolysis, after enzymolysis, enzymolysis solution is separated, collect supernatant, adopt chloroform extraction several times, merge chloroform extract, remove chloroform, obtain konjac alkaloid crude product, wherein, in described enzymolysis, total enzyme addition is 20~80U per gram konjac raw material, rotating speed is 160~200rpm, and the usage ratio of cellulase, pectinase and protease is 2~3:1:1, described konjac The mass volume ratio of the powder to the phosphate buffer is 1:10-15. In the enzymolysis, the initial setting pH value of the enzymolysis is 6.9-7.1. When the pH value is higher than 7.1, 0.01 mol / L hydrochloric acid solution is added dropwise for adjustment, and the amount of each addition does not exceed 0.5 mL. After the addition, stir evenly, and measure the pH value again after 5 minutes until the pH value returns to the set range; when the pH value is lower than 6.9, 0.01 mol / L sodium hydroxide solution is added dropwise, and the amount of each addition does not exceed 0.5 mL. After the addition, stir evenly, and measure the pH value again after 5 minutes until the pH value returns to the set range; Step 2: The crude konjac alkaloid product obtained in step 1 is loaded into an extraction kettle, and carbon dioxide is used as a supercritical fluid, and 50% to 70% by volume of an ethanol aqueous solution is used as an entrainer for extraction; Step 3: After the extraction is completed, the mixed fluid containing konjac alkaloids is introduced into a separation kettle to separate the supercritical carbon dioxide fluid from the alkaloids to obtain the konjac alkaloid extract.

[0007] Preferably, in the method for purifying konjac alkaloids, in the enzymolysis, the enzymolysis time is set by the following method: The initial estimated enzymatic hydrolysis time is 3 to 4 hours; During the enzymatic hydrolysis process, the amount of alkaloid release and the amount of by-products generated were monitored in real time: 1-2 mL of sample was taken from the enzymatic hydrolysis system every 30 minutes by high performance liquid chromatography-mass spectrometry, and centrifuged to remove solid impurities, and the supernatant was taken for HPLC-MS analysis to determine the amount of alkaloid release; For small molecule sugars such as oligosaccharides, galacturonic acid, peptides and amino acid byproducts, the corresponding monitoring methods are used: oligosaccharides use the anthrone-sulfuric acid method, galacturonic acid uses the m-hydroxybiphenyl method, peptides and amino acids use the ninhydrin colorimetric method, and 1 mL of enzymatic hydrolysis sample is taken every hour for detection; When the alkaloid release curve tends to be flat and no longer increases, and the by-product generation begins to rise, it is determined that the enzymatic hydrolysis has reached the optimal state, and the enzymatic hydrolysis is terminated at this time. If this state is reached within 3 to 4 hours, it is terminated in advance; If it is still not reached after 4 hours, continue monitoring for 1 hour. If it still does not meet the termination conditions, stop the enzymatic hydrolysis, cool the enzymatic hydrolysis solution, perform subsequent treatment on the enzymatic hydrolysis product, and analyze the cause.

[0008] Preferably, in the method for purifying konjac alkaloids, in the enzymolysis, the enzymolysis temperature is set by the following method: The initial enzymatic hydrolysis temperature was set to 36°C; Real-time monitoring of the temperature of the enzymatic hydrolysis system; When the temperature is higher than 36.5℃ and lasts for 10 minutes, the constant temperature water bath will automatically reduce the heating power by 20% of the current power; When the temperature is lower than 35.5℃ and lasts for 10 minutes, the constant temperature water bath will automatically increase the heating power by 20% of the current power; If the temperature fluctuates abnormally by more than 1°C, stop the enzymatic hydrolysis immediately and restart.

[0009] Preferably, in the purification method of described konjac alkaloid, when the alkaloid release curve tends to be flat and no longer increases, and the by-product generation amount starts to rise simultaneously, it is judged by the following method: Method for judging whether the alkaloid release curve is flattening and no longer increasing: assuming that the alkaloid release detected for the first time is set as 100%, in subsequent tests, if the increase in the alkaloid release detected twice in a row is less than 5%, it is considered that the curve is flattening and no longer increasing; The method for judging that the amount of by-product generation begins to increase is that any of the following is achieved: Oligosaccharides: Detected by anthrone-sulfuric acid method, at a wavelength of 620nm, if the absorbance rises from 0.2 to above 0.5 within 1 hour; Galacturonic acid: Detected by the m-hydroxybiphenyl method, at a wavelength of 520 nm, the absorbance increased from 0.1 to above 0.35 within 1 hour; Peptides and amino acids: Detected by ninhydrin colorimetry, at a wavelength of 570nm, the absorbance increased from 0.3 to above 0.6 within 1 hour.

[0010] Preferably, in the method for purifying konjac alkaloids, step one further comprises: After the enzymatic hydrolysis, an ultrasonic treatment is further performed, wherein the ultrasonic treatment is performed simultaneously at three frequencies of 15-25 kHz, 25-35 kHz, and 40-50 kHz, the ultrasonic power is 100-500 W, the ultrasonic time is 5-70 min, and the ultrasonic temperature is not more than 70° C., and then the enzymatic hydrolyzate is separated; In step 2, during extraction, the extraction temperature is 20-70°C, the extraction pressure is 10-60 MPa, the extraction time is 1-6 hours, the extraction fluid flow rate is 1.0-60 L / h, and the amount of entrainer ethanol is 10-200 mL / 100 g CO2; In step 3, in the separation kettle, the separation pressure is 6-10 MPa and the separation temperature is 32-38° C., so that the supercritical carbon dioxide fluid is separated from the alkaloid.

[0011] Preferably, the method for purifying konjac alkaloids further comprises: Step 4: Drying the konjac alkaloid extract to obtain konjac alkaloids.

[0012] Konjac alkaloids are prepared by any of the methods described above.

[0013] Application of the konjac alkaloids prepared in any one of the above methods in preventing and controlling insect pests.

[0014] Preferably, in the application, konjac alkaloids, chitinase and water are mixed to prepare a pesticide preparation which is sprayed on the surface of plants, and the mass ratio of the konjac alkaloids to the chitinase is 1:1.

[0015] Preferably, in the application, the pesticide preparation further comprises eugenol, and in parts by weight, the pesticide preparation comprises: 20 to 40 parts of konjac alkaloids, 20 to 40 parts of chitinase, 5 to 10 parts of eugenol and 60 to 100 parts of water.

[0016] Preferably, in the application, the pesticide preparation further comprises 5 to 10 parts of proline.

[0017] The present invention has at least the following beneficial effects: Because the konjac cell wall is complex and tough, it hinders the release of alkaloids. Therefore, in the present invention, cellulase, pectinase and protease are first used to specifically act on the cell wall components and macromolecular proteins, destroying their structure, making them loose and porous, opening up dissolution channels for alkaloids, and because of the gentle effect, the alkaloid structure and activity can be maintained. The cavitation effect of ultrasound causes the bubble to break and produce impact force, further breaking the fragile cell wall treated by biological enzymes; then using carbon dioxide as a supercritical fluid, supercritical extraction is carried out at a specific pressure and temperature, which has good solubility for alkaloids and weak solubility for impurities. The konjac material after deep processing by penetrability has greatly improved the konjac alkaloid extraction rate and purity, providing strong guarantee for its deep development and utilization. In addition, after cellulase decomposes cellulose, small molecule sugar substances such as oligosaccharides will be produced; pectinase hydrolysis of pectin will generate galacturonic acid, etc.; after the action of protease, polypeptides and amino acids will be produced. Therefore, in the present invention, by dynamic monitoring of the enzymolysis system and dynamic adjustment strategy of enzymolysis conditions, the presence of impurities in enzymolysis is reduced to avoid affecting the separation effect of chloroform extraction and the effect of supercritical extraction.

[0018] Eugenol is used in the present invention to make it easier for the eugenol to enter the body of the pest to exert a contact killing effect, and the konjac alkaloids and chitinase are easier to penetrate the epidermis of the pest, thereby improving their effects in the body of the pest. Proline enhances the physiological state of the plant, helps the plant to better absorb and transport components such as konjac alkaloids and chitinase, and transports these active substances to the affected part faster, thereby improving the overall control effect.

[0019] The konjac alkaloid purification method of the present invention has the advantages of simple operation, high extraction efficiency and good purification effect, and can significantly improve the purity of konjac alkaloids. The konjac alkaloids of the present invention have a significant effect in pest control, are environmentally friendly, do not produce drug resistance, and provide a new idea for the development of green plant pesticides.

[0020] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0022] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0023] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0024] The present invention provides a method for purifying konjac alkaloids, comprising the following steps: Step 1: Wash and peel the fresh konjac tubers, cut them into small pieces, and put them into a freeze dryer for drying. The dried konjac tubers are crushed to about 10-200 meshes by a pulverizer, and 200 g is accurately weighed. Then, the tubers are placed in 2400 mL of a phosphate buffer having a pH value of 7.0 and soaked for 1-5 hours to fully swell the raw materials and facilitate the subsequent biological enzymes to play a role.

[0025] A composite enzyme preparation is added to the soaked konjac raw material suspension, wherein the composite enzyme preparation is composed of cellulase, pectinase and protease in a ratio of 2-3:1:1, and the total enzyme addition amount is 20-80U per gram of raw material. The reaction system is transferred to a constant temperature shaker at a rotation speed of 160-200rpm. Under this condition, the composite enzyme can work synergistically to effectively decompose impurities such as cell walls, pectin and some proteins in konjac cells, and promote the release of alkaloids into the solution.

[0026] Among them, during enzymatic hydrolysis, real-time monitoring indicators and methods: Monitoring of alkaloid release: Using high performance liquid chromatography-mass spectrometry (HPLC-MS), 1-2 mL of sample was taken from the enzymatic hydrolysis system every 30 minutes, and the solid impurities were removed by rapid centrifugation (10,000 rpm, 5 minutes). The supernatant was taken for HPLC-MS analysis to determine the alkaloid release; Monitoring of by-product generation: For small molecule sugars such as oligosaccharides, take 1 mL of enzymatic hydrolysis sample every hour, use the anthrone-sulfuric acid method, add anthrone-sulfuric acid reagent, heat in a boiling water bath for 10 minutes, measure the absorbance at a wavelength of 620 nm after cooling, and calculate the oligosaccharide content through the standard curve; for galacturonic acid, use the m-hydroxybiphenyl method, take 1 mL of sample and react with m-hydroxybiphenyl reagent, and measure the absorbance at a wavelength of 520 nm for quantification; for peptides and amino acids, use the ninhydrin colorimetric method, take 1 mL of sample and heat with ninhydrin reagent, and measure the absorbance at a wavelength of 570 nm to determine the content; Enzyme hydrolysis system status monitoring: Use a temperature sensor with an accuracy of ±0.1°C and a pH electrode with an accuracy of ±0.01 to monitor the temperature and pH value of the enzymatic hydrolysis system in real time, record temperature data every 5 minutes, and record pH data every 10 minutes; Dynamic adjustment strategy of enzymatic hydrolysis conditions: Temperature adjustment: The initial enzymatic hydrolysis temperature is set to 36°C, and the allowable fluctuation range is 36°C±0.5°C. When the temperature is higher than 36.5°C and lasts for 10 minutes, the constant temperature water bath automatically reduces the heating power by 20% of the current power; when the temperature is lower than 35.5°C and lasts for 10 minutes, the constant temperature water bath automatically increases the heating power by 20% of the current power. If the temperature fluctuates abnormally by more than ±1°C, stop the enzymatic hydrolysis immediately, check the equipment, and restart after adjustment; pH value adjustment: The initial setting pH value is 7.0, and the allowable fluctuation range is 6.9-7.1. When the pH value is higher than 7.1, slowly add 0.01mol / L hydrochloric acid solution for adjustment. The amount of each drop should not exceed 0.5mL. Stir evenly after adding. Measure the pH value again after 5 minutes until the pH value returns to the set range. When the pH value is lower than 6.9, slowly add 0.01mol / L sodium hydroxide solution. The operation method is the same as when adjusting the pH value to be too high. Operation process: Preparation stage: calibrate HPLC-MS, spectrophotometer and other testing instruments, check constant temperature water bath, pH meter and other equipment and calibrate temperature and pH measurement functions, prepare pH 7.0 phosphate buffer, anthrone-sulfuric acid reagent, m-hydroxybiphenyl reagent, ninhydrin reagent and other experimental reagents, weigh appropriate amounts of konjac flour, cellulase, pectinase and protease, mix them evenly according to the proportion and set aside; Enzyme hydrolysis stage: adding konjac flour to a reaction vessel containing phosphate buffer, stirring evenly to make the konjac flour fully swell, adding enzyme preparation, starting a constant temperature water bath and a stirring device, starting an enzymolysis reaction, and simultaneously turning on a temperature sensor, a pH electrode and a data recording device to monitor the state of the enzymolysis system in real time, collecting enzymolysis samples on time according to the monitoring time interval requirements, and detecting the alkaloid release amount and the by-product generation amount; Adjustment and termination stage: According to the real-time monitoring data, the temperature and pH value are dynamically adjusted according to the adjustment strategy, the changes in the alkaloid release and by-product generation are continuously monitored, the rules are determined according to the enzymatic hydrolysis time, and the enzymatic hydrolysis reaction is terminated in time. After the reaction is terminated, the enzymatic hydrolyzate is quickly cooled and the enzymatic hydrolysis product is subsequently processed; Record and summarize: Record the monitoring data of each enzymatic hydrolysis experiment in detail, including the changes in alkaloid release, by-product generation, temperature, and pH value over time; record the time, adjustment method, and adjustment range of each adjustment of the enzymatic hydrolysis conditions; after the experiment, summarize the experience of dynamic adjustment of the enzymatic hydrolysis conditions, analyze the experimental results, provide reference for subsequent experiments and production, and continuously optimize the enzymatic hydrolysis process.

[0027] After the reaction is completed, the reaction solution is transferred to a centrifuge tube and centrifuged at a speed of 9600 rpm for 10 to 15 minutes to fully separate the solid and liquid. The supernatant is carefully collected and extracted with chloroform. The number of extractions is 3 to 4 times, and the amount of chloroform used each time is 400 mL. During the extraction process, the alkaloids are transferred to the chloroform phase. The chloroform extracts are combined and chloroform is recovered by a vacuum distillation apparatus to obtain a crude konjac alkaloid product.

[0028] Step 2, the konjac alkaloid crude product is loaded into the extraction kettle of supercritical fluid extraction device, with carbon dioxide as supercritical fluid, entrainer is the ethanol aqueous solution of volume fraction 50% ~ 70%, extraction temperature is 20 ~ 70 DEG C during extraction, extraction pressure is 10 ~ 60MPa, extraction time is 1 ~ 6 hours, extraction fluid flow is 1.0 ~ 60L / h, and the consumption of entrainer ethanol is 10 ~ 200mL / 100g CO2.

[0029] Step 3: After the extraction is completed, the mixed fluid containing konjac alkaloids is introduced into a separation kettle, and the supercritical carbon dioxide fluid is separated from the alkaloids at a separation pressure of 6-10 MPa and a separation temperature of 32-38° C. to obtain a konjac alkaloid extract.

[0030] Optionally, the enzymolysis time is set by the following method: The initial estimated enzymatic hydrolysis time is 3 to 4 hours; During the enzymatic hydrolysis process, the amount of alkaloid release and the amount of by-products generated were monitored in real time: 1-2 mL of sample was taken from the enzymatic hydrolysis system every 30 minutes using high performance liquid chromatography-mass spectrometry technology, and the sample was rapidly centrifuged at 10,000 rpm for 5 minutes to remove solid impurities, and the supernatant was taken for HPLC-MS analysis to determine the amount of alkaloid release; For small molecule sugars such as oligosaccharides, galacturonic acid, peptides and amino acid byproducts, the corresponding monitoring methods are used: oligosaccharides use the anthrone-sulfuric acid method, galacturonic acid uses the m-hydroxybiphenyl method, peptides and amino acids use the ninhydrin colorimetric method, and 1 mL of enzymatic hydrolysis sample is taken every hour for detection; When the alkaloid release curve tends to be flat and no longer increases, and the by-product generation begins to rise, it is determined that the enzymatic hydrolysis has reached the optimal state, and the enzymatic hydrolysis is terminated at this time. If this state is reached within 3 to 4 hours, it is terminated in advance; If it is not reached after 4 hours, continue monitoring for 1 hour. If it still does not meet the termination conditions, stop the enzymatic hydrolysis, quickly cool the enzymatic hydrolysis solution, perform subsequent treatment on the enzymatic hydrolysis product, and analyze the cause.

[0031] Optionally, the enzymolysis temperature is set by the following method: The initial enzymatic hydrolysis temperature was set to 36°C, and the allowed fluctuation range was 36°C ± 0.5; The temperature of the enzymatic hydrolysis system was monitored in real time using a temperature sensor with an accuracy of ±0.1°C, and the temperature reading was recorded every 5 minutes; When the temperature is higher than 36.5℃ and lasts for 10 minutes, the constant temperature water bath will automatically reduce the heating power by 20% of the current power; When the temperature is lower than 35.5℃ and lasts for 10 minutes, the constant temperature water bath will automatically increase the heating power by 20% of the current power; If the temperature fluctuates abnormally by more than ±1°C, stop the enzymatic hydrolysis immediately, check the equipment, make adjustments, and restart.

[0032] Optionally, when the alkaloid release curve tends to be flat and no longer increases, and the by-product generation amount begins to increase, it is judged by the following method: Method for judging whether the alkaloid release curve is flattening and no longer increasing: assuming that the alkaloid release detected for the first time is set as 100%, in subsequent tests, if the increase in the alkaloid release detected twice in a row is less than 5%, it is considered that the curve is flattening and no longer increasing; The method for judging that the amount of by-product generation begins to increase is that any of the following is achieved: Oligosaccharides: Using the anthrone-sulfuric acid method, at a wavelength of 620nm, if the absorbance rises rapidly from 0.2 to above 0.5 within 1 hour; Galacturonic acid: Using the m-hydroxybiphenyl method, at a wavelength of 520 nm, the absorbance quickly increased from 0.1 to above 0.35 within 1 hour; Peptides and amino acids: Using the ninhydrin colorimetric method, at a wavelength of 570nm, the absorbance quickly increased from 0.3 to above 0.6 within 1 hour.

[0033] Optionally, the method further comprises: step 4, drying the konjac alkaloid extract to obtain konjac alkaloid.

[0034] Optionally, step one also includes: after the enzymatic hydrolysis, ultrasonic treatment is performed, wherein the ultrasonic treatment is performed simultaneously at three frequencies of 15-25 kHz, 25-35 kHz, and 40-50 kHz, the ultrasonic power is 100-500 W, the ultrasonic time is 5-70 min, the ultrasonic temperature is not more than 70°C, and then the enzymatic hydrolyzate is separated.

[0035] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for further explanation: Example 1 A method for purifying konjac alkaloids comprises the steps of: 1) Wash and peel the fresh konjac tubers, cut them into small pieces, and put them into a freeze dryer for drying. The dried konjac tubers are crushed to about 100 meshes using a grinder, and 200 g are accurately weighed. Then, they are placed in 2400 mL of phosphate buffer with a pH value of 7.0 and soaked for 3 hours to fully swell the raw materials and facilitate the subsequent biological enzymes to work.

[0036] Add a composite enzyme preparation to the soaked konjac raw material suspension. The composite enzyme preparation consists of cellulase, pectinase and protease in a ratio of 2.5:1:1, and the total enzyme addition amount is 50U per gram of raw material. Transfer the reaction system to a constant temperature shaker, set the temperature to 35.5~36.5℃, and the speed to 180rpm. During enzymolysis, real-time monitoring indicators and methods: Monitoring of alkaloid release: Using high performance liquid chromatography-mass spectrometry (HPLC-MS), 1-2 mL of sample was taken from the enzymatic hydrolysis system every 30 minutes, and the solid impurities were removed by rapid centrifugation (10,000 rpm, 5 minutes). The supernatant was taken for HPLC-MS analysis to determine the alkaloid release; Monitoring of the production of small molecule sugars such as by-product oligosaccharides: Take 1 mL of enzymatic hydrolysis sample every hour, use the anthrone-sulfuric acid method, add anthrone-sulfuric acid reagent, heat in a boiling water bath for 10 minutes, measure the absorbance at a wavelength of 620 nm after cooling, and calculate the oligosaccharide content using the standard curve; pH value adjustment: The initial setting pH value is 7.0, and the allowable fluctuation range is 6.9-7.1. When the pH value is higher than 7.1, slowly add 0.01mol / L hydrochloric acid solution for adjustment. The amount of each addition should not exceed 0.5mL. Stir evenly after adding. Measure the pH value again after 5 minutes until the pH value returns to the set range; when the pH value is lower than 6.9, slowly add 0.01mol / L sodium hydroxide solution. The operation method is the same as when adjusting the pH value to be too high.

[0037] When the alkaloid release curve tends to be flat and no longer increases, and the by-product generation begins to rise, it is determined that the enzymatic hydrolysis has reached the optimal state, and the enzymatic hydrolysis is terminated at this time. In this embodiment, the enzymatic hydrolysis time is 3 hours; When the alkaloid release curve tends to be flat and no longer increases, and the by-product generation begins to increase, it can be judged by the following method: Method for judging whether the alkaloid release curve is flattening and no longer increasing: assuming that the alkaloid release detected for the first time is set as 100%, in subsequent tests, if the increase in the alkaloid release detected twice in a row is less than 5%, it is considered that the curve is flattening and no longer increasing; The method for judging that the amount of by-product generation begins to increase is: at a wavelength of 620nm, if the absorbance rises rapidly from 0.2 to above 0.5 within 1 hour.

[0038] After the enzymolysis reaction is terminated, the reaction solution is transferred to a centrifuge tube and centrifuged at 9600 rpm for 12 minutes to fully separate the solid and liquid. The supernatant is carefully collected and extracted with chloroform, the number of extractions is 3 to 4 times, and the amount of chloroform used each time is 400 mL. During the extraction process, the alkaloids are transferred to the chloroform phase. The chloroform extracts are combined, and the chloroform is recovered by a vacuum distillation apparatus to obtain a crude konjac alkaloid product.

[0039] 2) The crude konjac alkaloids were loaded into the extraction kettle of a supercritical fluid extraction device, with carbon dioxide as the supercritical fluid, an entrainer being an ethanol aqueous solution with a volume fraction of 60%, an extraction temperature of 45°C, an extraction pressure of 35 MPa, an extraction time of 3.5 hours, an extraction fluid flow rate of 30 L / h, and an entrainer volume fraction of 50%~70% ethanol in an amount of 100 mL / 100 g CO2.

[0040] 3) After the extraction is completed, the mixed fluid containing konjac alkaloids is introduced into a separation kettle, and the supercritical carbon dioxide fluid is separated from the alkaloids at a separation pressure of 8 MPa and a separation temperature of 35°C to obtain the konjac alkaloid extract.

[0041] Example 2 A method for purifying konjac alkaloids comprises the steps of: 1) Wash and peel the fresh konjac tubers, cut them into small pieces, and put them into a freeze dryer for drying. The dried konjac tubers are crushed to about 200 meshes using a grinder, and 200 g is accurately weighed. Then, they are placed in 2400 mL of phosphate buffer with a pH value of 7.0 and soaked for 1 hour to allow the raw materials to swell fully, which is conducive to the subsequent biological enzymes to work.

[0042] Add a composite enzyme preparation to the soaked konjac raw material suspension, the composite enzyme preparation is composed of cellulase, pectinase and protease, the ratio is 2:1:1, and the total enzyme addition amount is 20U per gram of raw material. Transfer the reaction system to a constant temperature shaker with a rotation speed of 160rpm. During the enzymolysis, real-time monitoring indicators and methods: Alkaloid release monitoring: same as in Example 1; Monitoring of the amount of byproduct galacturonic acid generated: using the m-hydroxybiphenyl method, take 1 mL of sample and react with the m-hydroxybiphenyl reagent, and measure the absorbance at a wavelength of 520 nm for quantification; for peptides and amino acids, use the ninhydrin colorimetric method, take 1 mL of sample and heat it with the ninhydrin reagent, and measure the absorbance at a wavelength of 570 nm to determine the content; Enzyme hydrolysis system status monitoring: Use a temperature sensor with an accuracy of ±0.1°C and a pH electrode with an accuracy of ±0.01 to monitor the temperature and pH value of the enzymatic hydrolysis system in real time, record temperature data every 5 minutes, and record pH data every 10 minutes; Dynamic adjustment strategy of enzymatic hydrolysis conditions: Temperature adjustment: The initial enzymatic hydrolysis temperature is set to 36°C, and the allowable fluctuation range is 36°C±0.5°C. When the temperature is higher than 36.5°C and lasts for 10 minutes, the constant temperature water bath automatically reduces the heating power by 20% of the current power; when the temperature is lower than 35.5°C and lasts for 10 minutes, the constant temperature water bath automatically increases the heating power by 20% of the current power; pH value adjustment: The initial setting pH value is 7.0, and the allowable fluctuation range is 6.9-7.1. When the pH value is higher than 7.1, slowly add 0.01mol / L hydrochloric acid solution for adjustment. The amount of each addition should not exceed 0.5mL. Stir evenly after adding. Measure the pH value again after 5 minutes until the pH value returns to the set range; when the pH value is lower than 6.9, slowly add 0.01mol / L sodium hydroxide solution. The operation method is the same as when adjusting the pH value to be too high.

[0043] When the alkaloid release curve tends to be flat and no longer increases, and the by-product generation begins to rise, it is determined that the enzymatic hydrolysis has reached the optimal state, and the enzymatic hydrolysis is terminated at this time. In this embodiment, the enzymatic hydrolysis time is 4 hours; When the alkaloid release curve tends to be flat and no longer increases, and the by-product generation begins to increase, it can be judged by the following method: Method for judging whether the alkaloid release curve is flattening and no longer increasing: assuming that the alkaloid release detected for the first time is set as 100%, in subsequent tests, if the increase in the alkaloid release detected twice in a row is less than 5%, it is considered that the curve is flattening and no longer increasing; The method for judging that the amount of by-product generation begins to increase is: the absorbance at a wavelength of 520nm increases from 0.1 to above 0.35 within 1 hour.

[0044] After enzymatic hydrolysis, ultrasonic treatment is carried out at three frequencies of 15 kHz, 25 kHz, and 40 kHz simultaneously. The ultrasonic power is 100 W. After 2 to 3 minutes of ultrasonic treatment, there is a 1 to 2 minute break. The ultrasonic time is 6 minutes. The ultrasonic temperature is not more than 70°C, and then the enzymatic hydrolysate is separated.

[0045] The reaction solution after ultrasound is transferred to a centrifuge tube and centrifuged at 9600 rpm for 10 minutes to fully separate the solid and liquid. The supernatant is carefully collected and extracted with chloroform. The number of extractions is 3 to 4 times, and the amount of chloroform used each time is 400 mL. During the extraction process, the alkaloids will be transferred to the chloroform phase. The chloroform extracts are combined and the chloroform is recovered by a vacuum distillation apparatus to obtain a crude konjac alkaloid product.

[0046] 2) The crude konjac alkaloids were loaded into the extraction kettle of a supercritical fluid extraction device, with carbon dioxide as the supercritical fluid, an entrainer as a 50% by volume ethanol aqueous solution, an extraction temperature of 20°C, an extraction pressure of 10 MPa, an extraction time of 1 hour, an extraction fluid flow rate of 1.0 L / h, and an entrainer ethanol dosage of 10 mL / 100 g CO2.

[0047] 3) After the extraction is completed, the mixed fluid containing konjac alkaloids is introduced into a separation kettle, and the supercritical carbon dioxide fluid is separated from the alkaloids at a separation pressure of 6 MPa and a separation temperature of 32° C. to obtain a konjac alkaloid extract.

[0048] 4) Drying the konjac alkaloid extract to obtain konjac alkaloids.

[0049] Example 3 A method for purifying konjac alkaloids comprises the steps of: 1) Wash and peel the fresh konjac tubers, cut them into small pieces, and put them into a freeze dryer for drying. The dried konjac tubers are crushed to about 10 meshes using a grinder, and 200 g is accurately weighed. Then, they are placed in 2400 mL of phosphate buffer with a pH value of 7.0 and soaked for 5 hours to allow the raw materials to fully swell and facilitate the subsequent biological enzymes to work.

[0050] Add a composite enzyme preparation to the soaked konjac raw material suspension, the composite enzyme preparation is composed of cellulase, pectinase and protease, the ratio is 3:1:1, and the total enzyme addition amount is 80U per gram of raw material. Transfer the reaction system to a constant temperature shaker with a rotation speed of 200rpm. During the enzymolysis, real-time monitoring indicators and methods: Alkaloid release monitoring: same as in Example 1; Monitoring of the production of by-product peptides and amino acids: Using the ninhydrin colorimetric method, take 1 mL of sample and heat it with the ninhydrin reagent, and detect the absorbance at a wavelength of 570 nm to determine the content; pH value adjustment: The initial setting pH value is 7.0, and the allowable fluctuation range is 6.9-7.1. When the pH value is higher than 7.1, slowly add 0.01mol / L hydrochloric acid solution for adjustment. The amount of each addition should not exceed 0.5mL. Stir evenly after adding. Measure the pH value again after 5 minutes until the pH value returns to the set range; when the pH value is lower than 6.9, slowly add 0.01mol / L sodium hydroxide solution. The operation method is the same as when adjusting the pH value to be too high.

[0051] When the alkaloid release curve tends to be flat and no longer increases, and the by-product generation begins to rise, it is determined that the enzymatic hydrolysis has reached the optimal state. In this embodiment, the enzymatic hydrolysis reaches the optimal state after 4.5 hours. Method for judging whether the alkaloid release curve is flattening and no longer increasing: assuming that the alkaloid release detected for the first time is set as 100%, in subsequent tests, if the increase in the alkaloid release detected twice in a row is less than 5%, it is considered that the curve is flattening and no longer increasing; The method for judging that the amount of by-product generation begins to increase is: at a wavelength of 570nm, the absorbance increases from 0.3 to above 0.6 within 1 hour.

[0052] After enzymatic hydrolysis, ultrasonic treatment was carried out at three frequencies of 25 kHz, 35 kHz, and 50 kHz simultaneously, with an ultrasonic power of 500 W. After 2 to 3 minutes of ultrasonic treatment, there was a 1 to 2 minute break, the ultrasonic time was 70 min, and the ultrasonic temperature was not more than 70°C. The enzymatic hydrolysate was then separated.

[0053] The reaction solution after ultrasound is transferred to a centrifuge tube and centrifuged at 9600 rpm for 15 minutes to fully separate the solid and liquid. The supernatant is carefully collected and extracted with chloroform. The extraction times are 3 to 4 times, and the amount of chloroform used each time is 400 mL. During the extraction process, the alkaloids are transferred to the chloroform phase. The chloroform extracts are combined and the chloroform is recovered by a vacuum distillation apparatus to obtain a crude konjac alkaloid product.

[0054] 2) The crude konjac alkaloids were loaded into the extraction kettle of a supercritical fluid extraction device, with carbon dioxide as the supercritical fluid, an entrainer being an ethanol aqueous solution with a volume fraction of 70%, an extraction temperature of 70°C, an extraction pressure of 60 MPa, an extraction time of 6 hours, an extraction fluid flow rate of 60 L / h, and an entrainer ethanol dosage of 200 mL / 100 g CO2.

[0055] 3) After the extraction is completed, the mixed fluid containing konjac alkaloids is introduced into a separation kettle, and the supercritical carbon dioxide fluid is separated from the alkaloids at a separation pressure of 10 MPa and a separation temperature of 38° C. to obtain a konjac alkaloid extract.

[0056] 4) Drying the konjac alkaloid extract to obtain konjac alkaloids.

[0057] Example 4 A method for purifying konjac alkaloids comprises the steps of: 1) Wash and peel the fresh konjac tubers, cut them into small pieces, and put them into a freeze dryer for drying. The dried konjac tubers are crushed to about 100 mesh using a grinder, and 200 g is accurately weighed. Then, they are placed in 2400 mL of phosphate buffer with a pH value of 7.0 and soaked for 3 hours to allow the raw materials to swell fully, which is conducive to the subsequent biological enzymes to work.

[0058] A composite enzyme preparation is added to the konjac raw material suspension after soaking, and the composite enzyme preparation is composed of cellulase, pectinase and protease, and the ratio is 2.5:1:1, and the total enzyme addition amount is 50U per gram of raw material. The reaction system is transferred to a constant temperature shaking table, and the pH value is set to 7.0, the temperature is 36 ° C, the rotating speed is 180rpm, and the reaction time is controlled at 3.5 hours. Under this condition, the composite enzyme can work synergistically, effectively decompose the impurities such as cell wall, pectin and part of protein in the konjac cell, and promote the release of alkaloids into the solution.

[0059] After enzymatic hydrolysis, ultrasonic treatment was carried out at three frequencies of 20 kHz, 30 kHz, and 45 kHz simultaneously. The ultrasonic power was 300 W. After 2 to 3 minutes of ultrasonic treatment, there was a 1 to 2 minute break. The total ultrasonic time was 35 min. The ultrasonic temperature was not more than 70°C, and then the enzymatic hydrolysate was separated.

[0060] The reaction solution after ultrasound is transferred to a centrifuge tube and centrifuged at 9600 rpm for 12 minutes to fully separate the solid and liquid. The supernatant is carefully collected and extracted with chloroform. The number of extractions is 3 to 4 times, and the amount of chloroform used each time is 400 mL. During the extraction process, the alkaloids are transferred to the chloroform phase. The chloroform extracts are combined and the chloroform is recovered by a vacuum distillation apparatus to obtain a crude konjac alkaloid product.

[0061] 2) The crude konjac alkaloids were loaded into the extraction kettle of a supercritical fluid extraction device, with carbon dioxide as the supercritical fluid, an entrainer being an ethanol aqueous solution with a volume fraction of 60%, an extraction temperature of 45°C, an extraction pressure of 35 MPa, an extraction time of 3.5 hours, an extraction fluid flow rate of 30 L / h, and an entrainer volume fraction of 50%~70% ethanol in an amount of 100 mL / 100 g CO2.

[0062] 3) After the extraction is completed, the mixed fluid containing konjac alkaloids is introduced into a separation kettle, and the supercritical carbon dioxide fluid is separated from the alkaloids at a separation pressure of 8 MPa and a separation temperature of 35°C to obtain the konjac alkaloid extract.

[0063] 4) Drying the konjac alkaloid extract to obtain konjac alkaloids Example 5 The application of konjac alkaloids in pest control is to prepare pesticide preparations according to the following components: 20 parts of konjac alkaloids, 20 parts of chitinase, 5 parts of eugenol, 5 parts of proline and 100 parts of water, wherein the mass ratio of the konjac alkaloids to the chitinase is 1:1.

[0064] Take the prepared konjac alkaloid powder and chitinase and add them to water, then add eugenol and proline in sequence, and then add surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerol) and other additives to form a uniform suspension or emulsion pesticide preparation, and spray it on the surface of plants.

[0065] Example 6 The application of konjac alkaloids in pest control is to prepare pesticide preparations according to the following components: 30 parts of konjac alkaloids, 30 parts of chitinase, 30 parts of eugenol, 7 parts of proline and 60 parts of water, wherein the mass ratio of the konjac alkaloids to the chitinase is 1:1.

[0066] Take the prepared konjac alkaloid powder and chitinase and add them to water, then add eugenol and proline in sequence, and then add surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerol) and other additives to form a uniform suspension or emulsion pesticide preparation, and spray it on the surface of plants.

[0067] Example 7 The application of konjac alkaloids in pest control is to prepare pesticide preparations according to the following components: 40 parts of konjac alkaloids, 40 parts of chitinase, 10 parts of eugenol, 10 parts of proline and 70 parts of water, wherein the mass ratio of the konjac alkaloids to the chitinase is 1:1.

[0068] Take the prepared konjac alkaloid powder and chitinase and add them to water, then add eugenol and proline in sequence, and then add surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerol) and other additives to form a uniform suspension or emulsion pesticide preparation, and spray it on the surface of plants.

[0069] Example 8 The application of konjac alkaloids in pest control is to prepare pesticide preparations according to the following components: 25 parts of konjac alkaloids, 25 parts of chitinase, 7 parts of eugenol, 8 parts of proline and 90 parts of water, wherein the mass ratio of the konjac alkaloids to the chitinase is 1:1.

[0070] Take the prepared konjac alkaloid powder and chitinase and add them to water, then add eugenol and proline in sequence, and then add surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerol) and other additives to form a uniform suspension or emulsion pesticide preparation, and spray it on the surface of plants.

[0071] Comparative Example 1 A method for purifying konjac alkaloids comprises the steps of: The fresh konjac tubers were washed, peeled, cut into small pieces, and placed in a freeze dryer for drying. The dried konjac tubers were crushed to about 100 meshes using a grinder, and refluxed at 60°C for 3 hours using a traditional solvent extraction method with ethanol as the solvent.

[0072] Comparative Example 2 Take 30 parts of konjac alkaloids and add 60 parts of water, then add eugenol and proline in sequence, and then add surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerol) and other auxiliary agents to form a uniform suspension or emulsion pesticide preparation.

[0073] Effect verification 1. The extraction rate of konjac alkaloids in measurement examples 1 to 4 and comparative examples is as shown in Table 1.

[0074] Table 1 Extraction rate of konjac alkaloids As can be seen from Table 1, the extraction rate and purification degree of the konjac bio-enzyme obtained by the enzymolysis and supercritical fluid extraction technology of the present invention have been greatly improved. Konjac cells are wrapped by a complex cell wall structure, which has formed an obstacle to the extraction of alkaloids. And the biological enzyme can specifically identify and act on the components of the cell wall. Cellulase can decompose the cellulose in the cell wall, pectinase is hydrolyzed for pectin, and protease can enzymolysis the protein of the macromolecule. The integrity of the enzymolysis cell wall is destroyed, and its structure becomes loose. After that, carbon dioxide is used as a supercritical fluid. Under certain pressure and temperature conditions, supercritical carbon dioxide can selectively dissolve the alkaloids in the konjac, and the dissolving ability to other impurities is weak. And further, after adding ultrasonic treatment after enzymolysis, the band ultrasonic treatment as a pretreatment step can provide higher quality raw materials for subsequent supercritical fluid extraction. After ultrasonic treatment, the konjac cell structure is destroyed, and the alkaloids are more easily contacted and dissolved by the supercritical fluid, thereby improving the efficiency and effect of supercritical fluid extraction. Further improve the extraction rate and purity of the product. These three technologies work synergistically in sequence, giving full play to their respective advantages and significantly improving the extraction rate and purity of konjac alkaloids.

[0075] 2. The experiment was carried out in a vegetable planting base, with tomatoes as the vegetable variety planted. The planting area was about 2 mu. The soil fertility of the experimental field was uniform and the cultivation and management conditions were consistent.

[0076] Take the pesticide preparations prepared by Examples 5, 6, 7, 8 and the pesticide preparations prepared by Comparative Example 2 as 5 treatment groups, each treatment group is provided with 3 repetition plots, each plot area is about 30 square meters, and the random block group is arranged. Simultaneously, a blank control group is set, and water is sprayed. The konjac alkaloids prepared in Example 4 are all adopted in Examples 5, 6, 7, 8 and Comparative Example 2.

[0077] Start applying pesticides at the early stage of tomato gray mold and the early stage of diamondback moth larvae. Use a backpack sprayer to spray. Dilute the pesticide preparation 500 times and spray it evenly on the front and back of the leaves and stems of tomato plants. The application rate is about 50 ml per square meter. Apply the pesticide once every 7 days for 3 consecutive times.

[0078] On the 3rd and 7th days after each application, the disease index of tomato gray mold and the population density of diamondback moth larvae were investigated and the control effect was calculated. The calculation formulas for the disease index and control effect are as follows: After three applications of the pesticide, the control effect of the compound preparation treatment group on tomato gray mold reached 85.4% at the time of the last survey, and the control effect on the larvae of the diamondback moth reached more than 90%, which were significantly higher than the single konjac alkaloid extract treatment group (Comparative Example 2) and the blank control water treatment group, and no obvious pesticide damage was caused to tomato plants, indicating that the compound preparation has good control effect and safety in the field.

[0079] Although the embodiments of the present invention are disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the embodiments of the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the embodiments of the present invention are not limited to the specific details and the embodiments shown and described here.

Claims

1. A method for purifying konjac alkaloids, characterized in that, The steps include: Step 1, take konjak raw material, be prepared into konjak powder of 10~200 mesh, place konjak powder in the phosphate buffer solution that pH value is 7.0 and soak, add cellulase, pectinase and protease in the konjak powder suspension after soaking and carry out enzymolysis, after enzymolysis, enzymolysis solution is separated, collect supernatant, adopt chloroform extraction several times, merge chloroform extract, remove chloroform, obtain konjak alkaloid crude product, wherein, in described enzymolysis, total enzyme addition is 20~80U per gram konjak raw material, rotating speed is 160~200rpm, the usage ratio of cellulase, pectinase and protease is 2~3:1:1, the mass volume ratio in described konjak powder and described phosphate buffer solution is 1:

1. :10~15, in the enzymolysis, the initial setting pH value of the enzymolysis is 6.9~7.1, when the pH value is higher than 7.1, 0.01mol / L hydrochloric acid solution is added dropwise for adjustment, the amount of each addition does not exceed 0.5mL, and the pH value is measured again after 5 minutes until the pH value returns to the set range; when the pH value is lower than 6.9, 0.01mol / L sodium hydroxide solution is added dropwise, the amount of each addition does not exceed 0.5mL, and the pH value is measured again after 5 minutes until the pH value returns to the set range; Step 2, the konjac alkaloid crude product obtained in step 1 is loaded into an extraction kettle, and carbon dioxide is used as a supercritical fluid, and an ethanol aqueous solution with a volume fraction of 50% to 70% is used as an entrainer to extract; Step 3: After the extraction is completed, the mixed fluid containing konjac alkaloids is introduced into a separation kettle to separate the supercritical carbon dioxide fluid from the alkaloids to obtain the konjac alkaloid extract.

2. The method for purifying konjac alkaloids as claimed in claim 1, wherein In the enzymolysis, the enzymolysis time is set by the following method: The initial estimated enzymatic hydrolysis time is 3 to 4 hours; During the enzymatic hydrolysis process, the amount of alkaloid release and the amount of by-products generated were monitored in real time: 1-2 mL of sample was taken from the enzymatic hydrolysis system every 30 minutes by high performance liquid chromatography-mass spectrometry, and centrifuged to remove solid impurities, and the supernatant was taken for HPLC-MS analysis to determine the amount of alkaloid release; For small molecule sugars such as oligosaccharides, galacturonic acid, peptides and amino acid byproducts, the corresponding monitoring methods are used: oligosaccharides use the anthrone-sulfuric acid method, galacturonic acid uses the m-hydroxybiphenyl method, peptides and amino acids use the ninhydrin colorimetric method, and 1 mL of enzymatic hydrolysis sample is taken every hour for detection; When the alkaloid release curve tends to be flat and no longer increases, and the by-product generation begins to rise, it is determined that the enzymatic hydrolysis has reached the optimal state, and the enzymatic hydrolysis is terminated at this time. If this state is reached within 3 to 4 hours, it is terminated in advance; If it is still not reached after 4 hours, continue monitoring for 1 hour. If it still does not meet the termination conditions, stop the enzymatic hydrolysis, cool the enzymatic hydrolysis solution, perform subsequent treatment on the enzymatic hydrolysis product, and analyze the cause.

3. The method for purifying konjac alkaloids as claimed in claim 2, wherein In the enzymolysis, the enzymolysis temperature is set by the following method: The initial enzymatic hydrolysis temperature was set to 36°C; Real-time monitoring of the temperature of the enzymatic hydrolysis system; When the temperature is higher than 36.5℃ and lasts for 10 minutes, the constant temperature water bath will automatically reduce the heating power by 20% of the current power; When the temperature is lower than 35.5℃ and lasts for 10 minutes, the constant temperature water bath will automatically increase the heating power by 20% of the current power; If the temperature fluctuates abnormally by more than 1°C, stop the enzymatic hydrolysis immediately and restart.

4. The method for purifying konjac alkaloids as claimed in claim 2, wherein When the alkaloid release curve tends to be flat and no longer increases, and the by-product generation begins to increase, it can be judged by the following method: Method for judging whether the alkaloid release curve is flattening and no longer increasing: assuming that the alkaloid release detected for the first time is set as 100%, in subsequent tests, if the increase in the alkaloid release detected twice in a row is less than 5%, it is considered that the curve is flattening and no longer increasing; The method for judging that the amount of by-product generation begins to increase is that any of the following is achieved: Oligosaccharides: Detected by anthrone-sulfuric acid method, at a wavelength of 620nm, if the absorbance rises from 0.2 to above 0.5 within 1 hour; Galacturonic acid: Detected by the m-hydroxybiphenyl method, at a wavelength of 520 nm, the absorbance increased from 0.1 to above 0.35 within 1 hour; Peptides and amino acids: Detected by ninhydrin colorimetry, at a wavelength of 570nm, the absorbance increased from 0.3 to above 0.6 within 1 hour.

5. The method for purifying konjac alkaloids as claimed in claim 1, wherein Step 1 also includes After the enzymatic hydrolysis, an ultrasonic treatment is further performed, wherein the ultrasonic treatment is performed simultaneously at three frequencies of 15-25 kHz, 25-35 kHz, and 40-50 kHz, the ultrasonic power is 100-500 W, the ultrasonic time is 5-70 min, and the ultrasonic temperature is not more than 70° C., and then the enzymatic hydrolyzate is separated; In step 2, during extraction, the extraction temperature is 20-70°C, the extraction pressure is 10-60 MPa, the extraction time is 1-6 hours, the extraction fluid flow rate is 1.0-60 L / h, and the amount of entrainer ethanol is 10-200 mL / 100 g CO2; In step 3, in the separation kettle, the separation pressure is 6-10 MPa and the separation temperature is 32-38° C., so that the supercritical carbon dioxide fluid is separated from the alkaloid.

6. Konjac alkaloids prepared by the method according to any one of claims 1 to 5.

7. Use of the konjac alkaloid prepared as claimed in any one of claims 1 to 5 in controlling insect pests.

8. The use according to claim 7, characterized in that The konjac alkaloids, chitinase and water are mixed to prepare a pesticide preparation which is sprayed on the surface of plants, wherein the mass ratio of the konjac alkaloids to the chitinase is 1:

1.

9. The use according to claim 8, characterized in that The pesticide preparation also includes eugenol. In parts by weight, the pesticide preparation includes: 20 to 40 parts of konjac alkaloids, 20 to 40 parts of chitinase, 5 to 10 parts of eugenol and 60 to 100 parts of water.

10. The use according to claim 8, characterized in that The pesticide preparation also includes 5 to 10 parts of proline.

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