Purification methods of konjac alkaloids and their application in pest control

Abstract

This invention discloses a method for purifying konjac alkaloids, comprising: soaking konjac powder in phosphate buffer, adding cellulase, pectinase, and protease for enzymatic hydrolysis to obtain crude konjac alkaloids; loading the crude konjac alkaloids into an extraction vessel and extracting using supercritical carbon dioxide as the supercritical fluid and an ethanol-water solution as the entrainer; after extraction, separating the supercritical carbon dioxide fluid from the alkaloids to obtain a konjac alkaloid extract. This invention also discloses the application of konjac alkaloids in pest control, by mixing konjac alkaloids with chitinase to prepare a pesticide formulation for spraying on the plant surface. This invention can significantly improve the purity of konjac alkaloids, has a significant effect in pest control, and is environmentally friendly, does not induce pesticide resistance, and provides a new approach for developing green plant-based pesticides.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction and application technology, specifically involving a purification method for konjac alkaloids and their application in pest control. Background Technology

[0002] Konjac, a plant with broad application prospects, contains alkaloids with significant biological activity and physiological functions. Konjac alkaloids are not only rapidly degradable without causing environmental pollution, but also exhibit high selectivity, are safe for humans and animals, and do not induce resistance. Therefore, konjac alkaloids have broad application prospects in pesticides, preservatives, medicine, and health care. However, existing methods for purifying konjac alkaloids suffer from low efficiency and low purity. Therefore, developing an efficient and feasible purification method for konjac alkaloids is of significant practical importance. Simultaneously, exploring the applications of konjac alkaloids is also one of the current research hotspots. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0004] Another objective of this invention is to provide a method for purifying konjac alkaloids.

[0005] Another objective of this invention is to provide the application of konjac alkaloids in pest control.

[0006] Therefore, the technical solution provided by this invention is as follows:

[0007] A method for purifying konjac alkaloids includes the following steps:

[0008] Step 1: Take konjac raw material and prepare konjac powder of 10-200 mesh. Soak the konjac powder in a phosphate buffer solution with a pH of 7.0. Add cellulase, pectinase, and protease to the soaked konjac powder suspension for enzymatic hydrolysis. After enzymatic hydrolysis, separate the hydrolysate, collect the supernatant, and extract several times with chloroform. Combine the chloroform extracts, remove the chloroform, and obtain crude konjac alkaloids. In the enzymatic hydrolysis, the total enzyme addition is 20-80 U per gram of konjac raw material, the rotation speed is 160-200 rpm, and the ratio of cellulase, pectinase, and protease is 2-3:1:1. The mass-to-volume ratio of the powder to the phosphate buffer is 1:10-15. During the enzymatic hydrolysis, the initial pH value is set to 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, with each addition not exceeding 0.5 mL. After addition, the solution is stirred thoroughly, 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, 0.01 mol / L sodium hydroxide solution is added dropwise, with each addition not exceeding 0.5 mL. After addition, the solution is stirred thoroughly, and the pH value is measured again after 5 minutes until the pH value returns to the set range.

[0009] Step 2: The crude konjac alkaloid obtained in Step 1 is loaded into an extraction vessel and extracted using carbon dioxide as a supercritical fluid and an ethanol aqueous solution with a volume fraction of 50%~70% as an entrainer.

[0010] Step 3: After extraction, the mixed fluid containing konjac alkaloids is introduced into the separation vessel to separate the supercritical carbon dioxide fluid from the alkaloids, thus obtaining the konjac alkaloid extract.

[0011] Preferably, in the purification method of konjac alkaloids, the enzymatic hydrolysis time is set by the following method:

[0012] The initial estimated enzymatic hydrolysis time is 3-4 hours;

[0013] During the enzymatic hydrolysis process, the release of alkaloids and the amount of byproducts were monitored in real time: using high performance liquid chromatography-mass spectrometry, 1-2 mL of sample was taken from the enzymatic hydrolysis system every 30 minutes, centrifuged to remove solid impurities, and the supernatant was taken for HPLC-MS analysis to determine the release of alkaloids.

[0014] For small molecule sugars such as oligosaccharides, galacturonic acid, peptides and amino acid byproducts, the corresponding monitoring methods are as follows: for oligosaccharides, the anthrone-sulfuric acid method is used; for galacturonic acid, the m-hydroxybiphenyl method is used; and for peptides and amino acids, the ninhydrin colorimetric method is used. 1 mL of enzymatically digested sample is taken every 1 hour for detection.

[0015] When the alkaloid release curve flattens out and stops increasing, while the amount of byproducts begins to rise, the enzymatic hydrolysis is considered to have reached its optimal state, and the hydrolysis is terminated at this point. If this state is reached within 3 to 4 hours, the hydrolysis is terminated earlier.

[0016] If the condition is not met after 4 hours, continue monitoring for another hour. If the termination condition is still not met, stop the enzymatic hydrolysis, cool the hydrolysate, and perform subsequent processing on the hydrolysate. At the same time, analyze the cause.

[0017] Preferably, in the purification method of konjac alkaloids, the enzymatic hydrolysis temperature is set by the following method:

[0018] The initial enzymatic hydrolysis temperature was set at 36℃;

[0019] Real-time monitoring of the temperature of the enzymatic hydrolysis system;

[0020] When the temperature exceeds 36.5℃ for 10 minutes, the constant temperature water bath will automatically reduce its heating power by 20% of the current power.

[0021] When the temperature is below 35.5℃ for 10 minutes, the constant temperature water bath will automatically increase the heating power by 20% of the current power.

[0022] If the temperature fluctuates abnormally by more than 1°C, stop the enzymatic hydrolysis immediately and start again.

[0023] Preferably, in the purification method for konjac alkaloids, the determination of when the alkaloid release curve flattens out and stops increasing, while the amount of by-products begins to rise, is made by the following method:

[0024] The method for judging that the alkaloid release curve tends to flatten and no longer increases is as follows: Assuming that the alkaloid release amount detected for the first time is set as 100%, if the increase in alkaloid release amount detected in two consecutive subsequent tests is less than 5%, the curve is considered to have flattened and no longer increased.

[0025] The determination method for the start of an increase in by-product generation is that any of the following conditions are met:

[0026] Oligosaccharides: Detected using the anthrone-sulfuric acid method, at a wavelength of 620 nm, if the absorbance increases from 0.2 to above 0.5 within 1 hour;

[0027] Galacturonic acid: Detected using the m-hydroxybiphenyl method, the absorbance at 520 nm wavelength increased from 0.1 to over 0.35 within 1 hour;

[0028] Peptides and amino acids: Detected using the ninhydrin colorimetric method, the absorbance increased from 0.3 to over 0.6 within 1 hour at a wavelength of 570 nm.

[0029] Preferably, in the purification method of konjac alkaloids, step one further includes:

[0030] After the enzymatic hydrolysis, an ultrasonic treatment is performed simultaneously at three frequencies: 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 does not exceed 70°C. The hydrolysate is then separated.

[0031] In step two, the extraction temperature is 20~70℃, the extraction pressure is 10~60MPa, the extraction time is 1~6 hours, the extraction fluid flow rate is 1.0~60L / h, and the amount of entrainer ethanol is 10~200mL / 100g CO2.

[0032] In step three, the separation pressure in the separation vessel is 6~10 MPa and the separation temperature is 32~38℃, so that the supercritical carbon dioxide fluid and alkaloids are separated.

[0033] Preferably, the purification method for konjac alkaloids further includes:

[0034] Step 4: The konjac alkaloid extract is dried to obtain konjac alkaloids.

[0035] Konjac alkaloids, which are prepared by any one of the methods described above.

[0036] For example, the application of konjac alkaloids prepared by any one of these methods in pest control.

[0037] Preferably, in the application, konjac alkaloids, chitinase and water are mixed to form a pesticide formulation which is then sprayed onto the plant surface, wherein the mass ratio of konjac alkaloids to chitinase is 1:1.

[0038] Preferably, in the application described, the pesticide formulation further includes eugenol, and by weight, the pesticide formulation comprises: 20-40 parts of konjac alkaloids, 20-40 parts of chitinase, 5-10 parts of eugenol, and 60-100 parts of water.

[0039] Preferably, in the application described, the pesticide formulation further includes 5 to 10 parts of proline.

[0040] The present invention has at least the following beneficial effects:

[0041] Because konjac cell walls are complex and tough, hindering the release of alkaloids, this invention first uses cellulase, pectinase, and protease to specifically target cell wall components and large protein molecules, disrupting their structure and making them porous, thus opening dissolution channels for alkaloids. Due to the gentle nature of this process, the structure and activity of the alkaloids are preserved. The cavitation effect of ultrasound causes bubble rupture, generating impact force that further breaks down the fragile cell walls treated with the enzymes. Then, using carbon dioxide as a supercritical fluid, supercritical extraction is performed under specific pressure and temperature. This method has good solubility for alkaloids but weak solubility for impurities, allowing for deep penetration into the treated konjac material, significantly improving the extraction rate and purity of konjac alkaloids, providing strong support for their in-depth development and utilization. Furthermore, cellulase decomposes cellulose to produce oligosaccharides and other small-molecule sugars; pectinase hydrolyzes pectin to produce galacturonic acid; and protease produces peptides and amino acids. Therefore, this invention uses dynamic monitoring of the enzymatic hydrolysis system and dynamic adjustment strategies for enzymatic hydrolysis conditions to reduce the presence of impurities during enzymatic hydrolysis, preventing them from affecting the separation effect of chloroform extraction and the effectiveness of supercritical extraction.

[0042] This invention utilizes eugenol, which allows it to more easily penetrate the insect's body and exert its contact-killing effect. Konjac alkaloids and chitinases also penetrate the insect's epidermis more readily, enhancing their effectiveness within the insect's body. Proline strengthens the plant's physiological state, helping it to better absorb and transport konjac alkaloids, chitinases, and other components, enabling these active substances to be transported more quickly to the affected areas, thereby improving the overall control effect.

[0043] The konjac alkaloid purification method of this invention has the advantages of simple operation, high extraction efficiency, and good purification effect, which can significantly improve the purity of konjac alkaloids. The konjac alkaloids of this invention have significant effects in controlling pests, are environmentally friendly, and do not induce pesticide resistance, providing a new approach for the development of green plant-based pesticides.

[0044] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0046] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0047] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0048] This invention provides a method for purifying konjac alkaloids, comprising the following steps:

[0049] Step 1: Wash and peel the fresh konjac tubers, then cut them into small pieces and freeze-dry them. After drying, grind the konjac tubers to approximately 10-200 mesh using a grinder, accurately weighing 200g. Then, place them in 2400mL of phosphate buffer solution (pH 7.0) and soak for 1-5 hours to allow the raw material to fully swell, facilitating the subsequent action of enzymes.

[0050] A compound enzyme preparation, consisting of cellulase, pectinase, and protease in a ratio of 2-3:1:1, is added to the soaked konjac raw material suspension, with a total enzyme addition of 20-80 U per gram of raw material. The reaction system is then transferred to a constant-temperature shaker at 160-200 rpm. Under these conditions, the compound enzymes work synergistically to effectively decompose cell walls, pectin, and some proteins in the konjac cells, promoting the release of alkaloids into the solution.

[0051] Among them, the real-time monitoring indicators and methods during enzymatic hydrolysis are as follows:

[0052] Alkaloid release monitoring: High performance liquid chromatography-mass spectrometry (HPLC-MS) was used. Every 30 minutes, 1-2 mL of sample was taken from the enzymatic hydrolysis system, centrifuged rapidly (10000 rpm, 5 minutes) to remove solid impurities, and the supernatant was taken for HPLC-MS analysis to determine the alkaloid release.

[0053] Byproduct generation monitoring: For small molecule sugars such as oligosaccharides, 1 mL of enzymatically hydrolyzed sample was taken every hour. The anthrone-sulfuric acid method was used, with the anthrone-sulfuric acid reagent added and the mixture heated in a boiling water bath for 10 minutes. After cooling, the absorbance was measured at 620 nm, and the oligosaccharide content was calculated using a standard curve. For galacturonic acid, the m-hydroxybiphenyl method was used, with 1 mL of sample reacting with the m-hydroxybiphenyl reagent and the absorbance measured at 520 nm for quantification. For peptides and amino acids, the ninhydrin colorimetric method was used, with 1 mL of sample being heated with the ninhydrin reagent and the absorbance measured at 570 nm to determine the content.

[0054] Enzymatic hydrolysis system status monitoring: The temperature and pH of the enzymatic hydrolysis system were monitored in real time using a temperature sensor with an accuracy of ±0.1℃ and a pH electrode with an accuracy of ±0.01℃. Temperature data was recorded every 5 minutes and pH data was recorded every 10 minutes.

[0055] Dynamic adjustment strategy for enzymatic hydrolysis conditions:

[0056] Temperature adjustment: The initial setting for enzymatic hydrolysis temperature is 36℃, with an allowable fluctuation range of 36℃±0.5℃. If the temperature exceeds 36.5℃ for 10 minutes, the constant temperature water bath will automatically reduce the heating power by 20% of the current power. If the temperature falls below 35.5℃ 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℃, the enzymatic hydrolysis will be stopped immediately, the equipment will be checked and adjusted, and then restarted.

[0057] pH adjustment: The initial pH setting is 7.0, with an allowable fluctuation range of 6.9-7.1. When the pH is higher than 7.1, slowly add 0.01 mol / L hydrochloric acid solution to adjust it, with each addition not exceeding 0.5 mL. Stir well after each addition, and measure the pH again after 5 minutes until the pH returns to the set range. When the pH is lower than 6.9, slowly add 0.01 mol / L sodium hydroxide solution, using the same method as when adjusting the pH for excessively high pH.

[0058] Operating procedures:

[0059] Preparation stage: calibrate detection instruments such as HPLC-MS and spectrophotometer, check equipment such as constant temperature water bath and pH meter and calibrate temperature and pH measurement functions, prepare experimental reagents such as pH 7.0 phosphate buffer, anthrone-sulfuric acid reagent, m-hydroxybiphenyl reagent, and ninhydrin reagent, weigh appropriate amounts of konjac powder, cellulase, pectinase and protease, and mix them evenly according to the proportion for later use.

[0060] Enzymatic hydrolysis stage: Add konjac powder to a reaction vessel containing phosphate buffer, stir evenly to allow the konjac powder to fully swell, add enzyme preparation, start the constant temperature water bath and stirring device to start the enzymatic hydrolysis reaction, and at the same time turn on the temperature sensor, pH electrode and data recording device to monitor the status of the enzymatic hydrolysis system in real time. Collect enzymatic hydrolysis samples on time according to the monitoring time interval requirements to detect the amount of alkaloid release and by-product generation.

[0061] Adjustment and Termination Stage: Based on real-time monitoring data, temperature and pH are dynamically adjusted according to the adjustment strategy. Changes in alkaloid release and by-product generation are continuously monitored. The enzymatic hydrolysis reaction is terminated in a timely manner according to the rules for determining the hydrolysis time. After the reaction is terminated, the hydrolysate is rapidly cooled and the hydrolysate is further processed.

[0062] Recording and summarizing: Record the monitoring data of each enzymatic hydrolysis experiment in detail, including the amount of alkaloids released, the amount of by-products generated, and the changes in temperature and pH over time. Record the time, method and magnitude of each adjustment to the enzymatic hydrolysis conditions. After the experiment, summarize the experience of dynamically adjusting the enzymatic hydrolysis conditions, analyze the experimental results, provide a reference for subsequent experiments and production, and continuously optimize the enzymatic hydrolysis process.

[0063] After the reaction is complete, transfer the reaction solution to a centrifuge tube and centrifuge at 9600 rpm for 10-15 minutes to ensure complete solid-liquid separation. Carefully collect the supernatant and extract with chloroform 3-4 times, using 400 mL of chloroform each time. During extraction, the alkaloids will transfer to the chloroform phase. Combine the chloroform extracts and recover the chloroform using a vacuum distillation apparatus to obtain crude konjac alkaloids.

[0064] Step 2: The crude konjac alkaloids are loaded into the extraction vessel of a supercritical fluid extraction device. Carbon dioxide is used as the supercritical fluid, and the entrainer is an aqueous ethanol solution with a volume fraction of 50%~70%. The extraction temperature is 20~70℃, the extraction pressure is 10~60MPa, the extraction time is 1~6 hours, the extraction fluid flow rate is 1.0~60L / h, and the amount of entrainer ethanol is 10~200mL / 100g CO2.

[0065] Step 3: After extraction, the mixed fluid containing konjac alkaloids is introduced into the separation vessel. 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℃ to obtain konjac alkaloid extract.

[0066] Optionally, the enzymatic hydrolysis time is set by the following method:

[0067] The initial estimated enzymatic hydrolysis time is 3-4 hours;

[0068] During the enzymatic hydrolysis process, the release of alkaloids and the amount of byproducts were monitored in real time: using high performance liquid chromatography-mass spectrometry, 1-2 mL of sample was taken from the enzymatic hydrolysis system every 30 minutes and centrifuged rapidly at 10,000 rpm for 5 minutes to remove solid impurities. The supernatant was then analyzed by HPLC-MS to determine the release of alkaloids.

[0069] For small molecule sugars such as oligosaccharides, galacturonic acid, peptides and amino acid byproducts, the corresponding monitoring methods are as follows: for oligosaccharides, the anthrone-sulfuric acid method is used; for galacturonic acid, the m-hydroxybiphenyl method is used; and for peptides and amino acids, the ninhydrin colorimetric method is used. 1 mL of enzymatically digested sample is taken every 1 hour for detection.

[0070] When the alkaloid release curve flattens out and stops increasing, while the amount of byproducts begins to rise, the enzymatic hydrolysis is considered to have reached its optimal state, and the hydrolysis is terminated at this point. If this state is reached within 3 to 4 hours, the hydrolysis is terminated earlier.

[0071] If the condition is not met after 4 hours, continue monitoring for another hour. If the termination condition is still not met, stop the enzymatic hydrolysis, quickly cool the hydrolysate, and perform subsequent processing on the hydrolysate while analyzing the cause.

[0072] Optionally, the enzymatic hydrolysis temperature is set by the following method:

[0073] The initial enzymatic hydrolysis temperature was set at 36℃, with an allowable fluctuation range of 36℃ ± 0.5℃.

[0074] The temperature of the enzymatic hydrolysis system was monitored in real time using a temperature sensor with an accuracy of ±0.1℃, and the temperature was recorded every 5 minutes.

[0075] When the temperature exceeds 36.5℃ for 10 minutes, the constant temperature water bath will automatically reduce its heating power by 20% of the current power.

[0076] When the temperature is below 35.5℃ for 10 minutes, the constant temperature water bath will automatically increase the heating power by 20% of the current power.

[0077] If the temperature fluctuates abnormally by more than ±1℃, stop the enzymatic hydrolysis immediately, check the equipment, adjust it, and then restart.

[0078] Optionally, when the alkaloid release curve flattens out and stops increasing, while the amount of byproducts begins to rise, this is determined by the following method:

[0079] The method for judging that the alkaloid release curve tends to flatten and no longer increases is as follows: Assuming that the alkaloid release amount detected for the first time is set as 100%, if the increase in alkaloid release amount detected in two consecutive subsequent tests is less than 5%, the curve is considered to have flattened and no longer increased.

[0080] The determination method for the start of an increase in by-product generation is that any of the following conditions are met:

[0081] Oligosaccharides: Using the anthrone-sulfuric acid method, if the absorbance rapidly increases from 0.2 to above 0.5 within 1 hour at a wavelength of 620nm;

[0082] Galacturonic acid: Using the m-hydroxybiphenyl method, the absorbance at a wavelength of 520 nm rapidly increased from 0.1 to over 0.35 within 1 hour;

[0083] Peptides and amino acids: Using the ninhydrin colorimetric method, the absorbance at a wavelength of 570 nm rapidly increased from 0.3 to over 0.6 within 1 hour.

[0084] Optionally, it also includes: step four, drying the konjac alkaloid extract to obtain konjac alkaloids.

[0085] Optionally, step one further includes: after the enzymatic hydrolysis, ultrasonic treatment is performed simultaneously at three frequencies: 15~25 kHz, 25~35 kHz, and 40~50 kHz, with an ultrasonic power of 100~500 W, an ultrasonic time of 5~70 min, and an ultrasonic temperature not exceeding 70℃, after which the enzymatic hydrolysate is separated.

[0086] 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:

[0087] Example 1

[0088] A method for purifying konjac alkaloids includes the following steps:

[0089] 1) Wash and peel the fresh konjac tubers, then cut them into small pieces and freeze-dry them. After drying, grind the konjac tubers to about 100 mesh using a grinder, and accurately weigh 200g. Then place them in 2400mL of phosphate buffer solution with a pH of 7.0 and soak for 3 hours to allow the raw material to fully swell, which is beneficial for the subsequent action of biological enzymes.

[0090] A compound enzyme preparation, consisting of cellulase, pectinase, and protease in a ratio of 2.5:1:1, was added to the soaked konjac raw material suspension at a total enzyme addition of 50 U per gram of raw material. The reaction system was transferred to a constant-temperature shaker, set at 35.5–36.5 °C and 180 rpm. During enzymatic hydrolysis, real-time monitoring of indicators and methods was conducted.

[0091] Alkaloid release monitoring: High performance liquid chromatography-mass spectrometry (HPLC-MS) was used. Every 30 minutes, 1-2 mL of sample was taken from the enzymatic hydrolysis system, centrifuged rapidly (10000 rpm, 5 minutes) to remove solid impurities, and the supernatant was taken for HPLC-MS analysis to determine the alkaloid release.

[0092] Monitoring of the production of byproduct oligosaccharides and other small molecule sugars: 1 mL of enzymatic hydrolysis sample was taken every 1 hour. The anthrone-sulfuric acid method was used. After adding the anthrone-sulfuric acid reagent, the sample was heated in a boiling water bath for 10 minutes. After cooling, the absorbance was measured at a wavelength of 620 nm. The oligosaccharide content was calculated through the standard curve.

[0093] pH adjustment: The initial pH setting is 7.0, with an allowable fluctuation range of 6.9-7.1. When the pH is higher than 7.1, slowly add 0.01 mol / L hydrochloric acid solution to adjust it, with each addition not exceeding 0.5 mL. Stir well after each addition, and measure the pH again after 5 minutes until the pH returns to the set range. When the pH is lower than 6.9, slowly add 0.01 mol / L sodium hydroxide solution, using the same method as when adjusting the pH for excessively high pH.

[0094] When the alkaloid release curve flattens out and stops increasing, and the amount of byproducts begins to rise, the enzymatic hydrolysis is considered to have reached its optimal state. At this point, the enzymatic hydrolysis is terminated. In this example, the enzymatic hydrolysis time is 3 hours.

[0095] When the alkaloid release curve flattens out and stops increasing, while the amount of byproducts begins to rise, this is determined by the following method:

[0096] The method for judging that the alkaloid release curve tends to flatten and no longer increases is as follows: Assuming that the alkaloid release amount detected for the first time is set as 100%, if the increase in alkaloid release amount detected in two consecutive subsequent tests is less than 5%, the curve is considered to have flattened and no longer increased.

[0097] The method for judging when the amount of by-products begins to increase is: at a wavelength of 620nm, if the absorbance rapidly increases from 0.2 to above 0.5 within 1 hour.

[0098] After terminating the enzymatic hydrolysis reaction, the reaction solution was transferred to a centrifuge tube and centrifuged at 9600 rpm for 12 minutes to ensure complete solid-liquid separation. The supernatant was carefully collected and extracted with chloroform 3-4 times, using 400 mL of chloroform each time. During extraction, the alkaloids transferred to the chloroform phase. The chloroform extracts were combined, and the chloroform was recovered by vacuum distillation to obtain crude konjac alkaloids.

[0099] 2) The crude konjac alkaloids were loaded into the extraction vessel of a supercritical fluid extraction device. Carbon dioxide was used as the supercritical fluid, and the entrainer was a 60% (v / v) aqueous ethanol solution. The extraction temperature was 45℃, the extraction pressure was 35 MPa, the extraction time was 3.5 hours, the extraction fluid flow rate was 30 L / h, and the amount of 50%~70% (v / v) ethanol used as the entrainer was 100 mL / 100 g CO2.

[0100] 3) After extraction, the mixed fluid containing konjac alkaloids is introduced into the separation vessel. The supercritical carbon dioxide fluid is separated from the alkaloids at a separation pressure of 8 MPa and a separation temperature of 35℃ to obtain konjac alkaloid extract.

[0101] Example 2

[0102] A method for purifying konjac alkaloids includes the following steps:

[0103] 1) Wash and peel the fresh konjac tubers, then cut them into small pieces and freeze-dry them. After drying, grind the konjac tubers to about 200 mesh using a grinder, and accurately weigh out 200g. Then place them in 2400mL of phosphate buffer solution with a pH of 7.0 and soak for 1 hour to allow the raw material to fully swell, which is beneficial for the subsequent action of biological enzymes.

[0104] A compound enzyme preparation, consisting of cellulase, pectinase, and protease in a 2:1:1 ratio, was added to the soaked konjac raw material suspension, with a total enzyme addition of 20 U per gram of raw material. The reaction system was transferred to a constant-temperature shaker at 160 rpm. During enzymatic hydrolysis, real-time monitoring of indicators and methods was conducted.

[0105] Alkaloid release monitoring: Same as in Example 1;

[0106] Monitoring of galacturonic acid production by-product: The m-hydroxybiphenyl method was used, where 1 mL of sample was reacted with m-hydroxybiphenyl reagent, and the absorbance was measured at 520 nm for quantification; for peptides and amino acids, the ninhydrin colorimetric method was used, where 1 mL of sample was heated with ninhydrin reagent, and the absorbance was measured at 570 nm to determine the content.

[0107] Enzymatic hydrolysis system status monitoring: The temperature and pH of the enzymatic hydrolysis system were monitored in real time using a temperature sensor with an accuracy of ±0.1℃ and a pH electrode with an accuracy of ±0.01℃. Temperature data was recorded every 5 minutes and pH data was recorded every 10 minutes.

[0108] Dynamic adjustment strategy for enzymatic hydrolysis conditions:

[0109] Temperature adjustment: The initial enzymatic hydrolysis temperature is set at 36℃, with an allowable fluctuation range of 36℃ ± 0.5℃. When the temperature exceeds 36.5℃ for 10 minutes, the constant temperature water bath automatically reduces the heating power by 20% of the current power. When the temperature falls below 35.5℃ for 10 minutes, the constant temperature water bath automatically increases the heating power by 20% of the current power.

[0110] pH adjustment: The initial pH setting is 7.0, with an allowable fluctuation range of 6.9-7.1. When the pH is higher than 7.1, slowly add 0.01 mol / L hydrochloric acid solution to adjust it, with each addition not exceeding 0.5 mL. Stir well after each addition, and measure the pH again after 5 minutes until the pH returns to the set range. When the pH is lower than 6.9, slowly add 0.01 mol / L sodium hydroxide solution, using the same method as when adjusting the pH for excessively high pH.

[0111] When the alkaloid release curve flattens out and stops increasing, and the amount of byproducts begins to rise, the enzymatic hydrolysis is considered to have reached its optimal state. At this point, the enzymatic hydrolysis is terminated. In this example, the enzymatic hydrolysis time is 4 hours.

[0112] When the alkaloid release curve flattens out and stops increasing, while the amount of byproducts begins to rise, this is determined by the following method:

[0113] The method for judging that the alkaloid release curve tends to flatten and no longer increases is as follows: Assuming that the alkaloid release amount detected for the first time is set as 100%, if the increase in alkaloid release amount detected in two consecutive subsequent tests is less than 5%, the curve is considered to have flattened and no longer increased.

[0114] The method for judging when the amount of by-products begins to increase is: at a wavelength of 520nm, the absorbance increases from 0.1 to above 0.35 within 1 hour.

[0115] After enzymatic hydrolysis, the solution is subjected to ultrasonic treatment at three frequencies of 15 kHz, 25 kHz, and 40 kHz simultaneously, with an ultrasonic power of 100W. After 2-3 minutes of ultrasonic treatment, there is a 1-2 minute interval, and the total ultrasonic time is 6 minutes. The ultrasonic temperature does not exceed 70℃. The hydrolysate is then separated.

[0116] The sonicated reaction solution was transferred to a centrifuge tube and centrifuged at 9600 rpm for 10 minutes to ensure complete solid-liquid separation. The supernatant was carefully collected and extracted with chloroform 3-4 times, using 400 mL of chloroform each time. During extraction, the alkaloids transferred to the chloroform phase. The chloroform extracts were combined and the chloroform was recovered by vacuum distillation to obtain crude konjac alkaloids.

[0117] 2) The crude konjac alkaloids were loaded into the extraction vessel of a supercritical fluid extraction device. Carbon dioxide was used as the supercritical fluid, and the entrainer was a 50% (v / v) aqueous ethanol solution. The extraction temperature was 20℃, the extraction pressure was 10MPa, the extraction time was 1 hour, the extraction fluid flow rate was 1.0L / h, and the amount of entrainer ethanol was 10mL / 100g CO2.

[0118] 3) After extraction, the mixed fluid containing konjac alkaloids is introduced into the separation vessel. 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 konjac alkaloid extract.

[0119] 4) Konjac alkaloids are obtained by drying the konjac alkaloid extract.

[0120] Example 3

[0121] A method for purifying konjac alkaloids includes the following steps:

[0122] 1) Wash and peel the fresh konjac tubers, then cut them into small pieces and freeze-dry them. After drying, grind the konjac tubers to about 10 mesh using a grinder, and accurately weigh 200g. Then place them in 2400mL of phosphate buffer solution with a pH of 7.0 and soak for 5 hours to allow the raw material to fully swell, which is beneficial for the subsequent action of biological enzymes.

[0123] A compound enzyme preparation, consisting of cellulase, pectinase, and protease in a 3:1:1 ratio, was added to the soaked konjac raw material suspension, with a total enzyme addition of 80 U per gram of raw material. The reaction system was transferred to a constant-temperature shaker at 200 rpm. During enzymatic hydrolysis, real-time monitoring of indicators and methods was conducted.

[0124] Alkaloid release monitoring: Same as in Example 1;

[0125] Monitoring of byproduct peptides and amino acids: The ninhydrin colorimetric method was used. 1 mL of sample was heated with ninhydrin reagent, and the absorbance was measured at a wavelength of 570 nm to determine the content.

[0126] pH adjustment: The initial pH setting is 7.0, with an allowable fluctuation range of 6.9-7.1. When the pH is higher than 7.1, slowly add 0.01 mol / L hydrochloric acid solution to adjust it, with each addition not exceeding 0.5 mL. Stir well after each addition, and measure the pH again after 5 minutes until the pH returns to the set range. When the pH is lower than 6.9, slowly add 0.01 mol / L sodium hydroxide solution, using the same method as when adjusting the pH for excessively high pH.

[0127] When the alkaloid release curve flattens out and stops increasing, while the amount of byproducts begins to rise, the enzymatic hydrolysis is considered to have reached its optimal state. In this example, the optimal state was achieved after 4.5 hours of enzymatic hydrolysis.

[0128] The method for judging that the alkaloid release curve tends to flatten and no longer increases is as follows: Assuming that the alkaloid release amount detected for the first time is set as 100%, if the increase in alkaloid release amount detected in two consecutive subsequent tests is less than 5%, the curve is considered to have flattened and no longer increased.

[0129] The method for judging when the amount of by-products begins to increase is: at a wavelength of 570nm, the absorbance increases from 0.3 to above 0.6 within 1 hour.

[0130] After enzymatic hydrolysis, the solution is subjected to ultrasonic treatment at three frequencies of 25 kHz, 35 kHz, and 50 kHz simultaneously, with an ultrasonic power of 500 W. After 2-3 minutes of ultrasonic treatment, there is a 1-2 minute interval, and the total ultrasonic time is 70 minutes. The ultrasonic temperature does not exceed 70℃. The hydrolysate is then separated.

[0131] The sonicated reaction solution was transferred to a centrifuge tube and centrifuged at 9600 rpm for 15 minutes to ensure complete solid-liquid separation. The supernatant was carefully collected and extracted with chloroform 3-4 times, using 400 mL of chloroform each time. During extraction, the alkaloids transferred to the chloroform phase. The chloroform extracts were combined and the chloroform was recovered by vacuum distillation to obtain crude konjac alkaloids.

[0132] 2) The crude konjac alkaloids were loaded into the extraction vessel of a supercritical fluid extraction device. Carbon dioxide was used as the supercritical fluid, and the entrainer was a 70% (v / v) aqueous ethanol solution. The extraction temperature was 70℃, the extraction pressure was 60MPa, the extraction time was 6 hours, the extraction fluid flow rate was 60L / h, and the amount of entrainer ethanol was 200mL / 100g CO2.

[0133] 3) After extraction, the mixed fluid containing konjac alkaloids is introduced into the separation vessel. 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 konjac alkaloid extract.

[0134] 4) Konjac alkaloids are obtained by drying the konjac alkaloid extract.

[0135] Example 4

[0136] A method for purifying konjac alkaloids includes the following steps:

[0137] 1) Wash and peel the fresh konjac tubers, then cut them into small pieces and freeze-dry them. After drying, grind the konjac tubers to about 100 mesh using a grinder, and accurately weigh 200g. Then place them in 2400mL of phosphate buffer solution with a pH of 7.0 and soak for 3 hours to allow the raw material to fully swell, which is beneficial for the subsequent action of biological enzymes.

[0138] A compound enzyme preparation, consisting of cellulase, pectinase, and protease in a ratio of 2.5:1:1, was added to the soaked konjac raw material suspension at a total enzyme addition of 50 U per gram of raw material. The reaction system was transferred to a constant-temperature shaker, with the pH set at 7.0, the temperature at 36°C, the rotation speed at 180 rpm, and the reaction time controlled at 3.5 hours. Under these conditions, the compound enzymes worked synergistically to effectively decompose cell walls, pectin, and some proteins in konjac cells, promoting the release of alkaloids into the solution.

[0139] After enzymatic hydrolysis, the solution is subjected to ultrasonic treatment at three frequencies of 20 kHz, 30 kHz, and 45 kHz simultaneously, with an ultrasonic power of 300 W. After 2-3 minutes of ultrasonic treatment, there is a 1-2 minute interval, with a total ultrasonic time of 35 minutes. The ultrasonic temperature does not exceed 70℃. The hydrolysate is then separated.

[0140] The sonicated reaction solution was transferred to a centrifuge tube and centrifuged at 9600 rpm for 12 minutes to ensure complete solid-liquid separation. The supernatant was carefully collected and extracted with chloroform 3-4 times, using 400 mL of chloroform each time. During extraction, the alkaloids transferred to the chloroform phase. The chloroform extracts were combined and the chloroform was recovered by vacuum distillation to obtain crude konjac alkaloids.

[0141] 2) The crude konjac alkaloids were loaded into the extraction vessel of a supercritical fluid extraction device. Carbon dioxide was used as the supercritical fluid, and the entrainer was a 60% (v / v) aqueous ethanol solution. The extraction temperature was 45℃, the extraction pressure was 35 MPa, the extraction time was 3.5 hours, the extraction fluid flow rate was 30 L / h, and the amount of 50%~70% (v / v) ethanol used as the entrainer was 100 mL / 100 g CO2.

[0142] 3) After extraction, the mixed fluid containing konjac alkaloids is introduced into the separation vessel. The supercritical carbon dioxide fluid is separated from the alkaloids at a separation pressure of 8 MPa and a separation temperature of 35℃ to obtain konjac alkaloid extract.

[0143] 4) Konjac alkaloids were obtained by drying the konjac alkaloid extract.

[0144] Example 5

[0145] The application of konjac alkaloids in pest control involves preparing pesticide formulations based on the following components:

[0146] The mixture contains 20 parts konjac alkaloids, 20 parts chitinase, 5 parts eugenol, 5 parts proline, and 100 parts water, wherein the mass ratio of konjac alkaloids to chitinase is 1:1.

[0147] Add the prepared konjac alkaloid powder and chitinase to water, then add eugenol and proline in sequence, followed by surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerol), and other adjuvants to form a uniform suspension or emulsion pesticide formulation. Spray onto the plant surface.

[0148] Example 6

[0149] The application of konjac alkaloids in pest control involves preparing pesticide formulations based on the following components:

[0150] The mixture contains 30 parts konjac alkaloids, 30 parts chitinase, 30 parts eugenol, 7 parts proline, and 60 parts water, wherein the mass ratio of konjac alkaloids to chitinase is 1:1.

[0151] Add the prepared konjac alkaloid powder and chitinase to water, then add eugenol and proline in sequence, followed by surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerol), and other adjuvants to form a uniform suspension or emulsion pesticide formulation. Spray onto the plant surface.

[0152] Example 7

[0153] The application of konjac alkaloids in pest control involves preparing pesticide formulations based on the following components:

[0154] The mixture contains 40 parts konjac alkaloids, 40 parts chitinase, 10 parts eugenol, 10 parts proline, and 70 parts water, wherein the mass ratio of konjac alkaloids to chitinase is 1:1.

[0155] Add the prepared konjac alkaloid powder and chitinase to water, then add eugenol and proline in sequence, followed by surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerol), and other adjuvants to form a uniform suspension or emulsion pesticide formulation. Spray onto the plant surface.

[0156] Example 8

[0157] The application of konjac alkaloids in pest control involves preparing pesticide formulations based on the following components:

[0158] The mixture contains 25 parts konjac alkaloids, 25 parts chitinase, 7 parts eugenol, 8 parts proline, and 90 parts water, with the mass ratio of konjac alkaloids to chitinase being 1:1.

[0159] Add the prepared konjac alkaloid powder and chitinase to water, then add eugenol and proline in sequence, followed by surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerol), and other adjuvants to form a uniform suspension or emulsion pesticide formulation. Spray onto the plant surface.

[0160] Comparative Example 1

[0161] A method for purifying konjac alkaloids includes the following steps:

[0162] Fresh konjac tubers were washed, peeled, and cut into small pieces, then placed in a freeze dryer for drying. The dried konjac tubers were then pulverized to about 100 mesh using a grinder, and extracted using a traditional solvent extraction method with ethanol as the solvent, refluxed at 60°C for 3 hours.

[0163] Comparative Example 2

[0164] Take 30 parts of konjac alkaloids and add 60 parts of water. Then add eugenol and proline in sequence, followed by surfactants (such as sodium dodecylbenzene sulfonate), stabilizers (such as glycerin), and other adjuvants to form a uniform suspension or emulsion pesticide formulation.

[0165] Effect verification

[0166] 1. The extraction rates of konjac alkaloids in Examples 1 to 4 and the comparative examples were determined, as shown in Table 1.

[0167] Table 1 Extraction rate of konjac alkaloids

[0168]

[0169] As shown in Table 1, the extraction rate and purification degree of konjac bio-enzymes obtained by the enzymatic hydrolysis and supercritical fluid extraction technology of this invention are significantly improved. Konjac cells are enclosed by a complex cell wall structure, which hinders the extraction of alkaloids. Bio-enzymes can specifically recognize and act on the components of the cell wall. Cellulase can decompose cellulose in the cell wall, pectinase hydrolyzes pectin, and protease can enzymatically decompose proteins encapsulating large molecules. Enzymatic hydrolysis disrupts the integrity of the cell wall, making its structure loose. Then, using carbon dioxide as a supercritical fluid, under certain pressure and temperature conditions, supercritical carbon dioxide can selectively dissolve alkaloids in konjac, while its solubility for other impurities is weak. Furthermore, after enzymatic hydrolysis, ultrasonic treatment is added as a pretreatment step, providing higher-quality raw materials for subsequent supercritical fluid extraction. After ultrasonic treatment, the konjac cell structure is disrupted, making it easier for alkaloids to contact and dissolve with the supercritical fluid, thereby improving the efficiency and effect of supercritical fluid extraction. This further improves the extraction rate and purity of the product. These three technologies work synergistically, giving full play to their respective advantages, and significantly improving the extraction rate and purity of konjac alkaloids.

[0170] 2. The experiment was conducted at a vegetable planting base, with tomatoes as the main vegetable variety. The planting area was about 2 mu (approximately 0.33 hectares). The soil fertility of the experimental field was uniform, and the cultivation and management conditions were consistent.

[0171] The pesticide formulations prepared in Examples 5, 6, 7, and 8, and the pesticide formulation prepared in Comparative Example 2, were used as five treatment groups. Each treatment group had three replicate plots, each approximately 30 square meters in size, arranged in a randomized block design. A blank control group was also included, sprayed with water. The konjac alkaloids prepared in Example 4 were used in Examples 5, 6, 7, 8, and Comparative Example 2.

[0172] Apply pesticides at the early stage of gray mold disease in tomatoes and at the early stage of diamondback moth larvae emergence. Use a backpack sprayer to spray the pesticide formulation. Dilute the pesticide formulation 500 times and spray it evenly on both sides of the leaves and the stem of the tomato plants. The application rate is about 50 ml per square meter. Apply the pesticide once every 7 days for 3 consecutive applications.

[0173] 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 to calculate the control effect. The formulas for calculating the disease index and control effect are as follows:

[0174]

[0175]

[0176] After three applications, the compound formulation treatment group achieved a control effect of 85.4% against tomato gray mold and over 90% against diamondback moth larvae in the final survey. Both were significantly higher than the single konjac alkaloid extract treatment group (Comparative Example 2) and the blank control water treatment group. Furthermore, it did not cause obvious phytotoxicity to tomato plants, indicating that the compound formulation has good control effect and safety in the field.

[0177] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for embodiments of the present invention. Other modifications can be readily implemented by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the embodiments of the present invention are not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for purifying konjac alkaloids, characterized in that, Includes the following steps: Step 1: Take konjac raw material and prepare konjac powder of 10-200 mesh. Soak the konjac powder in a phosphate buffer solution with a pH of 7.

0. Add cellulase, pectinase, and protease to the soaked konjac powder suspension for enzymatic hydrolysis. After enzymatic hydrolysis, separate the hydrolysate, collect the supernatant, and extract several times with chloroform. Combine the chloroform extracts, remove the chloroform, and obtain crude konjac alkaloids. In the enzymatic hydrolysis, the total enzyme addition is 20-80 U per gram of konjac raw material, the rotation speed is 160-200 rpm, the ratio of cellulase, pectinase, and protease is 2-3:1:1, and the mass-to-volume ratio of konjac powder to the phosphate buffer solution is 1:

1. 10~15, In the enzymatic hydrolysis, the initial pH value is set to 6.9~7.

1. When the pH value is higher than 7.1, 0.01mol / L hydrochloric acid solution is added dropwise for adjustment, with each dropwise addition not exceeding 0.5mL. After adding, the solution is stirred evenly, 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, with each dropwise addition not exceeding 0.5mL. After adding, the solution is stirred evenly, and the pH value is measured again after 5 minutes until the pH value returns to the set range. Step 2: The crude konjac alkaloid obtained in Step 1 is loaded into an extraction vessel and extracted using carbon dioxide as a supercritical fluid and an aqueous ethanol solution with a volume fraction of 50% to 70% as an entrainer. Step 3: After extraction, the mixed fluid containing konjac alkaloids is introduced into the separation vessel to separate the supercritical carbon dioxide fluid from the alkaloids, thus obtaining konjac alkaloid extract. Step one also includes: after the enzymatic hydrolysis, ultrasonic treatment is performed simultaneously at three frequencies: 15~25kHz, 25~35kHz, and 40~50kHz, with an ultrasonic power of 100~500W, an ultrasonic time of 5~70min, and an ultrasonic temperature not exceeding 70℃, after which the enzymatic hydrolysate is separated. In step two, the extraction temperature is 20~70℃, the extraction pressure is 10~60MPa, the extraction time is 1~6 hours, the extraction fluid flow rate is 1.0~60L / h, and the amount of entrainer ethanol is 10~200mL / 100g CO2. In step three, the separation pressure in the separation vessel is 6~10 MPa and the separation temperature is 32~38℃, so that the supercritical carbon dioxide fluid and alkaloids are separated.

2. The purification method for konjac alkaloids as described in claim 1, characterized in that, In the enzymatic hydrolysis, the hydrolysis time is set using the following method: The initial estimated enzymatic hydrolysis time is 3-4 hours; During the enzymatic hydrolysis process, the release of alkaloids and the amount of byproducts were monitored in real time: using high performance liquid chromatography-mass spectrometry, 1-2 mL of sample was taken from the enzymatic hydrolysis system every 30 minutes, centrifuged to remove solid impurities, and the supernatant was taken for HPLC-MS analysis to determine the release of alkaloids. For oligosaccharides, galacturonic acid, peptides and amino acid byproducts, the corresponding monitoring methods are as follows: for oligosaccharides, the anthrone-sulfuric acid method is used; for galacturonic acid, the m-hydroxybiphenyl method is used; and for peptides and amino acids, the ninhydrin colorimetric method is used. 1 mL of enzymatically digested sample is taken every 1 hour for detection. When the alkaloid release curve flattens out and stops increasing, while the amount of byproducts begins to rise, the enzymatic hydrolysis is considered to have reached its optimal state, and the hydrolysis is terminated at this point. If this state is reached within 3 to 4 hours, the hydrolysis is terminated earlier. If the condition is not met after 4 hours, continue monitoring for another hour. If the termination condition is still not met, stop the enzymatic hydrolysis, cool the hydrolysate, and perform subsequent processing on the hydrolysate. At the same time, analyze the cause.

3. The purification method for konjac alkaloids as described in claim 2, characterized in that, In the enzymatic hydrolysis, the enzymatic hydrolysis temperature is set using the following method: The initial enzymatic hydrolysis temperature was set at 36℃; Real-time monitoring of the temperature of the enzymatic hydrolysis system; When the temperature exceeds 36.5℃ for 10 minutes, the constant temperature water bath will automatically reduce the heating power by 20% of the current power. When the temperature is below 35.5℃ 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 start again.

4. The purification method of konjac alkaloids as described in claim 2, characterized in that, When the alkaloid release curve flattens out and stops increasing, while the amount of byproducts begins to rise, this is determined by the following method: The method for judging that the alkaloid release curve tends to flatten and no longer increases is as follows: Assuming that the alkaloid release amount detected for the first time is set as 100%, if the increase in alkaloid release amount detected in two consecutive subsequent tests is less than 5%, the curve is considered to have flattened and no longer increased. The determination method for the start of an increase in by-product generation is that any of the following conditions are met: Oligosaccharides: Detected using the anthrone-sulfuric acid method, at a wavelength of 620 nm, if the absorbance increases from 0.2 to above 0.5 within 1 hour; Galacturonic acid: Detected using the m-hydroxybiphenyl method, the absorbance at 520 nm wavelength increased from 0.1 to over 0.35 within 1 hour; Peptides and amino acids: Detected using the ninhydrin colorimetric method, the absorbance increased from 0.3 to over 0.6 within 1 hour at a wavelength of 570 nm.

5. Konjac alkaloids, characterized in that, It is prepared by the method as described in any one of claims 1 to 4.

6. The application of konjac alkaloids prepared according to any one of claims 1 to 4 in pest control, characterized in that, A pesticide formulation is prepared by mixing konjac alkaloids, chitinase, eugenol, proline, and water and then spraying it onto the plant surface. The pesticide formulation consists of 20-40 parts by weight of konjac alkaloids, 20-40 parts by weight of chitinase, 5-10 parts by weight of eugenol, 5-10 parts by weight of proline, and 60-100 parts by weight of water.

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