Garlic essential oil bacteriostatic microemulsion for prawn culture as well as preparation method and application of garlic essential oil bacteriostatic microemulsion

Abstract

The invention relates to the technical field of aquaculture, in particular to garlic essential oil bacteriostatic microemulsion for prawn culture and a preparation method and application thereof.The garlic essential oil bacteriostatic microemulsion is prepared from garlic essential oil, a surfactant, a cosurfactant and distilled water, and the ratio of the surfactant to the cosurfactant is 5: 1. The ratio of the garlic essential oil to the surfactant is 1: 8, after the garlic essential oil, the surfactant and the cosurfactant are mixed into a microemulsion, distilled water is dropwise added into the mixture while the microemulsion is magnetically stirred until the microemulsion is clarified, and the clarified microemulsion can be rapidly dispersed and dissolved in water after being dropwise added into water. According to the present invention, the surfactant and the co-surfactant are used to emulsify the garlic essential oil, such that the oily garlic essential oil is dissolved in water, and can be easily absorbed by the digestive system of prawns so as to effectively kill the vibrio harveyi infected by the prawns.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a garlic essential oil antibacterial microemulsion for shrimp farming, a preparation method and an application thereof. Background Art

[0002] Essential oils contain a series of plant secondary metabolites, which can directly act on microbial cells by inhibiting the growth of microbial cells, causing cytoplasmic membrane rupture, regulating intermediate metabolism, inhibiting enzymatic reactions, etc., thereby inhibiting the growth of harmful bacteria. Garlic oil is a natural product extracted from garlic Allium sativum. Essential oils are rarely directly extracted and used due to their poor stability and solubility and easy deterioration after long-term storage. They are often processed into various preparations to improve stability, extend the bactericidal efficacy of the product, and expand its application range. Microemulsions, as a spontaneously formed isotropic, transparent, thermodynamically stable homogeneous dispersion system, are good carriers for poorly soluble volatile essential oils. By selecting appropriate surfactants and co-surfactants, essential oils can be well solubilized in the microemulsion system, thereby improving their stability.

[0003] With the development of shrimp farming models and farming techniques, people have been overly pursuing farming output and increasing farming density, which has led to the massive reproduction of bacteria, deterioration of the water environment, and frequent diseases in the later stage of farming. Vibrio disease, a common bacterial disease, is one of the important diseases that endanger shrimp seedlings and farming. Vibrio harveyi is a conditionally pathogenic bacterium. The bacterium usually reproduces in large numbers at the lesion site, causing serious lesions in the internal organs, thereby leading to the death of farmed animals.

[0004] Therefore, in order to solve the problem of shrimp death caused by infection with Vibrio harveyi during shrimp farming, those skilled in the art are in urgent need of a green and pollution-free antibacterial agent that can be used directly in farming and will not cause harm to shrimp. Summary of the invention

[0005] In view of the problems existing in the existing shrimp farming process, the purpose of the present invention is to provide a green antibacterial agent which is pollution-free, can be directly used in shrimp farming, and will not cause harm to the cultured animals when used.

[0006] To achieve the above purpose, the technical scheme adopted by the present invention is: a garlic essential oil antibacterial microemulsion for shrimp farming, comprising garlic essential oil, a surfactant, a cosurfactant and distilled water, wherein the ratio of the surfactant to the cosurfactant is 5:1, and the ratio of the garlic essential oil to the surfactant is 1:8. After the garlic essential oil is mixed with the surfactant and the cosurfactant to form a microemulsion, the distilled water is dripped into the mixture while the microemulsion is stirred by magnetic force until the microemulsion becomes clear, and the clarified microemulsion can be quickly dispersed and dissolved in water when dripped into water.

[0007] The above-mentioned garlic essential oil antibacterial microemulsion for shrimp farming, the surfactant includes food grade Tween 60 and hydrogenated castor oil, and the mixing ratio of the food grade Tween 60 and hydrogenated castor oil is 3:1.

[0008] In the above-mentioned garlic essential oil antibacterial microemulsion for shrimp farming, the co-surfactant is glycerol.

[0009] The garlic essential oil antibacterial microemulsion for shrimp farming comprises diallyl sulfide, diallyl disulfide and diallyl trisulfide.

[0010] The garlic essential oil antibacterial microemulsion used for shrimp farming is of oil-in-water type.

[0011] The garlic essential oil antibacterial microemulsion for shrimp farming has an average particle size of 32.62±9.01 nm and a dispersion index of 0.38±0.025.

[0012] The garlic essential oil antibacterial microemulsion for shrimp farming has a zeta potential of -0.054±0.14 mV.

[0013] A method for preparing a garlic essential oil antibacterial microemulsion for shrimp farming, comprising: Step 1: Mix food grade Tween 60 and hydrogenated castor oil in a ratio of 3:1 to form a surfactant; Step 2: Add a co-surfactant to the mixed surfactant to prepare a mixed surfactant in a mixing ratio of 5:1; Step 3: Weigh garlic essential oil and mixed surfactant in a mass ratio of 1:8, put them into a beaker, and stir them evenly with a magnetic stirrer to obtain a microemulsion; Step 4: At room temperature, distilled water is added dropwise to the beaker until the microemulsion changes from turbid to clear and transparent, thereby obtaining a garlic essential oil antibacterial microemulsion.

[0014] In the above-mentioned method for preparing the garlic essential oil antibacterial microemulsion for shrimp farming, in step 4, distilled water is added dropwise to the beaker at a uniform speed using a pipette.

[0015] A garlic essential oil antibacterial microemulsion for inhibiting Vibrio harveyi, wherein the garlic essential oil antibacterial microemulsion is prepared using the preparation method of the garlic essential oil antibacterial microemulsion for shrimp farming according to any one of claims 8 to 9, and the antibacterial diameter of the garlic essential oil microemulsion on Vibrio harveyi is 21.67±2.05 mm.

[0016] The invention discloses a garlic essential oil antibacterial microemulsion for shrimp farming, a preparation method and an application thereof, and the beneficial effects are as follows: by using a surfactant and a co-surfactant, the garlic essential oil is emulsified, and the oily garlic essential oil is dissolved in water, making it easier for the digestive system of the shrimp to absorb, thereby more effectively disinfecting the Vibrio harveyi infected by the shrimp body. The garlic essential oil antibacterial microemulsion prepared by the invention can effectively inhibit Vibrio harveyi, is green and environmentally friendly, and has no toxic effects, can be used as an antibacterial agent mixed with bait and applied in aquaculture, and the antibacterial diameter of the garlic essential oil antibacterial microemulsion on Vibrio harveyi is 21.67±2.05 mm, which can effectively inhibit Vibrio harveyi. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a product diagram of the garlic essential oil microemulsion of the present invention; Figure 2 This is a diagram for identifying the types of garlic essential oil microemulsions of the present invention; Figure 3 is a transmission electron microscope image of the garlic essential oil microemulsion of the present invention; Figure 4 The particle size and polydispersity index (PDI) of the garlic essential oil microemulsion of the present invention are shown in FIG. Figure 5 is the zeta potential diagram of the garlic essential oil microemulsion of the present invention; Figure 6 This is a diagram showing the antibacterial effect of the plant essential oil microemulsion of the present invention; Figure 7 This is a diagram showing the antibacterial effect of the garlic essential oil microemulsion of the present invention in aquaculture. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the technical aspects of the present invention, the technical solutions of the present invention are described below in conjunction with specific implementations and drawings.

[0019] Example 1 A method for preparing a garlic essential oil antibacterial microemulsion for shrimp farming, comprising: Step 1: Mix food grade Tween 60 and hydrogenated castor oil in a ratio of 3:1 to form a surfactant.

[0020] Step 2: Add the co-surfactant to the mixed surfactant to prepare a mixed surfactant in a mixing ratio of 5:1.

[0021] Step 3: Weigh garlic essential oil and mixed surfactant in a mass ratio of 1:8, put them into a beaker, and stir them evenly using a magnetic stirrer to obtain a microemulsion.

[0022] Step 4: At room temperature, distilled water was added dropwise into the beaker until the microemulsion changed from turbid to clear and transparent, thereby obtaining the garlic essential oil antibacterial microemulsion.

[0023] The specific preparation method is to first mix the surfactant TW60 and PEG40 in a ratio of 3:1, with a total amount of 10g; and mix with the co-surfactant in a ratio of 5:1 to prepare a mixed surfactant (Smix), with a mixing amount of 12g, and then accurately weigh the essential oil and surfactant in a mass ratio of 1:8, with a total amount of 13.25g, put them in a beaker, and stir them evenly with a magnetic stirrer. At room temperature, distilled water is added to the system at a uniform speed with a 1mL pipette. The microemulsion system changes from turbid to clear and transparent at 30ml, that is, it reaches the critical point, thereby obtaining a microemulsion, until a clear and transparent microemulsion is formed, such as Figure 1 and Figure 3 shown.

[0024] Example 2 A garlic essential oil antibacterial microemulsion for shrimp farming comprises garlic essential oil, a surfactant, a cosurfactant and distilled water, wherein the ratio of the surfactant to the cosurfactant is 5:1, and the ratio of the garlic essential oil to the surfactant is 1:8. After the garlic essential oil, the surfactant and the cosurfactant are mixed into a microemulsion, the distilled water is dripped into the mixture while the microemulsion is stirred by magnetic force until the microemulsion becomes clear, and the clarified microemulsion can be quickly dispersed and dissolved in water when dripped into water.

[0025] The surfactant includes food grade Tween 60 and hydrogenated castor oil, and the mixing ratio of food grade Tween 60 and hydrogenated castor oil is 3:1.

[0026] The co-surfactant is glycerol.

[0027] The main components of garlic essential oil include diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS).

[0028] like Figure 2 As shown, the type of microemulsion is determined by dyeing. Sudan B (oil-soluble) and methylene blue (water-soluble) are dropped into two sample bottles containing about 10 mL of essential oil microemulsion, and the diffusion rate and color change of the dyed microemulsion are observed. The diffusion rate of Sudan B dye in garlic microemulsion is slower than that of methylene blue dyeing, which is determined to be oil-in-water type (O / W).

[0029] like Figure 4As shown, at 25 °C, the average particle size and PDI of the microemulsion droplets were determined by photon correlation spectroscopy based on the principle of dynamic light scattering (DLS). In DLS, the sample is irradiated with a laser beam, and the intensity of the scattered light generated by the particles fluctuates at a rate that depends on the particle size. Analysis of these intensity fluctuations can be used to derive the diffusion coefficient of the particles, and thus the particle size. The average particle size of the garlic essential oil microemulsion is 32.62±9.01nm, and the dispersion index (PDI) is 0.38±0.025.

[0030] like Figure 5 As shown, transmission electron microscopy (TEM) analysis was performed to determine the shape of the dispersed phase. A drop of the diluted emulsion was applied to a copper grid and left for 1 min. Then, keeping the grid inverted, a drop of phosphotungstic acid (PTA) was dropped onto the grid for 1 min, and the grid was analyzed using TEM operated at 200 kV. The zeta potential of the garlic essential oil microemulsion was (-0.054 ± 0.14) mV.

[0031] like Figure 6 As shown in the figure, the inhibitory effect of garlic essential oil microemulsion on Vibrio harveyi was measured by agar diffusion inhibition zone method. 150 μL of the 6 activated bacteria were evenly spread on the TSA plate, and then a sterile Oxford cup was placed, and 100 μL of microemulsion was added to each well. After incubation at 28 ℃ for 24 h, the inhibition zone was measured with a vernier caliper; each well was measured 3 times in different directions and the average value was taken. The diameter of the inhibition zone of garlic essential oil microemulsion on Vibrio harveyi was 21.67±2.05mm.

[0032] A greenhouse culture experiment of Penaeus monodon was carried out in shrimp ponds. The garlic essential oil antibacterial microemulsion was mixed with feed and fed at a feeding rate of 5g per kilogram of feed for 3 consecutive days and 7 days of rest. During the experiment, the intestinal contents of the shrimp were collected from time to time and spread on TCBS plates. The antibacterial effect of garlic essential oil was as follows: Figure 7 As shown, Figure 7 The left side of the figure is the experimental group, and the right side is the control group. The number of colonies in the control group is significantly less than that in the experimental group. The control group did not use garlic essential oil antibacterial microemulsion, while the experimental group used garlic essential oil antibacterial microemulsion.

[0033] The above embodiments are only for illustrating the inventive concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. A garlic essential oil antibacterial microemulsion for shrimp farming, characterized in that: The invention comprises garlic essential oil, a surfactant, a co-surfactant and distilled water, wherein the ratio of the surfactant to the co-surfactant is 5:1, and the ratio of the garlic essential oil to the surfactant is 1:

8. After the garlic essential oil, the surfactant and the co-surfactant are mixed into a microemulsion, the distilled water is dripped into the mixture while the microemulsion is stirred by magnetic force until the microemulsion becomes clear, and the clarified microemulsion can be quickly dispersed and dissolved in water when dripped into water.

2. The garlic essential oil antibacterial microemulsion for shrimp farming according to claim 1, characterized in that: The surfactant comprises food-grade Tween 60 and hydrogenated castor oil, and the mixing ratio of the food-grade Tween 60 and hydrogenated castor oil is 3:

1.

3. The garlic essential oil antibacterial microemulsion for shrimp farming according to claim 2, characterized in that: The co-surfactant is glycerol.

4. The garlic essential oil antibacterial microemulsion for shrimp farming according to claim 3, characterized in that: The garlic essential oil includes diallyl sulfide, diallyl disulfide and diallyl trisulfide.

5. The garlic essential oil antibacterial microemulsion for shrimp farming according to claim 4, characterized in that: The garlic essential oil antibacterial microemulsion is an oil-in-water type.

6. The garlic essential oil antibacterial microemulsion for shrimp farming according to claim 4, characterized in that: The average particle size of the garlic essential oil microemulsion is 32.62±9.01 nm, and the dispersion index is 0.38±0.

025.

7. The garlic essential oil antibacterial microemulsion for shrimp farming according to claim 4, characterized in that: The zeta potential of the garlic essential oil microemulsion is -0.054±0.14 mV.

8. A method for preparing a garlic essential oil antibacterial microemulsion for shrimp farming, characterized in that: include: Step 1: Mix food grade Tween 60 and hydrogenated castor oil in a ratio of 3:1 to prepare a surfactant; Step 2: Add a co-surfactant to the mixed surfactant to prepare a mixed surfactant in a mixing ratio of 5:1; Step 3: Weigh garlic essential oil and mixed surfactant in a mass ratio of 1:8, put them into a beaker, and stir them evenly with a magnetic stirrer to obtain a microemulsion; Step 4: At room temperature, distilled water is added dropwise to the beaker until the microemulsion changes from turbid to clear and transparent, thereby obtaining a garlic essential oil antibacterial microemulsion.

9. The method for preparing the garlic essential oil antibacterial microemulsion for shrimp farming according to claim 7, characterized in that: In step 4, distilled water is added dropwise into the beaker at a uniform speed using a pipette.

10. A garlic essential oil antibacterial microemulsion for inhibiting Vibrio harveyi, characterized in that: The garlic essential oil antibacterial microemulsion is prepared using the preparation method of the garlic essential oil antibacterial microemulsion for shrimp farming according to any one of claims 8 to 9, and the antibacterial diameter of the garlic essential oil microemulsion against Vibrio harveyi is 21.67±2.05 mm.

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