Aquaculture anti-vibrio microbial agent, application and preparation method thereof
Through the combination of fulriacic acid and copper salt preparation method, copper fulriacic acid was prepared as an antivibrio agent for aquaculture, which solved the problem of antibiotic pollution in aquaculture, achieved effective inhibition of hemolytic Vibrio, and replaced the use of traditional antibiotics. Reduce environmental and human harm.
Patent Information
- Application Number
- CN202411259241.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-09
- Publication Date
- 2024-12-31
AI Technical Summary
In aquaculture, excessive use of antibiotics leads to environmental pollution and antibiotic residues in aquatic products, and the existing antimicrobial agents have limited effects, making it difficult to effectively replace the use of antibiotics.
Using a combination of fulriacic acid and copper salt, copper fulriacic acid is prepared as an aquaculture anti-Vibiral agent to replace traditional antibiotics, and it has an inhibitory effect on hemolytic Vibrio. The method includes mixing sodium bicarbonate with peat, stirring, adding distilled water, adjusting the pH, and then adding copper sulfate to form a copper fulvicate precipitate.
Copper fulligate has a significant inhibitory effect on Vibrio hemolyticus, and its antibacterial effect is better than CuSO4 and fulligic acid. It can replace antibiotics for a long time and reduce harm to the environment and the human body.
Smart Images
Figure CN119215033A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of anti-vibrio, and in particular to an aquaculture anti-vibrio agent, an application thereof and a preparation method thereof. Background Art
[0002] Antibiotics have a strong ability to fight pathogenic bacteria and are widely used in animal and human medical care. They play a huge role in improving the health of humans and animals. However, due to people's excessive reliance on antibiotics and their large-scale use in daily life, antibiotics have become a pollutant.
[0003] In aquaculture, due to the irregular dosage of antibiotics used by breeders and the characteristics of the antibiotics themselves, most of the antibiotics used are lost to nearshore waters, polluting nearshore water sources, which in turn leads to antibiotic residues in aquatic products, generally in the form of compounds in nature.
[0004] It is known that copper has antibacterial and immune-enhancing effects. Fulvic acid, also known as fulvic acid, is a polyacid with low molecular weight extracted from humic acid. Fulvic acid is brown-red in color. It is soluble in acid, dilute alkali, ethanol, and water. Fulvic acid is a kind of acid with multiple active functional groups and a relatively special structure. It is mainly composed of benzene rings, which are rich in a large number of oxygen-containing functional groups, such as hydroxyl, phenolic hydroxyl, carboxyl, carbonyl, etc. In addition, it may also be conjugated with aromatic structures, olefin bonds, amides, etc. of carbonyl groups. The preparation and application of fulvic acid salts have been widely used.
[0005] Therefore, it is very necessary to use fulvic acid and copper salts to prepare antibacterial agents to replace the use of antibiotics and reduce the harm of antibiotics to the environment and human body. Summary of the invention
[0006] The present invention provides a method for preparing an anti-vibrio agent for aquaculture, which comprises the following steps: S1: Prepare fulvic acid, weigh 2-3 parts by weight of sodium bicarbonate solid, mix it with 8-21 parts by weight of pretreated peat, then place it in a three-necked flask, add distilled water according to a solid-liquid ratio of 1:10-30, and stir in a magnetic stirring device at an ambient temperature of 50-110°C for 30 minutes. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12-24 hours, take the upper layer of liquid and put it in a glass container, add HCL or H2SO4 to the glass container to adjust the pH to 1-2, let it stand for a while, and after precipitation, take the supernatant to obtain a fulvic acid solution; S2: preparing copper fulvic acid, taking 20 parts by weight of the fulvic acid solution prepared in step S1, adding 50 parts by weight of copper sulfate thereto, shaking well, placing in a water bath at 25-100° C. and standing for 10-200 minutes, and waiting for precipitation, the precipitate is copper fulvic acid.
[0007] The present invention also provides an anti-Vibrio agent for aquaculture prepared by the above-mentioned preparation method.
[0008] The present invention also provides an application of the above-mentioned anti-Vibrio agent for aquaculture in resisting Vibrio pathogens in aquaculture.
[0009] The beneficial effects of the present invention are as follows: copper fulvic acid can be prepared by the preparation method disclosed in the present invention, and copper fulvic acid has an inhibitory effect on hemolytic Vibrio, and the antibacterial effect is greater than that of CuSO4 and fulvic acid, and can achieve long-term replacement of antibiotics, thereby reducing the harm of antibiotics to the environment and human body. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 A curve graph showing the peak area of fulvic acid generated at different ratios of sodium bicarbonate to peat.
[0011] Figure 2 A graph showing the peak area of fulvic acid generated at different stirring temperatures.
[0012] Figure 3 A graph showing the peak area of fulvic acid generated at different HCL concentrations.
[0013] Figure 4 A graph showing the peak area of fulvic acid generated at different H2SO4 concentrations.
[0014] Figure 5 The content graph of the extracted fulvic acid peak area under different extraction acid types is shown.
[0015] Figure 6 A graph showing the peak area of fulvic acid consumption at different copper sulfate concentrations.
[0016] Figure 7 A graph showing the peak area of fulvic acid consumption at different water bath temperatures is shown.
[0017] Figure 8 A comparative experimental diagram of the antibacterial effect of copper fulvic acid in liquid culture medium is shown.
[0018] Fig. 9 The antibacterial comparison experiment diagram of three reagents under solid culture medium is shown. DETAILED DESCRIPTION
[0019] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.
[0020] Example 1
[0021] The present invention provides a method for preparing an anti-vibrio agent for aquaculture, comprising the following steps:
[0022] S1: To prepare fulvic acid, weigh 2-3 parts by weight of sodium bicarbonate solid, mix it with 8-21 parts by weight of pretreated peat, then place it in a three-necked flask, add distilled water at a solid-liquid ratio of 1:10-30, and stir it in a magnetic stirring device at an ambient temperature of 50-110°C for 30 minutes. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12-24 hours, take the upper layer of liquid and place it in a glass container, add HCL or H2SO4 to the glass container to adjust the pH to 1-2, let it stand for a while, and after precipitation, take the supernatant to obtain a fulvic acid solution.
[0023] S2: Prepare copper fulvic acid, take 20 parts by weight of the fulvic acid solution prepared in step S1, add 50 parts by weight of copper sulfate thereto, shake well, place in a water bath at 25-100°C and let stand for 10-200 minutes until a precipitate is precipitated, take the supernatant for liquid chromatography detection, and determine the unreacted fulvic acid content based on the peak area. The precipitate is copper fulvic acid.
[0024] The optimal ratio of sodium bicarbonate and peat in the preparation of fulvic acid was explored through experiments. The specific experiments are as follows:
[0025] Experiment 1
[0026] Peat and sodium bicarbonate are mixed in a ratio of 1:4. Take 20g of pretreated peat and mix it with 80g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device for 30min at a certain ambient temperature. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12h, take the supernatant, add H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0027] Experiment 2
[0028] Peat and sodium bicarbonate are mixed at a ratio of 1:5. Take 20g of pretreated peat and mix it with 100g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device for 30min at a certain ambient temperature. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12h, take the supernatant, add H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0029] Experiment 3
[0030] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of solid sodium bicarbonate. After mixing, place it in a 500mL three-necked flask. Add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device for 30min at a certain ambient temperature. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12h, take the supernatant, add H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained based on the peak area.
[0031] Experiment 4
[0032] Peat and sodium bicarbonate are mixed in a ratio of 1:7. Take 20g of pretreated peat and mix it with 1400g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device for 30min at a certain ambient temperature. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12h, take the supernatant, add H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0033] refer to Figure 1 , shows the experimental results of experiments 1-4, namely the fulvic acid peak area, where the peak area unit is mAU·S, according to Figure 1 It can be seen that when other variables are the same, the mixing ratio of peat and sodium bicarbonate is 1:6, which is the best ratio for preparing fulvic acid.
[0034] The optimal stirring temperature for the preparation of fulvic acid was explored through experiments. The specific experiments are as follows:
[0035] Experiment 5
[0036] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 50°C for 30min. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12h, take the supernatant and add H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0037] Experiment 6
[0038] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 70°C for 30min. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12h, take the supernatant and add H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0039] Experiment 7
[0040] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 90°C for 30min. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12h, take the supernatant and add H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0041] Experiment 8
[0042] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 110°C for 30min. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12h, take the supernatant and add H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0043] refer to Figure 2 , showing the experimental results of experiments 5-8, namely the fulvic acid peak area, where the peak area unit is mAU·S, according to Figure 2 It can be seen that when other variables are the same, the stirring environment temperature of 90°C is the optimal reaction temperature for preparing fulvic acid.
[0044] The best extraction acid type and extraction acid concentration for adjusting pH in the preparation of fulvic acid were explored through experiments. The specific experiments are as follows:
[0045] Experiment 9
[0046] Peat and sodium bicarbonate are mixed at a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 90°C for 30min. After stopping the reaction, remove the three-necked flask, and oxidize at room temperature for 12h, take the supernatant and add 3.5% HCL to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0047] Experiment 10
[0048] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 90°C for 30min. After stopping the reaction, remove the three-necked flask, and oxidize at room temperature for 12h, take the supernatant and add 4% HCL to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0049] Experiment 11
[0050] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 90°C for 30min. After stopping the reaction, remove the three-necked flask, and oxidize at room temperature for 12h, take the supernatant and add 4.5% HCL to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained according to the peak area.
[0051] Experiment 12
[0052] Peat and sodium bicarbonate were mixed at a ratio of 1:6. 20 g of pretreated peat was mixed with 120 g of sodium bicarbonate solid, and the mixture was placed in a 500 mL three-necked flask. Distilled water was added to the three-necked flask at a solid-liquid ratio of 1:20, and the mixture was stirred in a magnetic stirring device at an ambient temperature of 90°C for 30 min. After the reaction was stopped, the three-necked flask was removed, and after oxidation at room temperature for 12 h, the supernatant was taken and 5% HCL was added to adjust the pH to 1-2, and the mixture was allowed to stand for a while. After a precipitate was precipitated, the supernatant was the prepared fulvic acid solution, and the supernatant was subjected to liquid chromatography to obtain the fulvic acid content based on the peak area.
[0053] refer to Figure 3 , showing the experimental results of experiments 9-12, namely the fulvic acid peak area, where the peak area unit is mAU·S, according to Figure 3 As shown, when other variables are the same, when HCL is used to adjust the pH, the HCL concentration of 4.5% is the optimal concentration for preparing fulvic acid.
[0054] Experiment 13
[0055] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 90°C for 30min. After stopping the reaction, remove the three-necked flask, and oxidize at room temperature for 12h, take the clear liquid and add 1.5% H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained based on the peak area.
[0056] Experiment 14
[0057] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 90°C for 30min. After stopping the reaction, remove the three-necked flask, and oxidize at room temperature for 12h, take the clear liquid and add 2% H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained based on the peak area.
[0058] Experiment 15
[0059] Peat and sodium bicarbonate are mixed in a ratio of 1:6. Take 20g of pretreated peat and mix it with 120g of sodium bicarbonate solid. After mixing, place it in a 500mL three-necked flask, add distilled water into the three-necked flask at a solid-liquid ratio of 1:20, and stir in a magnetic stirring device at an ambient temperature of 90°C for 30min. After stopping the reaction, remove the three-necked flask, and oxidize at room temperature for 12h, take the clear liquid and add 2.5% H2SO4 to adjust the pH to 1-2, let it stand for a while, and after a precipitate is precipitated, the supernatant is the prepared fulvic acid solution, and the supernatant is detected by liquid chromatography, and the fulvic acid content is obtained based on the peak area.
[0060] Experiment 16
[0061] Peat and sodium bicarbonate were mixed at a ratio of 1:6. 20 g of pretreated peat was mixed with 120 g of sodium bicarbonate solid, and the mixed solution was placed in a 500 mL three-necked flask. Distilled water was added to the three-necked flask at a solid-liquid ratio of 1:20, and the mixture was stirred in a magnetic stirring device at an ambient temperature of 90°C for 30 min. After the reaction was stopped, the three-necked flask was removed, and after oxidation at room temperature for 12 h, the clear liquid was taken and 3% H2SO4 was added to adjust the pH to 1-2. The solution was allowed to stand for a while until a precipitate was precipitated. The supernatant was the prepared fulvic acid solution, and the supernatant was subjected to liquid chromatography to obtain the fulvic acid content based on the peak area.
[0062] refer to Figure 4 , shows the experimental results of experiments 13-16, namely the fulvic acid peak area, where the peak area unit is mAU·S, according to Figure 4 As shown, when other variables are the same, when H2SO4 is used to adjust the pH, the H2SO4 concentration of 2.5% is the optimal concentration for preparing fulvic acid.
[0063] according to Figure 3 and Figure 4As shown, the best concentrations of the two extraction acids were compared, that is, 2.5% H2SO4 and 4.5% HCL were used for experimental comparison, and chromatographic detection was performed to detect the peak area of fulvic acid when the two extraction acids were used. For details, refer to Figure 5 .
[0064] refer to Figure 5 It can be seen that in the process of preparing fulvic acid, H2SO4 is selected as the best extraction acid, and the H2SO4 concentration of 2.5% is the optimal concentration for preparing fulvic acid.
[0065] The optimal concentration of copper sulfate in the preparation of copper fulvic acid was explored through experiments. The specific experiments are as follows:
[0066] Experiment 17
[0067] Take 5mL of 4‰ fulvic acid in a test tube, then add 1.5% copper sulfate solution into the test tube, shake well, and place them in a water bath at a certain temperature for a certain period of time. After precipitation, take the supernatant for liquid chromatography detection, and the unreacted fulvic acid content is obtained according to the peak area. Then, the content of prepared copper fulvic acid is determined by the unreacted fulvic acid content, that is, the precipitate is copper fulvic acid.
[0068] Experiment 18
[0069] Take 5mL of 4‰ fulvic acid in a test tube, then add 2% copper sulfate solution into the test tube, shake well, and place them in a water bath at the same temperature as Experiment 17 for the same time as Experiment 17. After the precipitate is precipitated, take the supernatant for liquid chromatography detection, and the unreacted fulvic acid content is obtained according to the peak area. Then, the content of the prepared copper fulvic acid is determined by the unreacted fulvic acid content, that is, the precipitate is copper fulvic acid.
[0070] Experiment 19
[0071] Take 5mL of 4‰ fulvic acid in a test tube, then add 2.5% copper sulfate solution into the test tube, shake well, and place them in a water bath at the same temperature as Experiment 17 for the same time as Experiment 17. After the precipitate is precipitated, take the supernatant for liquid chromatography detection, and the unreacted fulvic acid content is obtained according to the peak area. Then, the content of the prepared copper fulvic acid is determined by the unreacted fulvic acid content, that is, the precipitate is copper fulvic acid.
[0072] Experiment 20
[0073] Take 5mL of 4‰ fulvic acid in a test tube, then add 3% copper sulfate solution into the test tube, shake well, and place them in a water bath at the same temperature as Experiment 17 for the same time as Experiment 17. After the precipitate is precipitated, take the supernatant for liquid chromatography detection, and the unreacted fulvic acid content is obtained according to the peak area. Then, the content of the prepared copper fulvic acid is determined by the unreacted fulvic acid content, that is, the precipitate is copper fulvic acid.
[0074] refer to Figure 6 , showing the experimental results of experiments 17-21, namely the fulvic acid peak area, where the peak area unit is mAU·S, according to Figure 6 As shown, when other variables are the same, when the concentration of copper sulfate is 2%, fulvic acid consumption is the highest and the remaining fulvic acid content is the lowest. At this time, the content of copper fulvic acid generated is the highest, which is the optimal concentration of copper sulfate for preparing copper fulvic acid.
[0075] The optimal water bath temperature for the preparation of copper fulvic acid was explored through experiments. The specific experiments are as follows:
[0076] Experiment 21
[0077] Take 5mL of 4‰ fulvic acid in a test tube, add 2% copper sulfate solution into the test tube, shake well, and place them in a 30°C water bath for a certain period of time. After the precipitate is precipitated, take the supernatant for liquid chromatography detection, and the unreacted fulvic acid content is obtained according to the peak area. Then, the content of the prepared copper fulvic acid is determined by the unreacted fulvic acid content, that is, the precipitate is copper fulvic acid.
[0078] Experiment 22
[0079] Take 5mL of 4‰ fulvic acid in a test tube, add 2% copper sulfate solution into the test tube, shake well, and place them in a 50°C water bath for the same time as experiment 22. After the precipitate is precipitated, take the supernatant for liquid chromatography detection, and the unreacted fulvic acid content is obtained according to the peak area. Then, the content of the prepared copper fulvic acid is determined by the unreacted fulvic acid content, that is, the precipitate is copper fulvic acid.
[0080] Experiment 23
[0081] Take 5mL of 4‰ fulvic acid in a test tube, add 2% copper sulfate solution into the test tube, shake well, and place them in a 70°C water bath for the same time as experiment 22. After the precipitate is precipitated, take the supernatant for liquid chromatography detection, and the unreacted fulvic acid content is obtained according to the peak area. Then, the content of the prepared copper fulvic acid is determined by the unreacted fulvic acid content, that is, the precipitate is copper fulvic acid.
[0082] Experiment 24
[0083] Take 5mL of 4% fulvic acid in a test tube, and then add 2% copper sulfate solution into the test tube. After shaking, place them in a 90°C water bath and let them stand for the same time as Experiment 22. After the precipitate is precipitated, take the supernatant for liquid chromatography detection, and the unreacted fulvic acid content is obtained based on the peak area. Then, the content of the prepared copper fulvic acid is determined by the unreacted fulvic acid content, that is, the precipitate is copper fulvic acid.
[0084] refer to Figure 7 , showing the experimental results of experiments 21-24, namely the fulvic acid peak area, where the peak area unit is mAU·S, according to Figure 7 As shown, when other variables are the same, when the water bath temperature is 50°C, fulvic acid consumption is the highest and the remaining fulvic acid content is the lowest. At this time, the content of copper fulvic acid generated is the highest, which is the optimal concentration of copper sulfate for the preparation of copper fulvic acid.
[0085] The antibacterial effect of copper fulvic acid was explored through experiments. The specific experiments are as follows:
[0086] Experiment 25
[0087] According to the structure of copper fulvic acid, in addition to copper, copper fulvic acid also contains oxygen-containing functional groups such as hydroxyl and phenolic hydroxyl. Copper has an antibacterial effect on bacterial growth, and oxygen-containing functional groups also have an inhibitory effect on bacterial growth.
[0088] Take two 150mL liquid culture media, add 2mL of bacterial solution to each of the two culture media, add the copper fulvic acid antibacterial agent prepared by the above method to one of the culture media, shake the flask and culture at an ambient temperature of 37°C for 12 hours, then take 3mL of the cultured bacterial solution from each of the two culture media for OD 600 Measurement, specific experimental results refer to Figure 8 and Table 1. Table 1 Antibacterial effect of copper fulvic acid in liquid culture No antimicrobial agent added Add antimicrobial agent OD value 2.015 0.255
[0089] It can be seen from Table 1 that the OD value with the addition of copper fulvic acid antibacterial agent is significantly lower than the OD value without the addition of antibacterial agent, indicating that copper fulvic acid has a certain antibacterial effect.
[0090] The antibacterial effect of copper fulvic acid compared with fulvic acid and copper sulfate was studied through experiments. The specific experiments are as follows:
[0091] Experiment 26
[0092] The OD of the bacterial solution was adjusted by 0.9% saline. 600 The value is adjusted so that the corresponding bacterial concentration reaches OD 600The value is 0.5. Then take three 30μL portions of bacterial solution and spread them on three solid culture media respectively. Use a 10mm diameter puncher to make three holes on each culture media. Drop 50μL of copper fulvic acid antibacterial agent, fulvic acid solution and copper sulfate solution into the three holes on one culture media respectively. Then place the three culture media in a constant temperature incubator at 37℃ for 24h, and then measure the size of the inhibition zone. The experimental results are as follows: Fig. 9 shown.
[0093] refer to Fig. 9 It can be seen that the diameters of the antibacterial zones around copper fulvic acid are 26mm, 23mm and 29mm respectively. The diameters of the antibacterial zones around copper sulfate are 19mm, 16mm and 24mm respectively. There is almost no antibacterial zone around fulvic acid. Therefore, it can be concluded that the antibacterial effect is: copper fulvic acid>copper sulfate>fulvic acid.
[0094] Electron microscopy was used to observe the effect of adding copper fulvic acid on the morphology of Vibrio parahaemolyticus in liquid culture medium. When no antimicrobial agent was added, Vibrio parahaemolyticus showed a regular oval shape, but after adding the antimicrobial agent, Vibrio parahaemolyticus had no obvious cell morphology, that is, the cell morphology was irregular, indicating that copper fulvic acid had a certain inhibitory effect on Vibrio parahaemolyticus.
[0095] It should be noted that the configuration of the culture medium in this application is as follows:
[0096] 3% sodium chloride alkaline peptone water medium (liquid medium): peptone 10.0 g, sodium chloride 30.0 g, final Ph 8.5±0.2 (1 L).
[0097] 3% sodium chloride tryptone soy agar medium (solid medium): tryptone 15.0 g, soy peptone 5.0 g, sodium chloride 30.0 g, agar 15.0 g, final pH 7.3±0.2 (1 L).
[0098] In summary, copper fulvic acid can be prepared by the preparation method disclosed in the present invention, and copper fulvic acid has an inhibitory effect on hemolytic Vibrio, and the antibacterial effect is greater than that of CuSO4 and fulvic acid, and can achieve long-term replacement of antibiotics, thereby reducing the harm of antibiotics to the environment and human body.
[0099] Example 2
[0100] This embodiment discloses an anti-Vibrio agent for aquaculture, which is prepared by the preparation method described in Example 1.
[0101] Example 3
[0102] This embodiment discloses the use of the anti-Vibrio bacterial agent for aquaculture as described in Example 2 in the fight against Vibrio pathogens in aquaculture.
[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be within the scope of protection of the present invention.
Claims
1. A method for preparing an anti-Vibrio agent for aquaculture, characterized in that: The following steps are involved: S1: Prepare fulvic acid, weigh 2-3 parts by weight of sodium bicarbonate solid, mix it with 8-21 parts by weight of pretreated peat, then place it in a three-necked flask, add distilled water according to a solid-liquid ratio of 1:10-30, and stir in a magnetic stirring device at an ambient temperature of 50-110°C for 30 minutes. After stopping the reaction, remove the three-necked flask, oxidize at room temperature for 12-24 hours, take the upper layer of liquid and put it in a glass container, add HCL or H2SO4 to the glass container to adjust the pH to 1-2, let it stand for a while, and after precipitation, take the supernatant to obtain a fulvic acid solution; S2: preparing copper fulvic acid, taking 20 parts by weight of the fulvic acid solution prepared in step S1, adding 50 parts by weight of copper sulfate thereto, shaking well, placing in a water bath at 25-100° C. and standing for 10-200 minutes, and waiting for precipitation, the precipitate is copper fulvic acid.
2. An anti-Vibrio agent for aquaculture prepared by the preparation method as claimed in claim 1.
3. Use of the anti-Vibrio agent for aquaculture as claimed in claim 2 in the fight against Vibrio pathogens in aquaculture.