Method for extracting azadirachtin dry powder from azadirachta indica kernels

Abstract

The invention provides a method for extracting dry powder of azadirachtin from azadirachta indica kernels, which comprises the following steps: S1, selecting mature fresh azadirachta indica fruits, ripening with an ethephon aqueous solution, and then peeling to obtain azadirachta indica kernels; s2, slicing the azadirachta indica kernels, and performing freeze-drying treatment to obtain freeze-dried slices; s3, carrying out intermittent CO2 supercritical extraction on the freeze-dried tablets by adopting an organic solvent to obtain extract liquor; s4, freezing, filtering and degreasing the extract for multiple times to obtain filtrate; s5, after the filtrate is subjected to vacuum concentration, liquid hydrocarbon is added for further oil removal, and a concentrated solution is obtained; and S6, performing vacuum concentration on the concentrated solution again, and performing vacuum drying to obtain powder, thereby obtaining the azadirachtin dry powder with the azadirachtin A content of 13-20wt%. The method disclosed by the invention is relatively simple in process, low in requirement on production equipment, less in used organic solvent, high in organic solvent recovery rate, relatively low in production cost and convenient for large-scale production, especially high in azadirachtin yield, and the prepared azadirachtin product is high in purity.

Description

Technical Field

[0001] The present invention belongs to the field of natural plant extraction and preparation, and particularly relates to an industrial production method for extracting azadirachtin dry powder from neem kernels. Background Art

[0002] Neem is an evergreen arbor plant of the genus Azadirachta in the family Meliaceae. Neem trees grow in tropical and subtropical regions, mainly distributed in the tropical regions of Southeast Asia. In China, the largest amount of neem trees are planted in the territory of Yunnan Province. The seeds, bark, fruits, leaves, etc. of neem trees have been used to extract various active ingredients, and the most popular one is to extract the tetracyclic triterpenoid azadirachtin from neem tree seeds. Azadirachtin mainly contains azadirachtin A and azadirachtin B; among them, azadirachtin A accounts for ~80% of the total azadirachtin and has the strongest insecticidal activity, and azadirachtin B accounts for ~20% of the total azadirachtin. Azadirachtin as a whole has strong contact toxicity, antifeedant, repellent, stomach toxicity and inhibitory effects on growth and development on insects. Azadirachtin pesticides show strong pest resistance to many harmful insects and can control more than 200 agricultural, forestry, storage and sanitary pests, so it is considered a natural insecticide. At the same time, azadirachtin can also be used as a microbial fungicide and is safer than traditional chemical pesticides. Azadirachtin is widely recognized as the safest biological pesticide and is an efficient and harmless alternative compared with other plant or microbial insecticides. Azadirachtin pesticides, as pure plant-derived organic pesticides, can solve the problem of excessive residues caused by inorganic pesticides and are friendly to humans and the environment.

[0003] Azadirachtin in neem dry kernels is only 0.5 - 3 wt‰. In addition, neem kernels contain rich lipid substances, carbohydrate substances, etc., and these substances are mixed with the active ingredients, thereby affecting the extraction efficiency, product purity and process difficulty. Moreover, the molecular structure of azadirachtin contains unstable groups such as epoxy groups, ester groups, and double bonds, resulting in azadirachtin being extremely prone to thermal decomposition, photolysis and hydrolysis, etc. Azadirachtin will immediately decompose under the working condition of 60°C or under sunlight irradiation or in a humid environment (air humidity ≥ 40%). Neem dry kernels contain ~30 wt% of oil, and the internal energy will increase and the temperature will rise when extracting oil by pressing; in an environment of ≥35°C, the degradation rate of azadirachtin increases significantly, and azadirachtin can be completely decomposed by maintaining at 65°C for 1 - 2 h. These make it difficult to extract azadirachtin from neem kernels, with problems such as complex extraction processes, low extraction efficiency, and difficulty in stably obtaining high-purity products.

[0004] The prior art mainly uses the solvent extraction method to extract azadirachtin, and the purity of the prepared azadirachtin dry powder product is relatively low. And the solvent waste is serious, the production cost is high, and the yield is low. Summary of the Invention

[0005] The object of the present invention is to solve the problems existing in the prior art, and to provide a method for extracting azadirachtin dry powder from neem kernels, which has a relatively simple process, low equipment requirements, low production costs, is convenient for large-scale production, and has a high azadirachtin yield and high purity of the azadirachtin product.

[0006] The technical solution adopted by the present invention is as follows: A method for extracting azadirachtin dry powder from neem kernels, the method steps are as follows: S1. Select mature neem fresh fruits, ripen them with an aqueous ethephon solution and then perform peeling treatment to obtain neem kernels; S2. Freeze-dry the neem kernels to obtain freeze-dried slices. This step can remove excess moisture to shorten the subsequent concentration time; S3. Use an organic solvent to perform intermittent CO 2 supercritical extraction on the freeze-dried slices to obtain an extraction solution; S4. Perform multiple freeze-filtration degreasing on the extraction solution to obtain a filtrate; S5. Concentrate the degreased extraction solution in vacuo at 35-50 °C to 1 / 3-1 / 4 of the original volume, and then add a liquid hydrocarbon to further remove oil to obtain a concentrated solution; S6. Concentrate the concentrated solution again in vacuo at 35-50 °C to 1 / 2-1 / 3 of the original volume, and then dry it into powder in vacuo at 25-30 °C to obtain azadirachtin dry powder with 13-20 wt% azadirachtin A.

[0007] Further, the peeling treatment method described in the above step S1 is to spray the mature neem fresh fruits with an aqueous ethephon solution with a mass concentration of 0.4-1 wt%, and the mass ratio of the aqueous ethephon solution to the fresh fruits is 1:100-1:500; after spraying, keep them in an environment of 15-25 °C for 2-4 days, and then rinse with water to remove the peel and pulp to obtain neem kernels.

[0008] Further, the freeze-drying treatment described in the above step S2 is to freeze the sliced neem kernels at -25 to -30 °C for 12-24 h, and sublime them at 25-30 °C for 12-48 h; among them, the cooling rate of freezing is 5 °C / min, and the heating rate of sublimation is 2 °C / min.

[0009] Further, the CO described in the above step S3 2Supercritical extraction is carried out 2 to 3 times. Among them, for the first extraction, the seeds and the organic solvent are mixed and extracted at a mass ratio of 1:0.5 to 4; for the second extraction, the extraction residue remaining after the first extraction and the dosage form organic solvent are mixed and extracted at a mass ratio of 1:0.5 to 2; for the third extraction, the extraction residue remaining after the second extraction and the organic solvent are mixed and extracted at a mass ratio of 1:0.5 to 1; the extraction liquids of the first extraction, the second extraction, and the third extraction are mixed; the extraction conditions for the first extraction, the second extraction, and the third extraction are all extraction at 30 to 40 °C for 2 to 6 h under a high-pressure environment of 30 to 45 Mpa; the organic solvent is one or more of alcohols, ketones, esters, and nitriles with a purity of more than 95%.

[0010] Furthermore, the organic solvent is one or more of methanol, ethanol, propanol, acetonitrile, methyl acetate, ethyl acetate, dichloromethane, and acetone.

[0011] Furthermore, the multiple degreasing of the extraction liquid by freezing in the above step S4 is to filter and degrease to obtain a filtrate after the extraction liquid is frozen at -5 to -20 °C for 6 to 24 h for 1 to 6 times.

[0012] Furthermore, the method for preparing the concentrated liquid in the above step S5 is to concentrate the filtrate obtained in step S4 to 1 / 3 to 1 / 4 of the original volume at 35 to 50 °C in a vacuum environment of -0.05 to -0.2 Mpa, then add 0.5 to 3 times the volume of liquid hydrocarbon for mixing and then separate the oil by stratification, and select the lower-layer concentrated liquid for use; the liquid hydrocarbon is one or more of pentane, heptane, octane, nonane, paraffin oil, petroleum ether, and n-hexane.

[0013] Furthermore, the method for preparing the azadirachtin dry powder in the above step S6 is to concentrate the lower-layer concentrated liquid obtained in step S5 to 1 / 2 to 1 / 3 of the original volume at 35 to 50 °C in a vacuum environment of -0.05 to -0.2 Mpa, and then perform vacuum drying. The vacuum drying conditions are to maintain at -0.05 to -0.2 Mpa at 20 to 30 °C for 12 to 24 h.

[0014] The method of the present invention, according to the characteristics of neem kernels, adopts a scientific and reasonable process, and preferably removes lipid substances, sugar substances, etc. that have an adverse effect in the process of extracting azadirachtin, effectively solving the problems of complex extraction process, low extraction efficiency, and difficulty in obtaining high-purity products caused by the easy pyrolysis, photolysis, and hydrolysis of azadirachtin. The process of the present invention is simple, and peeling, kernel slicing, freeze-drying, CO 2 supercritical extraction, freeze degreasing, vacuum concentration, vacuum drying, etc. can be completed by using existing technical equipment; CO 2The organic solvents used in the supercritical extraction and vacuum concentration processes can be recycled after simple treatment, and the waste rate is only 10-30%. The extraction process of the present invention has low requirements for production equipment, low process control difficulty, less solvent consumption, low production cost, and is convenient for large-scale production. In particular, when using the method of the present invention to extract azadirachtin, the extraction rate is as high as over 90%, the oil removal rate in the neem kernels is as high as over 95%, and very little organic solvent is used. Taking azadirachtin A as the detection sample for reference, the yield of azadirachtin A of the present invention can be as high as over 2%, and the purity of the obtained azadirachtin dry powder can reach 13-20 wt% and the quality is stable, far superior to the azadirachtin prepared by the existing solvent extraction method. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the RP-HPLC chart of the extraction sample of azadirachtin A prepared in the embodiment of the present invention and the azadirachtin A standard product. DETAILED DESCRIPTION OF THE INVENTION

[0016] The content of the present invention will be further elaborated below in conjunction with the embodiments and the drawings of the specification. Example 1

[0017] A method for extracting azadirachtin dry powder from neem kernels comprises the following steps: (1) Select ripe neem fresh fruits and spray them with an aqueous solution of ethephon with a mass concentration of 1 wt%. The mass ratio of the ethephon aqueous solution to the fresh fruits is 1:200. After spraying, keep them at 20°C for 3 days and then rinse with water to remove the pericarp and pulp outside the kernels to obtain neem kernels; (2) Cut the neem kernels into slices and conduct freeze-drying treatment for 72 h to obtain freeze-dried slices. The freeze-drying process is freezing at -30°C for 24 h and sublimating at 25°C for 48 h. The cooling rate of freezing is 5°C / min, and the heating rate of sublimation is 2°C / min; (3) Conduct two times of CO 2 supercritical extraction on the freeze-dried slices. The first extraction is to mix and extract the seeds and methanol with a purity of 95% at a mass ratio of 1:1. The second extraction is to mix and extract the extraction residue remaining after the first extraction and methanol with a purity of 95% at a mass ratio of 1:0.5. Mix the extraction liquids of the first extraction and the second extraction for later use. The extraction conditions for both extractions are to extract at 35°C for 4 h under a high-pressure environment of 45 Mpa. The CO 2 supercritical extraction adopts the existing technical equipment and methods; (4) Freeze the mixed extraction liquid at -18°C for 12 h and then filter to remove fat. Repeat the operation three times to obtain the filtrate; (5) Concentrate the filtrate to 1 / 3 of its original volume at 45°C under a vacuum of -0.2 Mpa to obtain a primary concentrate. Then add an equal volume of pentane for mixing and layering to remove oil, and select the lower-layer concentrate for use. (6) Concentrate the obtained lower-layer concentrate to 1 / 3 of its original volume at 45°C under a vacuum of -0.09 Mpa and then perform vacuum drying. The vacuum drying conditions are -0.09 Mpa maintained at 20°C for 24 h, and finally obtain azadirachtin dry powder. Weigh 0.1 g to prepare a sample and make up the volume to 10 ml with methanol. Use reverse-phase high-performance liquid chromatography (RP-HPLC) to detect the azadirachtin content in the azadirachtin dry powder, and the measured azadirachtin A content is 20 wt%.

[0018] Select a Shimadzu LC-2030, Kromasil-C18 chromatographic column analysis column (4.0 mm x 250 mm, 10 μm), use methanol:water (V:V) = 65:35 as the mobile phase, the flow rate is 0.8 mL / min, the detection wavelength is 213 nm, and the detection temperature is 30°C. Take samples of the azadirachtin A standard product and the azadirachtin dry powder prepared in this example for chromatographic analysis. The high-performance liquid chromatography of the azadirachtin dry powder sample and the standard product in this example is as Figure 1 shown. Azadirachtin A in the azadirachtin dry powder sample of this example is effectively separated and has good reproducibility. The retention time of azadirachtin A in the preparation is at ~11.68 min, and the retention time of the azadirachtin A standard product is at ~11.73 min. Example 2

[0019] A method for extracting azadirachtin dry powder from neem kernels, the steps are as follows: (1) Select mature neem fresh fruits and spray them with an aqueous solution of ethephon with a mass concentration of 1 wt%. The mass ratio of the ethephon aqueous solution to the fresh fruits is 1:200. After spraying, keep them at 25°C for 2 days and then rinse with water to remove the pericarp and pulp outside the kernels to obtain neem kernels. (2) Cut the neem kernels into slices and perform freeze-drying treatment to obtain freeze-dried slices. The freeze-drying process is freezing at -28°C for 18 h and sublimating at 30°C for 24 h. The cooling rate of freezing is 5°C / min, and the heating rate of sublimation is 2°C / min. (3) Perform two CO 2 supercritical fluid extractions on the freeze-dried slices. The first extraction is to mix the seeds with acetonitrile with a purity of 98% at a mass ratio of 1:1 for extraction. The second extraction is to mix the extraction residue remaining from the first extraction with acetonitrile with a purity of 98% at a mass ratio of 1:1 for extraction. Mix the extraction liquids from the first extraction and the second extraction for use. The extraction conditions for both extractions are extraction at 40 Mpa under a high-pressure environment at 40°C for 2 h. (4) Freeze the mixed extract at -20°C for 6 hours and then filter to remove fat. Repeat the operation four times to obtain the filtrate. (5) The filtrate was concentrated to 1 / 3 of the original volume at 50°C under a vacuum environment of -0.07 MPa to obtain a primary concentrated solution, and then 0.5 times the volume of paraffin oil was added for mixed and layered deoiling, and the lower concentrated solution was selected for standby use; (6) The lower concentrated liquid was concentrated to 1 / 3 of its original volume at 45°C under a vacuum environment of -0.07 Mpa and then vacuum dried. The vacuum drying conditions were -0.07 Mpa and 20°C for 24 hours to finally obtain azadirachtin dry powder. 0.1 g of the sample was weighed and the volume was made up to 10 ml with methanol. The azadirachtin content in the azadirachtin dry powder was detected by reverse phase high performance liquid chromatography (RP-HPLC). The azadirachtin A content was measured to be 17 wt%.

[0020] The same method as in Example 1 was used to sample the azadirachtin A standard product and the azadirachtin dry powder prepared in this example for chromatographic analysis. The HPLC results of the azadirachtin dry powder sample and the standard product in this example were as follows: Figure 1 As shown, azadirachtin A in the azadirachtin dry powder sample of this example was effectively separated with good reproducibility, the retention time of azadirachtin A in the preparation was ~11.60 min, and the retention time of azadirachtin A standard was ~11.73 min. Example 3

[0021] A method for extracting azadirachtin dry powder from neem kernels, comprising the following steps: (1) Selecting mature fresh neem fruits and spraying them with a 0.8 wt% ethephon aqueous solution, the mass ratio of the ethephon aqueous solution to the fresh fruit being 1:100, maintaining the temperature at 20°C for 4 days, and then rinsing with clean water to remove the peel and pulp outside the seed kernel, thereby obtaining the neem kernel; (2) Slice the neem kernels and freeze-dry them to obtain freeze-dried slices. The freeze-drying process is freezing at -25°C for 24 hours and sublimation at 28°C for 36 hours. The cooling rate of freezing is 5°C / min, and the heating rate of sublimation is 2°C / min; (3) The freeze-dried tablets were subjected to CO 2 Supercritical extraction. The first extraction is to mix the seeds with 96% pure ethyl acetate in a mass ratio of 1:2. The second extraction is to mix the remaining extraction residue of the first extraction with 96% pure ethyl acetate in a mass ratio of 1:1. The extracts of the first extraction and the second extraction are mixed for later use. The extraction conditions of both extractions are 30°C under a high pressure environment of 30Mpa for 6 hours; (4) Freeze the mixed extract at -15°C for 24 hours and then filter to remove fat. Repeat the operation three times to obtain the filtrate. (5) The filtrate is concentrated to 1 / 3 of the original volume at 40°C under a vacuum environment of -0.06 MPa to obtain a primary concentrated solution, and then 2 times the volume of petroleum ether is added for mixed stratification and oil removal, and the lower concentrated solution is selected for standby use; (6) The lower concentrated liquid was concentrated to 1 / 2 of its original volume at 50°C under a vacuum environment of -0.05Mpa and then vacuum dried. The vacuum drying conditions were -0.05Mpa and 25°C for 18 hours to finally obtain azadirachtin dry powder. 0.1g of the sample was weighed and the volume was made up to 10ml with methanol. The azadirachtin content in the azadirachtin dry powder was detected by reverse phase high performance liquid chromatography (RP-HPLC). The azadirachtin A content was measured to be 15wt%.

[0022] The same method as in Example 1 was used to sample the azadirachtin A standard product and the azadirachtin dry powder prepared in this example for chromatographic analysis. The HPLC results of the azadirachtin dry powder sample and the standard product in this example were as follows: Figure 1 As shown, azadirachtin A in the azadirachtin dry powder sample of this example was effectively separated with good reproducibility, the retention time of azadirachtin A in the preparation was ~11.64 min, and the retention time of azadirachtin A standard was ~11.73 min. Example 4

[0023] A method for extracting azadirachtin dry powder from neem kernels, comprising the following steps: (1) Selecting mature fresh neem fruits and spraying them with a 0.6 wt% ethephon aqueous solution, with the mass ratio of the ethephon aqueous solution to the fresh fruit being 1:200, spraying them continuously at 25°C for 4 days, then rinsing them with clean water to remove the peel and pulp outside the seed kernel, thereby obtaining neem kernels; (2) Slice the neem kernels and freeze-dry them for 72 hours to obtain freeze-dried slices. The freeze-drying process is freezing at -30°C for 12 hours and sublimation at 30°C for 12 hours. The cooling rate of freezing is 5°C / min, and the heating rate of sublimation is 2°C / min; (3) The freeze-dried slices were subjected to three CO 2 Supercritical extraction. The first extraction is to mix the seeds with 95% pure acetone at a mass ratio of 1:2. The second extraction is to mix the remaining extraction residue of the first extraction with 95% pure acetone at a mass ratio of 1:2. The third extraction is to mix the remaining extraction residue of the second extraction with 95% pure acetone at a mass ratio of 1:0.5. The extracts of the first extraction, the second extraction and the third extraction are mixed for use. The extraction conditions of the three extractions are all 45Mpa high pressure environment at 35℃ for 4 hours; (4) Freeze the mixed extract at -18°C for 24 hours and then filter to remove fat. Repeat the operation four times to obtain the filtrate. (5) The filtrate is concentrated to 1 / 4 of the original volume at 35°C under a vacuum environment of -0.05 MPa to obtain a primary concentrated solution, and then an equal volume of n-hexane is added to mix and separate the mixture for oil removal. The lower concentrated solution is selected for standby use; (6) The lower concentrated liquid was concentrated to 1 / 3 of its original volume at 35°C under a vacuum environment of -0.2Mpa and then vacuum dried. The vacuum drying conditions were -0.2Mpa and 30°C for 12 hours to finally obtain azadirachtin dry powder. 0.1g of the sample was weighed and the volume was made up to 10ml with methanol. The azadirachtin content in the azadirachtin dry powder was detected by reverse phase high performance liquid chromatography (RP-HPLC). The azadirachtin A content was measured to be 14wt%.

[0024] The same method as in Example 1 was used to sample the azadirachtin A standard product and the azadirachtin dry powder prepared in this example for chromatographic analysis. The HPLC results of the azadirachtin dry powder sample and the standard product in this example were as follows: Figure 1 As shown, azadirachtin A in the azadirachtin dry powder sample of this example was effectively separated with good reproducibility, the retention time of azadirachtin A in the preparation was ~11.68 min, and the retention time of azadirachtin A standard was ~11.73 min.

[0025] Unless otherwise specified, all percentages described in the present invention are by mass percentages.

[0026] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting azadirachtin dry powder from neem kernels, characterized in that: The steps are as follows: S1. Selecting mature fresh neem fruit, ripening with an ethephon aqueous solution and peeling the fruit to obtain neem kernels; S2. Slicing the neem kernels and freeze-drying them to obtain freeze-dried sheets; S3. Using an organic solvent to perform intermittent CO2 supercritical extraction on the freeze-dried sheet to obtain an extract; S4. The extract is subjected to multiple freeze filtration and fat removal to obtain a filtrate; S5. After the filtrate is concentrated under vacuum, a liquid hydrocarbon is added to further remove oil to obtain a concentrate; S6. The concentrated solution is vacuum concentrated again and then vacuum dried into powder to obtain azadirachtin dry powder containing 13-20 wt % of azadirachtin A.

2. The method for extracting azadirachtin dry powder from neem kernels according to claim 1, characterized in that: The peeling treatment method described in the above step S1 is to spray the ripe fresh neem fruit with an ethephon aqueous solution with a mass concentration of 0.4-1wt%, and the mass ratio of the ethephon aqueous solution to the fresh fruit is 1:100-1:500; after spraying, keep it in an environment of 15-25°C for 2-4 days, and then rinse it with clean water to remove the peel and pulp, so as to obtain the neem kernel.

3. The method for extracting azadirachtin dry powder from neem kernels according to claim 1, characterized in that: The freeze-drying treatment described in step S2 is to freeze the neem kernel slices at -25~-30℃ for 12~24 hours and sublimate at 25~30℃ for 12~48 hours; wherein the cooling rate of freezing is 5℃ / min, and the heating rate of sublimation is 2℃ / min.

4. The method for extracting azadirachtin dry powder from neem kernels according to claim 1, characterized in that: The CO2 supercritical extraction described in the above step S3 is performed twice or three times; wherein, the first extraction is to mix the seeds and the organic solvent in a mass ratio of 1:0.5~4 for extraction; the second extraction is to mix the remaining extraction residue of the first extraction with the dosage form organic solvent in a mass ratio of 1:0.5~2 for extraction; the third extraction is to mix the remaining extraction residue of the second extraction with the organic solvent in a mass ratio of 1:0.5~1 for extraction; the extracts of the first extraction, the second extraction and the third extraction are mixed; the extraction conditions of the first extraction, the second extraction and the third extraction are all 30~40℃ extraction for 2~6 hours under a high pressure environment of 30~45Mpa; the organic solvent is one or more of alcohols, ketones, lipids and nitriles with a purity of more than 95%.

5. The method for extracting azadirachtin dry powder from neem kernels according to claim 4, characterized in that: The organic solvent is one or more of methanol, ethanol, propanol, acetonitrile, methyl acetate, ethyl acetate, dichloromethane and acetone.

6. A method for extracting azadirachtin dry powder from neem kernels according to claim 1 or 4, characterized in that: The above-mentioned step S4 of subjecting the extract to multiple freeze filtration and fat removal is to freeze the extract at -5 to -20°C for 6 to 24 hours and then filter and remove fat, and obtain the filtrate after 2 to 5 cycles.

7. The method for extracting azadirachtin dry powder from neem kernels according to claim 1, characterized in that: The method for preparing the concentrated liquid in the above step S5 is to concentrate the filtrate obtained in step S4 to 1 / 3 to 1 / 4 of the original volume at 35 to 50° C. under a vacuum environment of -0.05 to -0.2 MPa, and then add 0.5 to 3 times the volume of liquid hydrocarbons to mix and then separate the layers for oil removal, and select the lower layer of concentrated liquid for standby use; the liquid hydrocarbons are one or more of pentane, heptane, octane, nonane, paraffin oil, petroleum ether, and n-hexane.

8. The method for extracting azadirachtin dry powder from neem kernels according to claim 1, characterized in that: The method for preparing azadirachtin dry powder in the above step S6 is to concentrate the lower concentrated liquid obtained in step S5 to 1 / 2~1 / 3 of the original volume under a vacuum environment of -0.05~-0.2 Mpa at 35~50°C, and then vacuum dry it. The vacuum drying conditions are -0.05~-0.2Mpa and maintained at 20~30°C for 12~24 hours.

Images