Preparation of citral preparation and application of citral preparation in aquaculture

Abstract

The invention relates to the technical field of aquaculture disease control, in particular to preparation of a citral preparation and application of the citral preparation in aquaculture, the preparation method comprises the following steps: step 1, adding a compound emulsifier into a co-solution, and uniformly stirring and mixing to obtain an emulsion; step 2, adding the active component into the emulsified liquid, and continuously stirring to form primary emulsion; and step 3, adding a stabilizer into the primary emulsion, stirring and mixing, homogenizing at high pressure, and cooling to room temperature to obtain the citral preparation. The citral preparation prepared by the invention has a strong antibacterial property, and the preparation process of the preparation is simple and convenient and does not need to depend on special equipment, so that the production cost is remarkably reduced, and the realization of large-scale mass production is facilitated; the device is flexible and convenient to operate, can be mixed into feed to be added for use, can also be sprayed through water, and can fully meet diversified scene requirements of aquaculture.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of disease prevention and treatment of aquaculture, and more particularly to a preparation of citral and application of the preparation in aquaculture. BACKGROUND

[0002] The existing prevention and treatment methods have the following defects: (1) long-term use of chemical disinfectants and antibiotics leads to problems such as increased drug resistance of pathogens, drug residues, decreased quality of aquatic products and environmental pollution; (2) traditional plant-derived drugs have the defects of unstable effective components, easy volatilization, poor dispersibility in water and slow effect, which limits their large-scale application; (3) citral is the main component of plant essential oils such as Chinese litse and lemon grass, and has broad-spectrum antibacterial, antifungal and insect repellent activities, however, it has poor water solubility, unstable chemical properties, easy oxidation and volatilization, which leads to low bioavailability, poor direct use effect and high cost. Based on the above statements, it is urgent to develop a preparation capable of improving the stability, water solubility and bioavailability of citral, so that it can be efficiently and safely applied to the prevention and treatment of diseases in aquaculture. SUMMARY

[0003] In order to solve the technical problems mentioned in the background, the application provides a preparation of citral and application of the preparation in aquaculture.

[0004] A preparation method of a citral preparation, comprising the following preparation steps: Step 1, adding a composite emulsifier to a cosolvent, stirring and mixing at a temperature of 30-45 DEG C for 10-20 min to obtain an emulsion; Step 2, adding an active ingredient to the emulsion, continuously stirring for 20-30 min to form a primary emulsion; Step 3, adding a stabilizer to the primary emulsion, stirring and mixing for 15-25 min, homogenizing 2-3 times under a pressure of 20-30 MPa, each homogenization time being 5-8 min, and cooling to room temperature to obtain the citral preparation.

[0005] Preferably, the composite emulsifier in step 1 is composed of polyglycerol fatty acid ester and sorbitan oleate with a mass ratio of 6-8:3-5.

[0006] Preferably, the mass ratio of the composite emulsifier and the cosolvent in step 1 is 10-20:100-150.

[0007] Preferably, the cosolvent in step 1 is composed of propylene glycol, polyethylene glycol 400 and deionized water with a mass ratio of 5-8:2-5:90-110.

[0008] Preferably, the active ingredients in step 2 are composed of citral, azone and tannic acid in a mass ratio of 10-12:1-3:1-2.

[0009] Preferably, the mass ratio of the active ingredients and the emulsion in step 2 is 15-20:80-120.

[0010] Preferably, the mass ratio of the colostrum and the stabilizer in step 3 is 20-30:1-3.

[0011] Preferably, the stabilizer in step 3 is one or more of ascorbic palmitate, rosemary acid and vitamin E.

[0012] A citral preparation prepared by the above preparation method is applied in aquaculture.

[0013] Preferably, the application mode is: when used for pathogenic bacteria prevention and control of aquatic animals, the feed adding mode is adopted, the citral preparation is added to the feed in a proportion of 0.5-1.0 g / kg of feed, and is continuously fed for 5-8 days; or the water body spraying mode is adopted, the citral preparation is used in an amount of 0.1-0.5 mg / L, once every 5-7 days, and is used continuously for 2-3 times. When used for aquaculture water purification, the water body spraying mode is adopted, the citral preparation is used in an amount of 0.3-0.8 mg / L, once every 7-10 days, and is used continuously for 2-3 times. When used for improving the stress resistance of aquatic animals, the feed adding mode is adopted, the citral preparation is added to the feed in a proportion of 0.5-1.0 g / kg of feed 24-48 hours before the release of aquatic animal fry, the water temperature difference is greater than or equal to 5℃, netting or transportation, and is continuously fed for 3-5 days. When used for the fry transportation temporary rearing stage, the citral preparation is used in an amount of 1.0-1.5 mg / L and is added to the temporary rearing water body 6-8 hours before transportation.

[0014] In summary, the present application has the following beneficial effects: The application can solve the problems of insufficient solubility and poor compatibility of citral in a single solvent system by compounding propylene glycol with polyethylene glycol 400, and finally realize the synergistic improvement of citral solubility and the strengthening of system stability. Propylene glycol can form hydrogen bonds with citral molecules, break the intermolecular forces of citral itself, and assist its rapid dispersion in the aqueous phase system, effectively avoiding the problem of crystal precipitation. Polyethylene glycol 400 can reduce the overall viscosity of the cosolvent and enhance the interfacial compatibility of citral and the aqueous phase. Under the synergistic action of the two, citral can be completely dissolved in the cosolvent without crystal precipitation, laying a foundation for subsequent mixing with emulsifiers and active ingredients. By compounding polyglycerol fatty acid ester with sorbitan oleate, the stability of the emulsifying system is significantly enhanced, avoiding the defects of demulsification and stratification caused by the absence of a single component. Polyglycerol fatty acid ester has excellent surface tension regulation ability, which can quickly reduce the oil-water interfacial tension and assist the initial dispersion of oily active ingredients such as citral; while sorbitan oleate can form a dense and elastic interfacial film at the oil-water interface to inhibit droplet aggregation.

[0015] The application can significantly enhance the bactericidal efficiency of the preparation by compounding citral, azone and tannic acid, realize the dual effect of rapid penetration and strong bacteriostatic effect. Citral, as the core bacteriostatic ingredient, can destroy the cell membrane structure of pathogenic bacteria, but when used alone, it is easily hindered by the cell membrane barrier and has low penetration efficiency. Azone, as a penetration enhancer, can quickly open the lipid channel of the cell membrane of pathogenic bacteria and assist citral to enter the bacteria to play a role. Tannic acid can further destroy the enzyme system of pathogenic bacteria and inhibit their metabolic activity in cooperation with citral. The three compounds can quickly improve the bactericidal rate of the preparation on common water-borne pathogenic bacteria (such as Vibrio and Edwardsiella) and make the effect more thorough, effectively reducing the residual pathogenic bacteria. Citral is easily oxidized and degraded during storage or use, leading to a decrease in bacteriostatic effect; tannic acid has excellent antioxidant properties and can inhibit the oxidation of citral by capturing free radicals. At the same time, its molecular structure can form a stable complex with citral, reducing the loss of active ingredients, significantly improving the bactericidal persistence and stability of the preparation, and avoiding the inactivation of active ingredients. After spraying or mixing, the preparation can continuously play a bacteriostatic role in the breeding environment without relying on traditional antibiotics, reducing the risk of pathogenic bacteria resistance and meeting the needs of green aquaculture. BRIEF DESCRIPTION OF DRAWINGS Fig. 1 is the MIC and MBC determination of citral on Aeromonas salmonicida.

[0016] Fig. 2 is the biofilm formation of Aeromonas salmonicida after citral treatment, wherein A is the control group and B is the experimental group.

[0017] Fig. 3is the scanning electron microscope image of Aeromonas salmonicida treated by citral, wherein A is a blank control group, BCD is an experimental group. DETAILED DESCRIPTION

[0018] The application will be further described in detail below in combination with examples.

[0019] The polyglycerol fatty acid ester used in the examples and comparative examples of the application was purchased from Chengdu Wanxiang Honglun Biological Technology Co., Ltd.; the sorbitan oleate (model number: S-80) was purchased from Nantong Huzhuo Chemical Co., Ltd.; the citral was purchased from Wuhan Kemike Biological Medicine Technology Co., Ltd.; the azone (model number: CP2020) was purchased from Rui Chengkang Medicine Technology (Shaanxi) Co., Ltd.; and the tannic acid was purchased from Wuhan Jiyesheng Chemical Co., Ltd.

[0020] Examples 1-3 provide a preparation of a citral preparation and its application in aquaculture.

[0021] Example 1 A preparation method of a citral preparation, comprising the following preparation steps: Step 1: according to the mass ratio of the composite emulsifier to the cosolvent of 10:100, the composite emulsifier is added to the cosolvent, stirred and mixed at a temperature of 30℃ and a stirring speed of 400 rpm for 10 min to obtain an emulsion, wherein the composite emulsifier is composed of polyglycerol fatty acid ester and sorbitan oleate at a mass ratio of 6:3, and the cosolvent is composed of propylene glycol, polyethylene glycol 400 and deionized water at a mass ratio of 5:2:90; Step 2: according to the mass ratio of the active ingredient to the emulsion of 15:80, the active ingredient is added to the emulsion, and stirred at a stirring speed of 800 rpm for 20 min to form a primary emulsion, wherein the active ingredient is composed of citral, azone and tannic acid at a mass ratio of 10:1:1; Step 3: according to the mass ratio of the primary emulsion to ascorbic acid palmitate of 20:1, ascorbic acid palmitate is added to the primary emulsion, stirred and mixed at a stirring speed of 500 rpm for 15 min, homogenized twice at a pressure of 20 MPa, each time for 5 min, and cooled to room temperature to obtain the citral preparation.

[0022] Example 2 A preparation method of a citral preparation, comprising the following preparation steps: Step 1: according to the mass ratio of the composite emulsifier to the cosolvent of 15:120, the composite emulsifier is added to the cosolvent, stirred and mixed at a temperature of 38℃ and a stirring speed of 500 rpm for 15 min to obtain an emulsion, wherein the composite emulsifier is composed of polyglycerol fatty acid ester and sorbitan oleate at a mass ratio of 7:4, and the cosolvent is composed of propylene glycol, polyethylene glycol 400 and deionized water at a mass ratio of 7:4:100; Step 2, the active ingredient is added to the emulsion according to the mass ratio of active ingredient to emulsion 18:100, the stirring speed is 1000 rpm, and the stirring is continuously conducted for 25 min to form a primary emulsion, wherein the active ingredient is composed of citral, azone and tannic acid with a mass ratio of 11:2:1.5; Step 3, the primary emulsion is added with ascorbic palmitate according to the mass ratio of primary emulsion to ascorbic palmitate 25:2, the stirring speed is 800 rpm, the stirring and mixing are conducted for 20 min, homogenization is conducted 3 times under the pressure of 25 MPa, the homogenization time is 7 min each time, and the preparation is cooled to room temperature to obtain the citral preparation.

[0023] Example 3 A preparation method of a citral preparation comprises the following preparation steps: Step 1, the composite emulsifier is added to the cosolvent according to the mass ratio of composite emulsifier to cosolvent 20:150, the stirring and mixing are conducted at the temperature of 45℃ and the stirring speed of 600 rpm for 20 min to obtain an emulsion, wherein the composite emulsifier is composed of polyglycerol fatty acid ester and sorbitan oleate with a mass ratio of 8:5, and the cosolvent is composed of propylene glycol, polyethylene glycol 400 and deionized water with a mass ratio of 8:5:110; Step 2, the active ingredient is added to the emulsion according to the mass ratio of active ingredient to emulsion 20:120, the stirring speed is 1200 rpm, and the stirring is continuously conducted for 30 min to form a primary emulsion, wherein the active ingredient is composed of citral, azone and tannic acid with a mass ratio of 12:3:2; Step 3, the primary emulsion is added with ascorbic palmitate according to the mass ratio of primary emulsion to ascorbic palmitate 30:3, the stirring speed is 1000 rpm, the stirring and mixing are conducted for 25 min, homogenization is conducted 3 times under the pressure of 30 MPa, the homogenization time is 8 min each time, and the preparation is cooled to room temperature to obtain the citral preparation.

[0024] Comparative Example 1 A preparation method of a citral preparation comprises the following preparation steps: Step 1, the sorbitan oleate is added to the cosolvent according to the mass ratio of sorbitan oleate to cosolvent 10:100, the stirring and mixing are conducted at the temperature of 30℃ and the stirring speed of 400 rpm for 10 min to obtain an emulsion, and the cosolvent is composed of propylene glycol, polyethylene glycol 400 and deionized water with a mass ratio of 5:2:90; Step 2, the active ingredient is added to the emulsion according to the mass ratio of active ingredient to emulsion 15:80, the stirring speed is 800 rpm, and the stirring is continuously conducted for 20 min to form a primary emulsion, wherein the active ingredient is composed of citral, azone and tannic acid with a mass ratio of 10:1:1; Step 3: Add ascorbate palmitate to colostrum at a mass ratio of 20:1. Stir at 500 rpm for 15 minutes, homogenize twice at 20 MPa for 5 minutes each time, and cool to room temperature to obtain citral preparation.

[0025] Comparative Example 2 A method for preparing a citral formulation includes the following preparation steps: Step 1: Add polyglycerol fatty acid ester to the co-solution at a mass ratio of 10:100. Stir and mix at 30°C and 400 rpm for 10 minutes to obtain an emulsion. The co-solution is composed of propylene glycol, polyethylene glycol 400 and deionized water at a mass ratio of 5:2:90. Step 2: Add the active ingredient to the emulsion at a mass ratio of 15:80 and stir continuously at 800 rpm for 20 minutes to form the primary emulsion. The active ingredient consists of citral, azone, and tannic acid in a mass ratio of 10:1:1. Step 3: Add ascorbate palmitate to colostrum at a mass ratio of 20:1. Stir at 500 rpm for 15 minutes, homogenize twice at 20 MPa for 5 minutes each time, and cool to room temperature to obtain citral preparation.

[0026] Comparative Example 3 A method for preparing a citral formulation includes the following preparation steps: Step 1: Add the composite emulsifier to the auxiliary solution at a mass ratio of 10:100. Stir and mix at 30°C and 400 rpm for 10 minutes to obtain an emulsion. The composite emulsifier consists of polyglycerol fatty acid ester and dehydrated sorbitan oleate at a mass ratio of 6:3, and the auxiliary solution consists of polyethylene glycol 400 and deionized water at a mass ratio of 7:90. Step 2: Add the active ingredient to the emulsion at a mass ratio of 15:80 and stir continuously at 800 rpm for 20 minutes to form the primary emulsion. The active ingredient consists of citral, azone, and tannic acid in a mass ratio of 10:1:1. Step 3: Add ascorbate palmitate to colostrum at a mass ratio of 20:1. Stir at 500 rpm for 15 minutes, homogenize twice at 20 MPa for 5 minutes each time, and cool to room temperature to obtain citral preparation.

[0027] Comparative Example 4 A method for preparing a citral formulation includes the following preparation steps: Step 1: Add the composite emulsifier to the auxiliary solution at a mass ratio of 10:100. Stir and mix at 30°C and 400 rpm for 10 minutes to obtain an emulsion. The composite emulsifier consists of polyglycerol fatty acid ester and dehydrated sorbitan oleate at a mass ratio of 6:3, and the auxiliary solution consists of propylene glycol and deionized water at a mass ratio of 7:90. Step 2: Add the active ingredient to the emulsion at a mass ratio of 15:80 and stir continuously at 800 rpm for 20 minutes to form the primary emulsion. The active ingredient consists of citral, azone, and tannic acid in a mass ratio of 10:1:1. Step 3: Add ascorbate palmitate to colostrum at a mass ratio of 20:1. Stir at 500 rpm for 15 minutes, homogenize twice at 20 MPa for 5 minutes each time, and cool to room temperature to obtain citral preparation.

[0028] Comparative Example 5 A method for preparing a citral formulation includes the following preparation steps: Step 1: Add the composite emulsifier to the auxiliary solution at a mass ratio of 10:100. Stir and mix at 30°C and 400 rpm for 10 minutes to obtain an emulsion. The composite emulsifier consists of polyglycerol fatty acid ester and dehydrated sorbitan oleate in a mass ratio of 6:3, and the auxiliary solution consists of propylene glycol, polyethylene glycol 400 and deionized water in a mass ratio of 5:2:90. Step 2: Add the active ingredient to the emulsion at a mass ratio of 15:80 and stir continuously at 800 rpm for 20 minutes to form the primary emulsion. The active ingredient consists of citral and tannic acid in a mass ratio of 10:1. Step 3: Add ascorbate palmitate to colostrum at a mass ratio of 20:1. Stir at 500 rpm for 15 minutes, homogenize twice at 20 MPa for 5 minutes each time, and cool to room temperature to obtain citral preparation.

[0029] Comparative Example 6 A method for preparing a citral formulation includes the following preparation steps: Step 1: Add the composite emulsifier to the auxiliary solution at a mass ratio of 10:100. Stir at 400 rpm for 10 minutes at 30°C to obtain an emulsion. The composite emulsifier consists of polyglycerol fatty acid ester and sorbitan oleate at a mass ratio of 6:3, and the auxiliary solution consists of propylene glycol, polyethylene glycol 400, and deionized water at a mass ratio of 5:2:90. Step 2: Add the active ingredient to the emulsion at a mass ratio of 15:80. Stir at 800 rpm for 20 minutes to form a primary emulsion. The active ingredient consists of citral and azone at a mass ratio of 10:1. Step 3: Add ascorbate palmitate to colostrum at a mass ratio of 20:1. Stir at 500 rpm for 15 minutes, homogenize twice at 20 MPa for 5 minutes each time, and cool to room temperature to obtain citral preparation.

[0030] Performance testing The comprehensive performance of the citral preparations obtained in Examples 1-3 and Comparative Examples 1-6 was tested respectively, as follows: The citral preparations prepared in Examples 1-3 and Comparative Examples 1-6 were applied to water by spraying at a dosage of 0.3 mg / L, once every 7 days, for two consecutive times. The blank control group was not sterilized. The CFU values ​​of the target pathogens (Vibrio parahaemolyticus and Aeromonas hydrophila) in 1 mL of water were counted using the plate count method (Vibrio parahaemolyticus on TCBS medium and Aeromonas hydrophila on TSA medium) before the first application of the preparation (0 day), and 1 day, 7 days, and 14 days after application.

[0031] Table 1. Antibacterial properties of citral preparations prepared in Examples 1-3 and Comparative Examples 1-6 The bactericidal effect of citral on Aeromonas salmonii was determined using OD200. 600 =0.1 Aeromonas salmonicidae bacterial suspension, diluted 100 times and then diluted 2 times with 100 μL of citral solution, and incubated at 30℃ for 24 h, the results showed that the MIC and MBC of citral against Aeromonas salmonicidae were both 0.195 μL / mL.

[0032] Using OD 600 =0.1% Aeromonas salmonicidae bacterial suspension, 50 μL of bacterial suspension per plate, spread on BHI plates, and after punching, 100 μL of different concentrations of citral solution were added to each well. The plates were incubated at 30℃ for 24 h. The results showed that different concentrations of citral had an inhibitory effect on Aeromonas salmonicidae. (See attached figures). Fig. 1 ; Table 2. Inhibition zones of different concentrations of citral against Aeromonas salmonicida. Crystal violet (CV) staining was used to assess the effect of citral on biofilm formation. Aeromonas salmonicida suspension was adjusted to OD... 600 The concentration was 0.5. The experimental group used 2MIC citral, and the blank group used bacterial suspension. These were added to 24-well plates and cultured at 30℃ for 72 h to form a biofilm. The culture medium was gently removed, and the wells were rinsed twice with PBS. Methanol was then added to each well for fixation for 30 min. The methanol was removed, and the samples were washed with sterile PBS. The samples were dried for 30 min, and 0.1% (w / v) crystal violet solution was added to the wells. All samples were incubated for 20 min. The results showed that citral treatment reduced biofilm formation in *Aeromonas salmonicida*, inhibiting bacterial biofilm formation. This indicates that citral treatment inhibits biofilm formation in *Aeromonas salmonicida*, helping to prevent bacterial colonization and infection of the host, interrupting signaling communication between bacterial communities, and reducing bacterial biofilm resistance. The results are shown in the table below. Fig. 2 ; Adjust the bacterial concentration to OD 600 =0.5, divided into a blank group and an experimental group. The blank group contained only bacterial culture, while the experimental group was incubated with 2MIC citral. After incubation at 30℃ for 30 min, the cells were centrifuged, washed twice with PBS, and fixed with electron microscopy fixative. After adding the fixative, the cells were promptly agitated and mixed. The results showed that citral treatment reduced biofilm formation in *Aeromonas salmonicida*, inhibiting bacterial biofilm formation. This indicates that citral treatment inhibits biofilm formation in *Aeromonas salmonicida*, helping to prevent bacterial colonization and infection of the host, interrupting signaling communication between bacterial communities, and reducing bacterial biofilm resistance. The blank A group showed clear, plump rod-shaped cells with smooth surfaces and obvious cell integrity. 2MIC citral treatment caused cell surface roughness, shrinkage, collapse, pore formation, rupture, breakage, and leakage, showing severe damage to cell structure. These conclusions suggest that the significant changes in cell morphology further demonstrate that citral exerts its antibacterial activity against *Aeromonas salmonicida* by altering the bacterial membrane structure. (See results below.) Fig. 3 .

[0033] From Table 1-2 and Figs. 1-3 As can be seen, the citral formulation prepared in this application uses natural citral as the core active ingredient, which can effectively replace traditional antibiotics and disinfectants, highly aligning with the development direction of green aquaculture, and also possesses strong antibacterial properties. Furthermore, the formulation has a simple preparation process, requiring no special equipment, which not only significantly reduces production costs but also facilitates large-scale production. In terms of application, it is flexible and convenient to use, either by spraying into water or by mixing into feed, fully adapting to the diverse needs of aquaculture scenarios.

[0034] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a citral preparation, characterized in that, The preparation steps include the following: Step 1: Add the composite emulsifier to the auxiliary solution and stir for 10-20 minutes at a temperature of 30-45℃ to obtain the emulsion; Step 2: Add the active ingredients to the emulsion and stir continuously for 20-30 minutes to form the colostrum; Step 3: Add stabilizer to colostrum, stir and mix for 15-25 minutes, homogenize 2-3 times under 20-30 MPa pressure, each time for 5-8 minutes, cool to room temperature to obtain citral preparation.

2. The method for preparing citral formulation according to claim 1, characterized in that, In step 1, the composite emulsifier is composed of polyglycerol fatty acid ester and dehydrated sorbitan oleate in a mass ratio of 6-8:3-5.

3. The method for preparing citral formulation according to claim 1, characterized in that, In step 1, the mass ratio of the composite emulsifier to the co-solution is 10-20:100-150.

4. The method for preparing citral formulation according to claim 1, characterized in that, In step 1, the auxiliary solution is composed of propylene glycol, polyethylene glycol 400 and deionized water in a mass ratio of 5-8:2-5:90-110.

5. The method for preparing citral formulation according to claim 1, characterized in that, In step 2, the active ingredients consist of citral, azone, and tannic acid in a mass ratio of 10-12:1-3:1-2.

6. The method for preparing citral formulation according to claim 1, characterized in that, In step 2, the mass ratio of active ingredient to emulsion is 15-20:80-120.

7. The method for preparing citral formulation according to claim 1, characterized in that, In step 3, the mass ratio of colostrum to stabilizer is 20-30:1-3.

8. The method for preparing citral formulation according to claim 1, characterized in that, The stabilizer in step 3 is one or more of ascorbyl palmitate, rosmarinic acid, and vitamin E.

9. The application of a citral preparation obtained by the preparation method according to any one of claims 1-8 in aquaculture.

10. The application of the citral preparation according to claim 9 in aquaculture, characterized in that, The application method is as follows: When used for the prevention and control of pathogens in aquatic animals, it is added to the feed at a ratio of 0.5-1.0 g / kg of citral preparation, and fed continuously for 5-8 days; or it is sprayed on water at a ratio of 0.1-0.5 mg / L of citral preparation, every 5-7 days, for 2-3 consecutive times. When used for purifying aquaculture water, it is applied by water spraying, with a dosage of citral at 0.3-0.8 mg / L, every 7-10 days, for 2-3 consecutive applications. When used to enhance the stress resistance of aquatic animals, it is added to the feed as a feed additive. 24-48 hours before the release of aquatic animal seedlings, when the water temperature difference is ≥5℃, or before netting or transportation, the citral preparation is added to the feed at a ratio of 0.5-1.0g / kg of feed and fed continuously for 3-5 days. When used for the temporary holding stage of seedling transportation, 1.0-1.5 mg / L of citral preparation should be added to the holding water 6-8 hours before transportation.

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