Application of gallic acid in treatment of glass seedling diseases of penaeus vannamei and experimental method of gallic acid

Abstract

The invention relates to application of gallic acid in treatment of penaeus vannamei boone glass seedling diseases and an experimental method of gallic acid in treatment of penaeus vannamei boone glass seedling diseases, and belongs to the technical field of experimental animal production. Experiments determine that when the minimum inhibitory concentration of gallic acid to glass seedling disease pathogenic vibrio V.Para.TS-GE is 1000 [mu] g / mL and the minimum bactericidal concentration of gallic acid is 2000 [mu] g / mL, and the concentration of gallic acid is 200 [mu] g / mL, the survival rate of penaeus vannamei seedlings infected with glass seedling diseases can be effectively improved, the death rate can be reduced, and the problem of drug resistance existing in current antibiotic prevention and control can be effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of experimental animal production and relates to an application of gallic acid in the treatment of vibrio disease in white shrimp seedlings and an experimental method thereof, and specifically relates to an application of gallic acid in the treatment of vibrio disease in white shrimp seedlings and an experimental method thereof, and more particularly to an application of gallic acid in the treatment of vibrio disease in white shrimp seedlings and the treatment of vibrio disease in white shrimp seedlings. Background Art

[0002] Vannamei shrimp, a highly economically valuable aquaculture species, are susceptible to Vibrio disease during the shrimp larval stage. This disease, caused by Vibrio bacteria, typically occurs between P5 and P7, with a short onset and a high mortality rate. Current antibiotics for prevention and control pose resistance challenges. Gallic acid (GA), a botanical drug with diverse bioactivities, including antioxidant and antibacterial properties, is widely available and inexpensive, making its development as a treatment for Vibrio disease of great significance. Summary of the Invention

[0003] The present invention aims to address the current problems of shrimp seedlings being susceptible to vibrio-induced vibrio disease, resulting in a high shrimp seedling mortality rate, and the existence of drug resistance in antibiotic prevention and control. The present invention proposes an application of gallic acid in the treatment of vibrio disease in whiteleg shrimp seedlings and an experimental method thereof. By determining the in vitro inhibitory concentration of GA on the pathogenic Vibrio TS-GE of vibrio disease, combined with shrimp seedling toxicity tests and in vivo animal feeding experiments, the resistance of whiteleg shrimp seedlings after infection with vibrio disease is enhanced and the mortality rate is reduced.

[0004] The application of gallic acid provided in this application in the treatment of diseases of white shrimp seedlings and glass seedlings adopts the following technical solution: The invention discloses an application of gallic acid in the treatment of vitreous disease of white shrimp seedlings. The invention is characterized in that the gallic acid is used to improve the survival rate of white shrimp seedlings infected with vitreous disease, and the concentration of the gallic acid is 200 μg / mL.

[0005] By adopting the above technical solution, the specific concentration and efficacy of gallic acid in the treatment of glass seedling disease of white shrimp seedlings were clarified. When its concentration is 200μg / mL, it can enhance the ability of white shrimp seedlings to resist glass seedling disease and improve the survival rate after infection with the disease.

[0006] Furthermore, the glass seedling disease is caused by pathogenic Vibrio V.Para.TS-GE, and the gallic acid can effectively inhibit the growth and reproduction of the Vibrio, with a minimum inhibitory concentration of 1000 μg / mL and a minimum bactericidal concentration of 2000 μg / mL.

[0007] By adopting the above technical solution, the pathogenic cause of glass seedling disease and the effect of gallic acid on pathogenic bacteria are limited, thereby enhancing the credibility of the targeted treatment of this application.

[0008] Furthermore, the preparation process of the gallic acid is as follows: (1) Select analytically pure gallic acid >98%; (2) Weigh a certain amount of gallic acid and dissolve it in sterile phosphate buffer solution to prepare a working concentration of 2 mg / mL; (3) Filter and sterilize using a 0.22 μm microporous filter membrane.

[0009] By adopting the above technical solution, gallic acid is screened for purity, prepared into a solution of specific concentration, and sterilized to obtain a gallic acid solution that meets the application requirements. This standardizes the preparation process of gallic acid, helps to ensure the consistency of product quality, and provides practical guidance for the actual promotion of this application, ensuring its safety and effectiveness during the treatment process.

[0010] The experimental method provided in this application for treating diseases of white shrimp seedlings and glass seedlings with gallic acid adopts the following technical scheme: An experimental method for treating diseases of white shrimp seedlings and glass seedlings with gallic acid, characterized in that the experimental method comprises the following steps: (1) After 2 days of temporary culture in sterile seawater, healthy whiteleg shrimp fry were randomly divided into 4 groups, with 20 shrimp in each group, including the negative control group containing 200 μg / mL GA, the V.Para.TS-GE with a final concentration of 1.04×10 6 The positive control group contained 200 μg / mL GA + 1.04×10 6 The experimental group of V.Para.TS-GE and the blank control group without bacterial solution and GA were included; (2) Pick a single colony of V.Para.TS-GE and inoculate it into LB liquid medium at 37°C and 120 rpm overnight. Centrifuge the obtained bacterial solution at 6°C and 6000 rpm for 10 min. Wash it three times with sterile phosphate buffer solution, resuspend it with sterile phosphate buffer solution, and adjust the concentration to about 10. 8 CFU / mL was used as the working bacterial solution, and then confirmed by plate colony counting method; (3) The experimental group was treated by mixing 10 mL of the working bacterial solution with 100 mL of GA solution and soaking for 30 min; the negative control group was treated by mixing 10 mL of sterile phosphate buffer solution with 100 mL of GA solution and soaking for 30 min; the positive control group was treated by mixing 10 mL of the working bacterial solution with 100 mL of sterile phosphate buffer solution and soaking for 30 min; (4) Place the soaked mixture and 20 shrimps into 890 mL of oxygenated water in a 1 L bucket and feed them normally for 2 days. Record the death rate and appearance changes of the shrimp fry every 2 hours.

[0011] By adopting the above technical solution, healthy white shrimp fry were cultured in groups, different control groups and experimental groups were set up, and the shrimp fry were soaked in a specific concentration of gallic acid solution mixed with pathogenic Vibrio. The survival and appearance changes of the shrimp fry were observed over a certain period of time, thereby verifying the therapeutic effect of gallic acid on Vibrio fry disease. Through the steps of experimental grouping, Vibrio culture, mixed soaking and feeding observation, it is easy for others to repeat and verify the feasibility and effectiveness of this application. Furthermore, the salinity of the sterile seawater is 15‰, and the preparation method is to add 20g of sea salt to each liter of sterile pure water, and use stabilizers and detergents to balance the water body for 5-7 days to form a stable water environment with a pH of ≈ 7.5.

[0012] By adopting the above technical solution, the configuration method and requirements of sterile seawater are clarified, a stable breeding environment is provided for the experiment, the importance of controlling experimental conditions is highlighted, and it helps to improve the accuracy and repeatability of the experiment.

[0013] Furthermore, the preparation method of the phosphate buffer solution is as follows: (1) Weigh 8 g of sodium chloride, 1.44 g of disodium hydrogen phosphate, 0.2 g of potassium chloride, and 0.24 g of potassium dihydrogen phosphate and dissolve them in 800 mL of distilled water; (2) Adjust the pH to 7.2 with hydrochloric acid and make the volume to 1 L; (3) Sterilize with high-pressure steam at 121°C for 20 min, then store at 4°C after cooling.

[0014] By adopting the above technical solution, the phosphate buffer solution can maintain a relatively stable pH environment, which is important for the stability of the gallic acid solution and the cultivation of Vibrio. Through the specific preparation method and sterilization treatment, the quality and applicability of the buffer solution are guaranteed. This ensures that the quality and performance of the buffer solution used in the experiment meet the requirements, thereby ensuring the smooth implementation of the entire application method.

[0015] Furthermore, the preparation method of the LB liquid culture medium is as follows: (1) Weigh 10 g of peptone, 5 g of yeast extract, and 10 g of sodium chloride; (2) Adjust the pH to 7.0 with 5 mol / L sodium hydroxide and then dilute to 1 L with distilled water; (3) Sterilize with high-pressure steam at 121°C for 15 minutes, let it cool, and then store it at room temperature.

[0016] By adopting the above technical solution, LB liquid culture medium is a commonly used culture medium for culturing pathogenic Vibrio V.Para.TS-GE. Through specific preparation methods and sterilization treatment, it can provide a good growth environment for Vibrio, ensure the number and activity of Vibrio, and provide sufficient pathogens for subsequent experiments, thereby improving the accuracy and repeatability of the experiments.

[0017] Furthermore, the final concentration of GA in the test group and the negative control group was 200 μg / mL, and the final concentration of V.Para.TS-GE in the positive group and the test group was 1.04×10 6 CFU / mL.

[0018] By adopting the above technical scheme, the final concentrations of gallic acid and pathogenic Vibrio in the experimental group and the control group were limited, ensuring the consistency and comparability of the experimental conditions, facilitating the accurate evaluation of the therapeutic effect of gallic acid on Vitreous seedling diseases, and helping to improve the reliability and credibility of the experimental conclusions.

[0019] In summary, the present invention includes at least one of the following beneficial technical effects: (1) Improve the survival rate: Gallic acid is used to improve the survival rate of white shrimp seedlings infected with glass seedling disease, and its effective concentration is determined to be 200 μg / mL. When its concentration is 200 μg / mL, it can enhance the ability of white shrimp seedlings to resist glass seedling disease and effectively reduce the mortality rate, providing a safe and effective treatment plan for the shrimp seedling farming industry.

[0020] (2) Effective inhibition of pathogens: Gallic acid can effectively inhibit the growth and reproduction of pathogenic Vibrio V.Para.TS-GE that causes vitreous seedling disease. Its minimum inhibitory concentration is 1000 μg / mL and its minimum bactericidal concentration is 2000 μg / mL. This provides a scientific basis for the targeted treatment of vitreous seedling disease and enhances the credibility of this application.

[0021] (3) Standardized preparation and application methods: By standardizing the preparation process of gallic acid and detailed experimental operation procedures, including the preparation of sterile seawater, phosphate buffer solution, and LB liquid culture medium, the quality stability of gallic acid and the safety and effectiveness of its application are ensured, which makes it easier for others to repeat and verify the feasibility and effectiveness of the application method, provides reliable guidance for actual promotion, helps to improve the reliability and credibility of the experiment, and has important practical significance for the shrimp fry farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the minimum inhibitory and bactericidal concentration results of GA against V.Para.TS-GE pathogens in the present invention.

[0023] Figure 2This is a graph showing the test results of the toxicity of GA to whiteleg shrimp fry in the present invention.

[0024] Figure 3 This is a diagram showing the therapeutic effect of GA on glass seedling diseases in the present invention.

[0025] Figure 4 This is the histopathological section result diagram of the present invention. DETAILED DESCRIPTION

[0026] The present invention is further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art fall within the scope defined by the claims attached to this application.

[0027] Example 1 Healthy whiteleg shrimp (Penaeus vannamei) were temporarily cultured in sterile seawater at 26-28°C for two days and then randomly divided into four groups (n=20) (as described above). The shrimp had an average body length of 6 mm and a PL of 5-7. For the negative control group, 10 mL of sterile phosphate buffer and 100 mL of GA solution were mixed and soaked for 30 minutes. For the positive control group, 10 mL of working bacterial solution and 100 mL of sterile phosphate buffer were mixed and soaked for 30 minutes. The soaked mixture and 20 shrimp were then placed in 890 mL of oxygenated water, resulting in a final GA concentration of 200 μg / mL in both the experimental and negative control groups. The shrimp were then reared normally for two days, and shrimp mortality and physical changes were recorded every two hours.

[0028] The living individuals of the experimental shrimp groups and the dead individuals of the positive control group, as well as the surviving individuals of the negative control group, were fixed in 4% paraformaldehyde for 24 h for subsequent frozen section and histopathological analysis.

[0029] The minimum inhibitory and bactericidal concentrations of GA against V.Para.TS-GE pathogens are shown in the results Figure 1 The minimum inhibitory concentration of GA against V.Para.TS-GE pathogens was 1000 μg / mL. Further plate coating verification showed that its minimum bactericidal concentration was 2000 μg / mL, indicating that GA has good antibacterial and bactericidal effects on pathogens of glass seedling diseases. The results of GA toxicity test on white shrimp fry are shown in Figure 2 GA toxicity test results showed that when the concentration was greater than 200 μg / mL, the toxicity to shrimp fry became more obvious with the increase of GA treatment time. After 48 h of treatment, the LC 50=588μg / mL. When the final concentration of GA was 200μg / mL, the toxicity to shrimp fry was significantly reduced, with almost no lethal toxicity. Therefore, in the subsequent treatment experiments, 200μg / mL of GA was selected to improve the ability of shrimp fry to resist glass seedling disease; See the effect Figure 3 : GA had a significant protective effect when used to treat shrimp challenged with V.Para.TS-GE at a final concentration of 200 μg / mL. The mortality rate of shrimp after V.Para.TS-GE challenge was reduced to 15% (p<0.05).

[0030] Histopathological results are shown in Figure 4 Figures A, B, and D show hepatopancreas sections from healthy shrimp, shrimp from the negative group, and shrimp from the experimental group. The hepatopancreas structure of the shrimp larvae is relatively intact, with clearly visible hepatopancreatic ductules, showing no significant differences. Figures E, F, and H show intestinal sections from healthy shrimp, shrimp from the negative group, and shrimp from the experimental group. The intestinal structure is essentially intact and clear, with intestinal epithelial cells arranged in a relatively neat and dense pattern, showing no significant differences. Figure C shows a hepatopancreas section from the positive group, showing severe lesions in the shrimp larvae, with desquamated hepatopancreas cells, partial structural loss, and serous internal tissue. Figure G shows a section from the intestinal section from the positive group, showing severe damage to the intestinal structure, detached epithelial cells, and a large amount of debris within the intestine.

[0031] Example 2 Test of the inhibitory effect of gallic acid on pathogenic Vibrio V.Para.TS-GE Experimental purpose: To determine the minimum inhibitory concentration and minimum bactericidal concentration of gallic acid against Vibrio parasitica TS-GE, the pathogenic bacterium of Vibrio parasitica TS-GE.

[0032] Experimental materials: The pathogenic bacteria V.Para.TS-GE, which is the cause of vibrio disease, was obtained from Huanghua Hairen Aquatic Seed Technology Co., Ltd. Vibrio was cultured in LB liquid medium, and gallic acid solution was prepared using sterile phosphate buffer solution.

[0033] Experimental steps: Prepare gallic acid solution: Weigh a certain amount of analytically pure (>98%) gallic acid and dissolve it in sterile phosphate buffer solution to a working concentration of 2 mg / mL. Filter and sterilize using a 0.22 μm microporous filter.

[0034] Cultivation of pathogenic Vibrio TS-GE: Pick a single colony and inoculate it into LB liquid culture medium, and culture it at 37°C and 120 rpm overnight.

[0035] The overnight culture was centrifuged at 6000 r / min for 10 min at 6°C, washed three times with sterile phosphate buffer solution, resuspended and diluted to about 10 8The CFU / mL was used as the working bacterial solution, and the concentration was confirmed by plate colony counting method.

[0036] Gallic acid solutions of different concentrations were mixed with the working bacterial solution, and the effects of gallic acid on the growth of Vibrio were observed and recorded to determine the minimum inhibitory concentration and minimum bactericidal concentration.

[0037] Experimental results: The minimum inhibitory concentration of gallic acid against V.Para.TS-GE pathogenic bacteria is 1000μg / mL, and the minimum bactericidal concentration is 2000μg / mL, indicating that gallic acid has significant inhibitory and bactericidal effects on pathogens of glass seedling diseases.

[0038] Example 3 Verification of the therapeutic effect of gallic acid on diseases of white shrimp seedlings Experimental purpose: To verify the effect of gallic acid in improving the survival rate of whiteleg shrimp seedlings infected with glass seedling disease.

[0039] Materials: Healthy whiteleg shrimp (PL5-7) fry, averaging 6 mm in length and sterile as determined by a shrimp fry disease test kit. Also prepared were pathogenic Vibrio spp. V.Para.TS-GE, gallic acid solution, and related laboratory instruments and reagents.

[0040] Experimental steps: After culturing white shrimp fry temporarily in sterile seawater at 26-28℃ for 2 days, they were randomly divided into 4 groups, with 20 shrimp in each group, including a negative control group containing 200 μg / mL GA, a V.Para.TS-GE with a final concentration of 1.04×10 6 CFU / mL of positive control group, containing 200μg / mLGA + 1.04×10 6 The experimental group was composed of V.Para.TS-GE and the blank control group was composed of no bacterial solution and GA.

[0041] Gallic acid solution and pathogenic Vibrio working bacterial solution were prepared according to the method in Example 2.

[0042] The experimental group mixed 10 mL of working bacterial solution with 100 mL of GA solution and soaked it for 30 min; the negative control group mixed 10 mL of sterile phosphate buffer solution with 100 mL of GA solution and soaked it for 30 min; the positive control group mixed 10 mL of working bacterial solution with 100 mL of sterile phosphate buffer solution and soaked it for 30 min.

[0043] The soaked mixture and 20 shrimps were placed in 890 mL of oxygenated water in a 1 L bucket and raised normally for 2 days. The mortality and appearance changes of the shrimp fry were recorded every 2 hours.

[0044] Experimental results: GA significantly reduced its toxicity to shrimp fry at a final concentration of 200 μg / mL, with virtually no lethal toxicity. After challenge with V.Para.TS-GE, the mortality rate of the experimental group was reduced to 15% (p < 0.05), while the mortality rate in the positive control group was higher. Histopathological sections showed that the hepatopancreas and intestinal structures of the experimental group shrimp fry were essentially intact and clear, with no significant differences from healthy shrimp, while the hepatopancreas and intestinal structures of the shrimp fry in the positive control group showed severe lesions. This indicates that GA at a concentration of 200 μg / mL can effectively improve the ability of white shrimp fry to resist glass seedling disease, reduce mortality, and has low toxicity to shrimp fry.

Claims

1. An application of gallic acid in the treatment of diseases of whiteleg shrimp seedlings and glass seedlings, characterized in that: The gallic acid is used to improve the survival rate of whiteleg shrimp seedlings infected with glass seedling disease, and the concentration of the gallic acid is 200 μg / mL.

2. The application of gallic acid according to claim 1 in the treatment of diseases of Penaeus vannamei seedlings and glass seedlings is characterized in that: The glass seedling disease is caused by pathogenic Vibrio V.Para.TS-GE, and the gallic acid can effectively inhibit the growth and reproduction of the Vibrio, with a minimum inhibitory concentration of 1000 μg / mL and a minimum bactericidal concentration of 2000 μg / mL.

3. The application of gallic acid in the treatment of diseases of Penaeus vannamei seedlings and glass seedlings according to claim 2, is characterized in that, The preparation process of described gallic acid is as follows: (1) Select analytically pure gallic acid >98%; (2) Weigh a certain amount of gallic acid and dissolve it in sterile phosphate buffer solution to prepare a working concentration of 2 mg / mL; (3) Filter and sterilize using a 0.22 μm microporous filter membrane.

4. An experimental method for treating diseases of white shrimp seedlings and glass seedlings with gallic acid, characterized in that: The experimental method comprises the following steps: (1) After 2 days of temporary culture in sterile seawater, healthy whiteleg shrimp fry were randomly divided into 4 groups, with 20 shrimp in each group, including the negative control group containing 200 μg / mL GA, the V.Para.TS-GE with a final concentration of 1.04×10 6 CFU / mL positive control group, containing 200 μg / mL GA + 1.04×10 6 CFU / mL of V.Para.TS-GE experimental group and blank control group without bacterial solution and GA; (2) Pick a single colony of V.Para.TS-GE and inoculate it into LB liquid medium at 37°C and 120 rpm overnight. Centrifuge the obtained bacterial solution at 6°C and 6000 rpm for 10 min. Wash it three times with sterile phosphate buffer solution, resuspend it with sterile phosphate buffer solution, and adjust the concentration to about 10. 8 CFU / mL was used as the working bacterial solution, and then confirmed by plate colony counting method; (3) In the experimental group, 10 mL of the working bacterial solution was mixed with 100 mL of GA solution and soaked for 30 min; in the negative control group, 10 mL of sterile saline was mixed with 100 mL of GA solution and soaked for 30 min; in the positive control group, 10 mL of the working bacterial solution was mixed with 100 mL of sterile phosphate buffer solution and soaked for 30 min; (4) Place the soaked mixture and 20 shrimps into 890 mL of oxygenated water in a 1 L bucket and feed them normally for 2 days. Record the death rate and appearance changes of the shrimp fry every 2 hours.

5. The experimental method of gallic acid in the treatment of diseases of whiteleg shrimp seedlings and glass seedlings according to claim 4, characterized in that: The salinity of the sterile seawater is 15‰, and the preparation method is to add 20g of sea salt per liter of sterile pure water, and use stabilizers and detergents to balance the water body for 5-7 days to form a stable water environment with a pH of ≈ 7.

5.

6. The experimental method of gallic acid in the treatment of diseases of whiteleg shrimp seedlings and glass seedlings according to claim 4, characterized in that, The preparation method of the phosphate buffer solution is as follows: (1) Weigh 8 g of sodium chloride, 1.44 g of disodium hydrogen phosphate, 0.2 g of potassium chloride, and 0.24 g of potassium dihydrogen phosphate and dissolve them in 800 mL of distilled water; (2) Adjust the pH to 7.2 with hydrochloric acid and make the volume to 1 L; (3) Sterilize with high-pressure steam at 121°C for 20 min, then store at 4°C after cooling.

7. The experimental method of gallic acid in the treatment of diseases of Penaeus vannamei seedlings and glass seedlings according to claim 4, characterized in that, The preparation method of the LB liquid culture medium is as follows: (1) Weigh 10 g of peptone, 5 g of yeast extract, and 10 g of sodium chloride; (2) Adjust the pH to 7.0 with 5 mol / L sodium hydroxide and then dilute to 1 L with distilled water; (3) Sterilize with high-pressure steam at 121°C for 15 minutes, let it cool, and then store it at room temperature.

8. The experimental method of using gallic acid in the treatment of diseases of Penaeus vannamei seedlings and glass seedlings according to any one of claims 4 to 7, characterized in that: The final concentration of GA in the experimental group and the negative control group was 200 μg / mL, and the final concentration of V.Para.TS-GE in the positive group and the experimental group was 1.04×10 6 CFU / mL.

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