6% ginkgolic acid microemulsion and application thereof

Abstract

The invention discloses a 6% ginkgolic acid microemulsion and application thereof, and belongs to the technical field of biological control. The ginkgolic acid microemulsion is prepared from the following raw materials in percentage by mass: 6% of ginkgolic acid, 2% of absolute ethyl alcohol, 2% of n-butyl alcohol, 10% of OP10 (alkylphenol polyoxyethylene ether), 10% of DBS-Ca (calcium dodecyl benzene sulfonate) and 70% of distilled water. The 6% ginkgolic acid microemulsion is developed according to the formula. When the 6% ginkgolic acid microemulsion is diluted by 200 times and 100 times to be sprayed, the prevention and control effects of the pepper anthracnose are 76.47% and 75.73%, the prevention and control effects of the 6% ginkgolic acid microemulsion are equivalent to those of chemical pesticides, and meanwhile the 6% ginkgolic acid microemulsion has a good prevention and control effect on various plant pathogenic fungi.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and particularly relates to a 6% ginkgolic acid microemulsion and application thereof. Background Art

[0002] In recent years, the research and development of green pesticides using plant extracts as raw materials has received extensive attention in the research of various plant disease prevention and control due to its advantages of low production cost, safety for humans and animals, and low environmental pollution. Ginkgo is a precious medicinal plant resource unique to my country and belongs to gymnosperms. Its seeds, ginkgo nuts, have three layers of exocarps, of which the outermost one is called the fleshy exocarp. Because of its smelly taste and easy rot, it is usually discarded as solid waste. Ginkgo acid is a phenolic acid with important biological activity in the exocarp of ginkgo, which is composed of 5 monomers. Ginkgo acid has selective bactericidal, antibacterial, and insecticidal effects, low production cost, and safety for humans and animals. Recycling can solve the environmental pollution problem caused by direct disposal. It is a high-quality raw material in the research and development of plant-based pesticides.

[0003] Ginkgo biloba acid, as a natural product with significant biological activity, can effectively inhibit a variety of plant pathogens due to its unique selective bactericidal and antibacterial effects. Existing public literature reports that ginkgo biloba preparations have certain preventive and control effects on crop downy mildew, powdery mildew, gray mold and blight, pear black spot, rice blast, rice sheath blight and tomato leaf blight. Ginkgo biloba acid, as a plant extract, is used for plant disease prevention and control, and its greatest advantages are green, safe and efficient. However, ginkgo biloba acid has problems such as poor water solubility and low utilization rate, which limits its application in disease prevention and control in actual production. Therefore, developing a new type of ginkgo biloba acid microemulsion to improve its stability and bioavailability is of great significance to the prevention and control of plant diseases. Summary of the invention

[0004] The invention provides a 6% ginkgolic acid microemulsion, which is prepared from the following raw materials in percentage by mass: 6% ginkgolic acid, 2% anhydrous ethanol, 2% n-butanol, 10% OP10 (alkylphenol polyoxyethylene ether), 10% DBS-Ca (calcium dodecylbenzene sulfonate), and 70% distilled water.

[0005] The present invention also provides application of the 6% ginkgolic acid microemulsion in preventing and controlling plant pathogenic fungi.

[0006] Further, the plant pathogenic fungus is at least one of Sclerotium rolfsii, Globisporangium irregulare, G. spinosum, Botryosphaeria dothidea, Colletotrichum gloeosporioide, C. musae, C. fructicola, Colletotrichum sp., Rhizoctonia solani, Phomopsis sp., Fusarium oxysporum, F. solani, Phlebiopsis crassa, and C. truncatum.

[0007] Furthermore, the plant is at least one of kiwi fruit, pomegranate, scoparia, tea, banana, tobacco, peanut, citrus, gastrodia elata, cabbage, leek, ginger, and pepper.

[0008] The invention also provides a product for preventing and controlling plant pathogenic fungi, wherein the product contains the 6% ginkgolic acid microemulsion.

[0009] The present invention also provides a method for controlling plant pathogenic fungi using the 6% ginkgolic acid microemulsion, the method comprising the following steps:

[0010] (1) diluting 6% ginkgolic acid microemulsion;

[0011] (2) Spray the 6% ginkgolic acid microemulsion diluted in step (1) on the leaves of the plants.

[0012] Furthermore, the dilution factor of the 6% ginkgolic acid microemulsion in step (1) is 100 or 200.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention tests the 6% original drug content of the ginkgo acid microemulsion through 6 items such as stability, transparent temperature range, thermal stability, low temperature stability, pH value and continuous foaming. According to experimental research, the 6% ginkgo acid microemulsion has no precipitation when diluted 50 times, 100 times and 200 times, the transparent temperature range is 0-95.87°C, and the low temperature stability and thermal stability tests are in compliance with the standards; the pH value of the 6% microemulsion is 5.23, and the foaming volume is 32.83mL / 180mL. The above results all meet the national pesticide standards. The ginkgo acid microemulsion of the present invention has good stability, the appearance does not change when stored at room temperature, and the microemulsion has no stratification and no precipitation; the ginkgo acid microemulsion of the present invention can be diluted with water as needed when used, with a dilution multiple of 100 being the best, the diluted liquid has no turbidity and no precipitation, and can be sprayed or irrigated according to the user, which is conducive to uniform and quantitative application, saves the cost of medication, and provides convenience for the application of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The antibacterial activity of ginkgo acid against 15 different pathogenic fungi was determined in Example 1;

[0016] Figure 2 The transparent temperature range of the ginkgo acid microemulsion in Example 2;

[0017] Figure 3 is the pH value of the ginkgo acid microemulsion in Example 2;

[0018] Figure 4 is the sustained foaming volume of the ginkgo acid microemulsion in Example 2;

[0019] Figure 5 The 6% ginkgolic acid microemulsion prepared in Example 2 is prepared from the following raw materials in percentage by mass: 6% ginkgolic acid, 2% anhydrous ethanol, 2% n-butanol, 10% OP10 (alkylphenol polyoxyethylene ether), 10% DBS-Ca (calcium dodecylbenzene sulfonate), and 70% distilled water;

[0020] Figure 6 The control effect of 6% ginkgolic acid microemulsion on pepper anthracnose in Example 3 is shown. DETAILED DESCRIPTION

[0021] Example 1 Determination of the antibacterial effect of ginkgolic acid on 15 plant pathogens

[0022] 1.1 Test materials

[0023] As shown in Table 1, a total of 15 plant pathogens, including Sclerotium rolfsii GUSJ, Globisporangium irregulare GUBC, G. spinosum GUJC, Botryosphaeria dothidea GUMHT, Colletotrichum gloeosporioideGUCS02, C. musae GUXJ, C. fructicola GUTC, Colletotrichum sp.GUGJ, Colletotrichum sp.GUHS, Rhizoctonia solani GUYC01, Phomopsis sp.GUSL01, Fusarium oxysporumGUYC02, F. solani GUTM, Phlebiopsis crassa GUCS01, and C. truncatum GULJ01, were used in the indoor toxicity determination experiment of ginkgo acid.

[0024] Table 1 Information on strains used in the present invention

[0025]

[0026] 1.2 Test agents

[0027] (1) Ginkgo biloba acid, provided by the team of Professor He Jun from Guizhou Biochemical Engineering Center; the preparation method of ginkgo biloba acid is as follows: take dried ginkgo seed coat, extract with methanol at 40°C for one and a half hours, extract twice, combine the two extracts after extraction, filter the extract into a concentration tank, concentrate to extract, add the extract into an extraction tank, extract twice with ethyl acetate, combine the ethyl acetate layers and concentrate to extract, dissolve the extract with 70% ethanol and apply HPD-5000 resin, remove impurities with 2BV 70% ethanol solution, elute with 4BV 90% ethanol, collect the 90% ethanol eluate, concentrate to extract, transfer to a 60°C vacuum drying oven until constant weight, and obtain ginkgo biloba acid extract.

[0028] (2) Anhydrous ethanol (Chongqing Chuandong Chemical (Group) Co., Ltd.)

[0029] 1.3 Test culture medium

[0030] PDA medium (potato dextrose solid medium): 200.0 g / L potato, 20 g / L glucose, 15-20 g / L agar powder, distilled water to 1 L, sterilized at 121°C for 15 min; PDA medium was used for plate confrontation experiments to culture 15 plant pathogens.

[0031] 1.4 Effects of Ginkgo Biloba on the Growth Rate of Mycelium of Plant Pathogens

[0032] The effect of ginkgolic acid on the growth rate of different fungi was determined by the mycelial growth rate method. Five geometric gradient concentrations of ginkgolic acid were set. 1 mL of each concentration of ginkgolic acid was added to 9 mL of PDA medium to make a drug-containing plate. Three replicates were set, with sterile water as the blank control and an equal amount of anhydrous ethanol as the negative control. A 6 mm diameter bacterial cake was taken from the edge of the activated fungal colony and inoculated onto the drug-containing plate and cultured at 28 °C. When the growth of the blank control colony exceeded 2 / 3 of the entire plate, the colony diameter was measured by the cross method, and the inhibition rate was calculated according to the following formula:

[0033] Inhibition rate (%) = (colony diameter of control group – colony diameter of treatment group) / colony diameter of control group × 100%

[0034] The results showed that different concentrations of ginkgolic acid had different inhibitory effects on 15 plant pathogens ( Figure 1 ). When its concentration is 0.06 mg·mL -1 The inhibition rates of 15 pathogens were less than 45% when the concentration was 0.11 mg·mL -1 The inhibition rates of 13 pathogens including Colletotrichum sp. GUGJ, R. solani GUYC01, and C. fructicola GUTC were less than 20%, the inhibition rates of C. musae GUXJ and C. truncatum GULJ01 were 35.08%, and 43.87% respectively. When the concentration was 0.23 mg·mL -1 The inhibition rate of 4 pathogens including S.rolfsii GUSJ, G.irregulare GUBC, G.spinosumGUJC was less than 20%, the inhibition rate of 8 pathogens including F.solan GUTMi, Colletotrichum sp.GUHS, C.fructicola GUTC was between 20% and 50%, and the inhibition rate of C.musae GUXJ, R.solaniGUYC01 and C.truncatum GULJ01 was more than 50%; when its concentration was 0.46 mg·mL -1When the concentration was 0.92 mg·mL -1 The inhibition rate against five pathogens including S. rolfsii GUSJ, G. irregulare GUBC, G. spinosum GUJC was less than 20%, the inhibition rate against eight pathogens including C. gloeosporioides GUCS02, Colletotrichum sp. GUGJ, C. fructicola GUTC was between 20% and 50%, and the inhibition rate against C. musae GUXJ and R. solani GUYC01 was more than 50% (Table 2).

[0035] Table 2 Inhibitory effect of ginkgolic acid on 15 plant pathogens

[0036]

[0037]

[0038] Example 2 Preparation of a Ginkgo Acid Microemulsion

[0039] 2.1 Experimental agents

[0040] Methanol, ethyl acetate, anhydrous ethanol, dimethylformamide, dimethyl sulfoxide, n-butanol, xylene, isopropanol, n-hexane, glycerol, n-propanol, acetonitrile, ether, acetone, calcium dodecylbenzenesulfonate (DBS-Ca), styrylphenol polyoxyethylene ether (Nongru 600#), castor oil polyoxyethylene ether (BY-110), alkylphenol polyoxyethylene ether (OP-7), alkylphenol polyoxyethylene ether (OP-9), alkylphenol polyoxyethylene ether (OP-10), alkylphenol polyoxyethylene ether (OP-15), phenethylphenol polyoxyethylene ether (601#), phenethylphenol polyoxyethylene ether (602#), phenethylphenol polyoxyethylene ether (603#), phenethylphenol polyoxyethylene polyoxypropylene ether (1602#), alcohol ether glycoside, trifluoroacetic acid, etc.

[0041] 2.2 Experimental instruments

[0042] Analytical balance; digital display constant temperature water bath (model: HH-6); CNC ultrasonic cleaner (JOYN-22600A); high-speed refrigerated centrifuge (D1524R).

[0043] 2.3 Microemulsion preparation method (10 mL system)

[0044] 2.3.1 Solvent screening

[0045] According to the property that ginkgolic acid is insoluble in water but soluble in organic solvents, methanol, ethyl acetate, anhydrous ethanol, dimethylformamide, dimethyl sulfoxide, n-butanol, xylene, isopropanol, n-hexane, glycerol, n-propanol, acetonitrile, ether and acetone were used as alternative solvents. 0.6 g of ginkgolic acid was added to a 10 mL centrifuge tube, and the solvent was added in small amounts and multiple times until the ginkgolic acid in the tube was completely dissolved. At this time, the solution was clear and transparent.

[0046] 2.3.2 Microemulsion system and transparent temperature range screening

[0047] Microemulsions cannot lack nonionic or anionic surfactants for emulsification and stabilization of emulsions. Dodecylbenzenesulfonate calcium (DBS-Ca), styrylphenol polyoxyethylene ether (Agricultural milk 600#), castor oil polyoxyethylene ether (BY-110), alkylphenol polyoxyethylene ether (OP-7), alkylphenol polyoxyethylene ether (OP-9), alkylphenol polyoxyethylene ether (OP-10), alkylphenol polyoxyethylene ether (OP-15), phenethylphenol polyoxyethylene ether (601#), phenethylphenol polyoxyethylene ether (602#), phenethylphenol polyoxyethylene ether (603#), phenethylphenol polyoxyethylene polyoxypropylene ether (1602#), alcohol ether glycosides, etc. were used as alternative emulsifiers to prepare microemulsions containing more ginkgo acid active ingredients. According to the ratio requirements between the components of the microemulsion, the emulsifier should not exceed 20%. Weigh 0.6g of ginkgolic acid and dissolve it in a solvent, then add 0.5, 1.0, and 1.5g of DBS-Ca, and then add various non-ionic surfactants to mix. After stirring, observe that the whole system is clear and transparent. The clear and transparent system is further screened and its state at different temperatures is observed. Generally speaking, the wider the transparency range of the emulsion, the better.

[0048] 2.3.3 Cosolvent Screening

[0049] Cosolvents can reduce the amount of solvent used. Using low-cost cosolvents is beneficial to saving the cost of emulsion development. Several commonly used cosolvents, such as anhydrous ethanol, acetone, methanol, n-butanol, and isopropanol, were used to explore whether the same dissolution effect could be achieved. Weigh 0.6 g of ginkgolic acid into a centrifuge tube, add the main solvent in amounts of 100, 200, and 300 μL, and then add cosolvents in small amounts multiple times, with the total amount not exceeding the total amount of the original solvent used.

[0050] 2.3.4 Water quality screening

[0051] Water is an important component of microemulsions. Different water qualities will affect the microemulsion system. Therefore, the determination of water quality is also crucial in the research and development of microemulsions, and has important theoretical guidance for subsequent actual production. Deionized water, tap water, distilled water, and hard water are used for water quality screening. These four different water qualities are slowly added to the clear and transparent emulsion. The water that can still maintain clear and transparent emulsion in the end is selected for the preparation of the microemulsion.

[0052] 2.3.5 National Standard Test for Microemulsion

[0053] For stability determination, refer to "Method for Determination of Stability of Pesticide Emulsions" GB / T 1603-2001; for low temperature stability determination, refer to "Specification for the Preparation of Pesticide Emulsion Product Standards" HG / T 2467.2-2003; for hot storage stability determination, refer to "Specification for the Preparation of Pesticide Emulsion Product Standards" HG / T 2467.2-2003; for pH value determination, refer to "Method for Determination of Pesticide pH" GB / T 1601-93; for continuous foaming volume, refer to "Specification for the Preparation of Pesticide Product Standards" HG / T 2467.5-2003; for transparent temperature range, refer to "Specification for the Preparation of Pesticide Product Standards" HG / T 2467.10-2003.

[0054] The results show that: as shown in Table 3, ethyl acetate, anhydrous ethanol, n-hexane and acetone have the best solubility. Considering safety, economy and sustainability, anhydrous ethanol was selected as the main solvent of the ginkgo acid microemulsion. According to the results of Tables 3, 4, 5, 6 and 7, it was finally determined that anhydrous ethanol was the main solvent of the ginkgo acid microemulsion, DBS-Ca / OP10 (1:1) was the emulsifier, n-butanol was the cosolvent, and distilled water was the water quality to prepare a 6% ginkgo acid microemulsion. The stability of the 6% ginkgo acid microemulsion was determined with reference to the GB / T 1603 method. As shown in Table 8, the 6% ginkgo acid microemulsion had no precipitation at three dilution multiples. Figure 2 As shown in Table 9, the transparent temperature range of 6% ginkgolic acid microemulsion is 0-95.87℃. The refrigerated and hot storage stability tests of 6% ginkgolic acid microemulsion were carried out, as shown in Table 9 and Table 10. The results are in line with the expected assumptions. The pH value of ginkgolic acid microemulsion was measured. The difference of the data of three parallel measurements was not greater than 0.1, so the pH value of 6% ginkgolic acid microemulsion was 5.23 ( Figure 3 The foaming volume of ginkgolic acid microemulsion was measured, and the foaming volume of 6% ginkgolic acid was 32.83mL / 180mL ( Figure 4 ).

[0055] Table 3 Screening results of solvent systems

[0056]

[0057]

[0058] Table 4 Screening test results of emulsifiers

[0059]

[0060] Table 5 Temperature screening test results of emulsifiers

[0061]

[0062]

[0063] Table 6 Screening of cosolvents

[0064]

[0065] Table 7 Water quality screening of ginkgo acid microemulsion

[0066]

[0067] Table 8 Stability determination of ginkgo acid microemulsion

[0068]

[0069] Table 9 Physical properties of cold storage (0℃, 7d)

[0070]

[0071] Table 10 Physical properties of hot storage (54±2℃, 14d)

[0072]

[0073]

[0074] Example 3 Indoor protective effect of ginkgolic acid microemulsion in controlling plant diseases

[0075] 3.1 Test materials

[0076] The tested pepper varieties were Guishu Jiamanfeng (registration number: GPD Pepper (2021) 520756); 6% original drug content of ginkgolic acid microemulsion (6% ginkgolic acid, 2% ethanol, 2% n-butanol, 10% OP10 (alkylphenol polyoxyethylene ether), 10% DBS-Ca (calcium dodecylbenzene sulfonate), 70% distilled water), 20% dicyandiamide (Mingdelida Mingshun), and 1% osthole microemulsion (Yunnan Nanbao Biotechnology Co., Ltd.).

[0077] 3.2 Indoor protection test

[0078] (1) Pepper seedling cultivation

[0079] After sterilizing the seedling medium at high temperature, put it into the seedling pot, evenly sow the precious vegetables, keep the medium moist, and place it in a greenhouse (temperature 25℃, light-dark ratio 2:1, relative humidity 60%) for cultivation. Transplant it when it is a seedling, and wait until the seedling grows to the fruiting period for use;

[0080] (2) Preparation of anthrax spore suspension

[0081] The test strain GULJ01 was inoculated on a PDA plate and cultured at 28°C with 12 h light / 12 h dark for 7-14 days. The conidia were washed with sterile water and prepared with a hemocytometer to a concentration of 1×10 6 CFU / mL spore suspension is ready for use.

[0082] (3) Anthrax inoculation treatment

[0083] Use a 500mL small spray bottle to evenly inoculate the spore suspension onto healthy pepper fruits in the form of spray, and spray an equal amount of water as a blank control. The temperature is 28°C, the relative humidity is 60%, and the light / dark period is 12h. The symptoms of disease are observed every day. When sporadic lesions appear on the inoculated fruits, the next step of experimental treatment is carried out.

[0084] (4) Ginkgo acid treatment settings

[0085] When pepper plants enter the fruiting period, dilute the ginkgolic acid microemulsion at different times (the dilution times of 6% ginkgolic acid microemulsion are: 25, 50, 100, 200, 400 times) to the leaves and spray until water drips (apply once every 7 days in the early stage of the disease, apply once every 3 days during the high incidence period, and apply 3 times in succession). Spray an equal amount of clean water as a blank control, and spray the botanical pesticide 1% osthole microemulsion (each 30 ml is diluted with 15 kg of water, and every 7 days in the early stage of the disease is applied). 1 time, apply once every 3 days during the peak period, and apply it three times in succession) and 20% dicyandiamide·pyraclostrobin suspension chemical pesticide (each 15 grams is diluted with 15 kg of water, and the agent is sprayed immediately after the disease occurs, and it is applied once every 7-10 days, and sprayed twice in succession) as positive controls, 10 plants are treated each time, and repeated 3 times; the manufacturer of the above-mentioned 1% osthole microemulsion is Yunnan Nanbao Biotechnology Co., Ltd., and the manufacturer of 20% dicyandiamide·pyraclostrobin suspension is Mingdelida Mingshun.

[0086] (5) Disease investigation and control effect investigation

[0087] Disease investigation, classification standards and efficacy investigation calculation methods were carried out in accordance with the "Guidelines for Field Efficacy Tests of Pesticides (1)" (Compiled by the Pesticide Testing Laboratory of the Ministry of Agriculture and Rural Affairs, 2000) and the "Guizhou Provincial Local Standard DB52 / T 1501.14-2020" Technical Specifications for Identification of Disease Resistance of Crops Part 14: Pepper Anthracnose Resistance. Data were analyzed for variance using Excel spreadsheets and the Duncan new multiple range method of the DPS 7.05 data processing system.

[0088] Incidence rate (%) = (number of diseased plants / total number of plants investigated) × 100;

[0089] Disease index (DI) = ∑ (number of diseased plants at each level × disease level value) / [(total number of plants surveyed × highest disease level) × 100]

[0090] Relative prevention effect (%) = [(CK disease index - treatment disease index) / CK disease index] × 100

[0091] The results show that: from Table 11 and Figure 6 It can be seen that the diseased fruit rate of the blank control group was 23.44%, and the disease index was 15.45; the diseased fruit rate of the group treated with 20% dicyandiamide / pyraclostrobin dropped to 6.45, the disease index was 3.23, and the relative prevention effect was 79.12%; the diseased fruit rate of the group treated with 1% Cnidium monnieri microemulsion was 10.53, the disease index was 4.51, and the relative prevention effect was 70.80%; when the 6% ginkgo acid microemulsion was diluted 200 times and 100 times for spraying, the control effect was 76.47% and 75.73%, respectively, and its control effect is equivalent to that of chemical pesticides.

[0092] Table 11 The control effect of ginkgo acid microemulsion on pepper anthracnose

[0093]

[0094] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A 6% ginkgolic acid microemulsion, characterized in that: The 6% ginkgolic acid microemulsion is prepared from the following raw materials in percentage by mass: 6% ginkgolic acid, 2% anhydrous ethanol, 2% n-butanol, 10% OP10 (alkylphenol polyoxyethylene ether), 10% DBS-Ca (calcium dodecylbenzene sulfonate), and 70% distilled water.

2. A method for preparing the 6% ginkgolic acid microemulsion according to claim 1, characterized in that: The method comprises the following steps: firstly dissolving ginkgolic acid in ethanol and n-butanol, then adding emulsifiers OP10 and DBS-Ca, and finally adding distilled water.

3. Use of the 6% ginkgolic acid microemulsion according to claim 1 in preventing and controlling plant pathogenic fungi.

4. The use according to claim 3, characterized in that: The plant pathogenic fungus is at least one of Sclerotium rolfsii, Globisporangium irregulare, G. spinosum, Botryosphaeria dothidea, Colletotrichum gloeosporioide, C. musae, C. fructicola, Colletotrichum sp., Colletotrichum sp., Rhizoctonia solani, Phomopsis sp., Fusarium oxysporum, F. solani, Phlebiopsis crassa, and C. truncatum.

5. The use according to claim 3 or 4, characterized in that: The plant is at least one of kiwi fruit, pomegranate, euphorbia pulcherrima, tea, banana, tobacco, peanut, citrus, gastrodia elata, cabbage, leek, ginger and pepper.

6. A product for controlling plant pathogenic fungi, characterized in that: The product contains the 6% ginkgolic acid microemulsion described in claim 1.

7. A method for controlling plant pathogenic fungi using the 6% ginkgolic acid microemulsion according to claim 1, characterized in that: The method comprises the following steps: (1) diluting 6% ginkgolic acid microemulsion; (2) Spray the 6% ginkgolic acid microemulsion diluted in step (1) on the leaves of the plants.

8. The method according to claim 7, characterized in that The dilution multiple of the 6% ginkgolic acid microemulsion in step (1) is 100 or 200.

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