Use of allicin e for preventing and treating vibrio parahemolyticus infection in giant freshwater prawns

Abstract

The present invention provides a use of allicin E for preventing and treating Vibrio parahemolyticus infection in giant freshwater prawns, comprising the step of: adding a compound as shown in formula (I) to giant freshwater prawn feed. This can improve Vibrio parahemolyticus infection in giant freshwater prawns, reduce the mortality rate of giant freshwater prawns, and reduce the threat to food and environmental safety caused by long-term use of antibiotics.

Description

Uses of garlic E in the prevention and treatment of Vibrio parahaemolyticus infection in giant freshwater prawns. Technical Field

[0001] This invention relates to the field of Vibrio parahaemolyticus infection prevention and control technology, and proposes the use of garlic E to prevent Vibrio parahaemolyticus infection in giant freshwater prawns. Background Technology

[0002] Vibrio parahemolyticus is a Gram-negative bacillus. Ingestion of food containing this bacterium can cause food poisoning, also known as halophilic bacteria food poisoning. Clinically, the main symptoms are acute onset, abdominal pain, vomiting, diarrhea, and watery stools. Vibrio parahemolyticus is a marine bacterium, primarily found in seafood such as fish, shrimp, crabs, shellfish, and seaweed. Infection with Vibrio parahemolyticus causes giant freshwater prawns to feed sparingly or not at all, resulting in empty stomachs, significant atrophy of the hepatopancreas, and a reddened, more transparent body. Infected prawns rarely swim at the surface, often staying near the edge of the pond, and exhibit weakened vitality. Vibrio parahemolyticus produces hemolytic toxins. These toxins have direct hemolytic properties, causing hemolysis of various shrimp cells, leading to the dissolution and death of blood cells. Vibrio parahemolyticus also has enterotoxic effects, causing damage to the digestive tract tissues of shrimp and even disrupting the digestive function of the hepatopancreas.

[0003] Currently, the prevention and control of Vibrio parahaemolyticus mainly relies on antibiotics, such as enrofloxacin, doxycycline hydrochloride, and florfenicol. However, long-term use of antibiotics easily leads to drug resistance, and the amount of antibiotics used is increasing year by year, posing a significant threat to food and environmental safety. Therefore, there is an urgent need in this field to provide novel and effective ingredients for the prevention and control of Vibrio parahaemolyticus.

[0004] Summary of the Invention

[0005] The purpose of this invention is to provide a novel effective ingredient for the prevention and treatment of Vibrio parahaemolyticus.

[0006] A first aspect of the present invention provides a use of garlic E, characterized in that it is used to prepare a feed composition for inhibiting Vibrio parahaemolyticus in Macrobrachium rosenbergii; wherein the garlic E has a structure as shown in the following formula:

[0007] In another preferred embodiment, the feed composition is used to treat or improve diseases or symptoms caused by Vibrio parahaemolyticus infection in giant freshwater prawns.

[0008] In another preferred embodiment, the amount of garlic E in the feed composition is 100-2000 mg / kg of feed.

[0009] In another preferred embodiment, the amount of garlic E in the feed composition is 900-1100 mg / kg of feed.

[0010] In another preferred embodiment, the feed composition is used to increase bacterial cell membrane permeability.

[0011] In another preferred embodiment, the feed composition is used to inhibit the formation of Vibrio parahaemolyticus biofilm.

[0012] In another preferred embodiment, the feed composition is used to increase vacuolation of Vibrio parahaemolyticus cells. In yet another preferred embodiment, the feed composition is used to reduce mortality in Macrobrachium rosenbergii infected with Vibrio parahaemolyticus.

[0013] In another preferred embodiment, the garlic E ingredient is prepared as a stock solution with a concentration of 5-10 mg / L.

[0014] In another preferred embodiment, the garlic E ingredient is prepared as a stock solution with a concentration of 30-35 mg / L for use.

[0015] In a second aspect, the present invention provides a feed composition for giant freshwater prawns, characterized in that the feed composition comprises garlic E.

[0016] In another preferred embodiment, the content of allicin E in the feed composition is 100-2000 mg / kg feed.

[0017] In another preferred embodiment, the amount of garlic E in the feed composition is 900-1100 mg / kg of feed.

[0018] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0019] Figure 1 shows the MIC (a) and MBC (b) of allicin E against Vibrio parahaemolyticus.

[0020] Figure 2 shows the growth curves of Vibrio parahaemolyticus under different concentrations of garlic E.

[0021] Figure 3 shows the effects of allicin E on the activities of extracellular lactate dehydrogenase (a), β-D-galactosidase (b), and protein content (c) of Vibrio parahaemolyticus.

[0022] Figure 4 shows a transmission electron microscope image of Vibrio parahaemolyticus after treatment with garlic E. As shown in Figure 4, a is the control group, with intact cell structure and normal growth; b to d are the 1 / 4 MIC treatment groups, where the cell wall and membrane are dissolved, cytoplasm is leaked, and vacuolization occurs.

[0023] Figure 5 shows the staining pattern (a) and absorbance (b) of Vibrio parahaemolyticus biofilm after treatment with different concentrations of garlic E. Detailed Implementation

[0024] Through long-term and in-depth research, the inventors discovered that adding garlic E to feed can effectively improve Vibrio parahaemolyticus infection in giant freshwater prawns, exhibiting good in vitro antibacterial activity against Vibrio parahaemolyticus, disrupting its cell structure, and increasing cell membrane permeability. Based on these findings, the inventors completed this invention.

[0025] Vibrio parahaemolyticus infection in giant freshwater prawns

[0026] The giant freshwater prawn (Macrobrachium rosenbergii), also known as the white-legged prawn, long-armed prawn, or Malaysian prawn, is a large freshwater shrimp. Due to its rapid growth, large size, broad diet, and short growth cycle, it is an important aquaculture species in many regions. Infection with Vibrio parahaemolyticus can cause giant freshwater prawns to feed less or not at all, reducing their activity. Furthermore, the hemolytic toxins produced by Vibrio parahaemolyticus can cause hemolysis of various shrimp cells, leading to the death of blood cells. Its enterotoxins can also damage the digestive tract tissues and even disrupt the digestive function of the hepatopancreas. Therefore, developing methods to prevent and control Vibrio parahaemolyticus infection in giant freshwater prawn farming is of paramount importance in aquaculture technology.

[0027] Garlic E

[0028] ALE (Allicin E) is a single compound synthesized biomimetically by modifying the structure outside the antibacterial active group of ordinary allicin. Its structure is shown in the following formula:

[0029] Garlic E has broad-spectrum bactericidal and green safety characteristics, and can effectively replace traditional antibiotics, supporting environmental disinfection, animal and plant protection, preservation, and upgrading of the biopharmaceutical industry.

[0030] Feed composition with added garlic E

[0031] This invention provides a feed composition; the feed composition is fortified with garlic E, the garlic E having the structure shown in the following formula:

[0032] Because garlic E has good bactericidal properties, the feed composition described above is used to reduce or improve the infection of giant freshwater prawns with Vibrio parahaemolyticus. In a preferred embodiment, the amount of garlic E in the feed composition is 100-2000 mg / kg feed, more preferably 900-1100 mg / kg feed.

[0033] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0034] Experimental materials

[0035] The experiment was conducted at the Comprehensive Experimental Base of the Zhejiang Provincial Freshwater Fisheries Research Institute. Garlic E was provided by Shanghai Laishen Biotechnology Co., Ltd. Vibrio parahaemolyticus (VP) was purchased from the China Industrial Microbial Culture Collection Center. 2216E broth and agar medium were purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd. The giant freshwater prawns were collected from the Comprehensive Experimental Base of the Zhejiang Provincial Freshwater Fisheries Research Institute; the prawns weighed approximately 5g and were healthy and vigorous.

[0036] Example 1: Strain Culture

[0037] Remove the Vibrio parahaemolyticus VP strain from the refrigerator and streak it onto 2216E agar medium. Incubate statically at 28°C for 1–2 days until uniformly morphological colonies appear, indicating successful bacterial activation and resuscitation. Pick a single colony and inoculate it into a test tube containing 5 mL of 2216E broth. Incubate at 28°C with shaking at 150 rpm for 24 hours. Determine the concentration of Vibrio parahaemolyticus in the culture using a turbidimetric method. In subsequent experiments, dilute the culture to the desired concentration using sterilized 2216E broth medium.

[0038] Example 2: Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

[0039] The MIC of allicin E against Vibrio parahaemolyticus was determined using the resazurin method. 100 μL (1 x 10⁻⁶) of allicin was added to each well of a 96-well plate. 5 CFU / mL Vibrio parahaemolyticus bacterial suspension was added to the first row of wells with the required amount of garlic E stock solution. A 2-fold serial dilution was performed row by row to achieve final drug concentrations of 7.8, 3.9, 1.95, 1, 0.5, and 0.25 mg / L. A positive control (100 μL bacterial suspension + 100 μL 2216E broth) and a negative control (200 μL 2216E broth) were also included, with three wells per group (three replicates). The 96-well plate was incubated at 28°C for 24 hours. 50 μL of resveratrol blue solution was added to each well, and the plate was incubated for another 24 hours. The color of the bacterial suspension was observed (pink indicates bacterial growth, blue indicates inhibition). The lowest drug concentration at which no bacterial growth was observed was the MIC.

[0040] Based on the MIC experiment, 100 μL of bacterial suspension was taken from the clear well and spread onto 2216E agar plates. The plates were incubated at 28°C for 24 h. The lowest concentration at which no colonies grew on the plate was taken as the MBC of the drug for that strain.

[0041] The resazurin assay showed that when the concentration of allicin E was greater than 7.8 mg / L, the bacterial culture turned blue (no cell activity), indicating that the MIC (minimum inhibitory concentration) of allicin E against Vibrio parahaemolyticus was 7.8 mg / L (Figure 1a). When bacterial cultures with concentrations greater than 7.8 mg / L were spread from each well of a 96-well plate, no colonies grew when the allicin E concentration was greater than 31.25 mg / L (Figure 1b), indicating that its MBC (minimum bactericidal concentration) was 31.25 mg / L. These results indicate that allicin E has a strong inhibitory effect on Vibrio parahaemolyticus.

[0042] Example 3 Growth Curve Measurement

[0043] The concentration of Vibrio parahaemolyticus in the test tubes was adjusted to 1 x 10⁻⁶ using 2216E broth medium. 7 CFU / mL of allicin was added and serially diluted twofold to achieve final concentrations of MIC, 1 / 2MIC, and 1 / 4MIC. The test tubes were incubated at 28℃ and 180 rpm in a shaker, and the OD of the culture medium was measured every 2 hours. 600 nm value. The OD value of the culture medium is plotted on the x-axis as incubation time. 600 The growth curve is plotted with nm as the ordinate.

[0044] Based on bacterial OD 600 The growth of Vibrio parahaemolyticus in each group was compared and analyzed using nm absorbance values. The growth curves showed that the OD values ​​of the bacterial cultures in the three garlic E-treated groups (MIC, 1 / 2MIC, 1 / 4MIC) were significantly different. 600 The nm values ​​were all lower than those of the control group, and the OD values ​​were also lower. 600 The absorbance at nm was negatively correlated with the drug concentration, with the OD of the bacterial culture at MIC being... 600 The nm value remained at a low level throughout (Figure 2). These results indicate that the use of garlic E can continuously inhibit the growth of Vibrio parahaemolyticus.

[0045] Example 4: Cell membrane permeability measurement

[0046] The concentration of Vibrio parahaemolyticus culture in the test tubes was adjusted to 1 x 10⁻⁶ using 2216E broth medium. 7CFU / mL of allicin was added and serially diluted 2-fold to achieve final concentrations of MIC, 1 / 2 MIC, and 1 / 4 MIC. A control group was also included, with an equal volume of PBS added. All tubes were incubated at 28°C and 180 rpm for 6 hours. Protein leakage (Coomassie Brilliant Blue assay) and lactate dehydrogenase activity (LDH kit, Nanjing Jiancheng Bioengineering Institute) were measured. β-galactosidase activity (ONPG reagent, Beijing Solarbio Science & Technology Co., Ltd.) was measured after 12 hours of incubation.

[0047] The effects of garlic E on the cell membrane permeability of Vibrio parahaemolyticus were characterized based on extracellular lactate dehydrogenase (LDH), β-D-galactosidase (β-D-galactosidase) activity, and protein content. The results showed that LDH activity was negatively correlated with garlic E concentration. LDH activity in the MIC, 1 / 2MIC, and 1 / 4MIC concentration groups was significantly lower than that in the control group (P<0.05) (Figure 3a), indicating that garlic E treatment inhibited the growth of Vibrio parahaemolyticus. With increasing drug concentration, both β-D-galactosidase activity and protein content increased. The MIC group showed significantly higher β-D-galactosidase activity and protein content than the control group (P<0.05), and the 1 / 2MIC group showed significantly higher β-D-galactosidase activity than the control group (P<0.05) (Figures 3b and 3c). This indicates that garlic E treatment increased bacterial cell membrane permeability, leading to leakage of intracellular macromolecules.

[0048] Example 5: Observation using transmission electron microscopy (TEM)

[0049] In containing 1x10 7 In test tubes containing CFU / mL Vibrio parahaemolyticus bacterial suspension, allicin E (i.e., drug treatment group) was added to a final concentration of 1 / 2 MIC. A control group with an equal amount of PBS was also set up. The suspensions were cultured at 28℃ and 180 rpm for 24 h. 1.5 mL of the bacterial suspension was taken from each tube and centrifuged at 8000 rpm for 5 min at 4℃. The supernatant was discarded, and the precipitate was washed three times with sterile PBS. The precipitate was collected, and 2.5% glutaraldehyde was added. The tubes were then fixed at 4℃ for 12 h and sent to Hangzhou Yanqu Information Technology Co., Ltd. The morphology and structure of the bacteria were observed by transmission electron microscopy (TEM).

[0050] Transmission electron microscopy images show that the control group bacteria have intact structures, smooth surfaces, and uniform distribution of cell contents (Fig. 4a); while the Vibrio parahaemolyticus treated with 1 / 4 MIC allicin showed a significant reduction in cytoplasm, a lighter color (Fig. 4b), and partial ablation of cell walls and membrane edges (Fig. 4c). The bacterial cells also showed some bright pores and vacuolization (Fig. 4d).

[0051] Example 6: Determination of Biofilm Content

[0052] In 1x10 7 CFU / mL Vibrio parahaemolyticus bacterial suspension was incubated with allicin at final concentrations of 1 / 4 MIC, 1 / 2 MIC, and MIC, and cultured at 28℃ and 180 rpm for 6 h, followed by static incubation for 12 h. The bacterial suspension was discarded, and 1 mL of 2% (v / v) crystal violet was used for staining for 2 min. The solution was rinsed repeatedly with tap water 3–5 times, then dissolved in 1 mL of glacial acetic acid, and the absorbance at 570 nm was measured.

[0053] Untreated Vibrio parahaemolyticus formed a biofilm on the tube wall after shaking, which stained darkly with crystal violet. However, the tube wall treated with MIC concentration of Vibrio parahaemolyticus E showed a lighter color (Fig. 5a). Absorbance was measured by dissolving the biofilm on the tube wall with glacial acetic acid. The results showed that the absorbance at 570 nm in the 1 / 2 MIC and MIC groups was significantly lower than that in the control group (Fig. 5b). These results indicate that Vibrio parahaemolyticus E treatment can inhibit biofilm formation.

[0054] Example 7: In vivo antibacterial efficacy experiment

[0055] Before the experiment, all giant freshwater prawns were temporarily held for one week at a water temperature maintained at 25±1℃. They were fed twice daily, and approximately 30% of the water was changed according to water quality indicators. The mortality rate during the temporary holding period was less than 0.5%. Ten prawns were randomly selected before the experiment for pathogen testing to ensure they did not carry common pathogens. Before the formal experiment began, medicated feed was prepared by first crushing the feed, adding the medication, mixing thoroughly, pressing it into pellets using a pelleting machine, and then drying it before use.

[0056] Healthy freshwater prawns were randomly divided into four groups: a test drug group (garlic E, 1000, 500, and 250 mg / kg feed), a control drug group (enrofloxacin, 1000 mg / kg feed), a challenge control group, and a blank control group. Each group had three replicates, with 20 prawns per replicate. The acclimation period lasted 2–3 days, and the experiment began after all prawns were feeding normally. During the experiment, prawns were fed twice daily (9:00 AM and 4:00 PM) until they were satiated (based on statistics and calculations during the temporary holding period, a daily feed intake of 2% of the prawn's body weight was considered appropriate, as the prawns could finish feeding in about 10 minutes). Considering that prawns would eat less after challenge, this study used a pre-challenge drug administration method. The test drug group and the control drug group were fed feed containing garlic E and enrofloxacin, respectively, while the challenge control group and the blank control group were fed ordinary feed. Water was changed according to water quality indicators, with approximately 30% of the water being replaced. Water quality parameters such as temperature, dissolved oxygen, pH, ammonia nitrogen, and nitrite nitrogen were measured daily. During the experiment, the water temperature was maintained at 25 ± 1℃, dissolved oxygen was greater than 5.0 mg / L, pH was 7.8–9.0, ammonia nitrogen was less than 0.5 mg / L, and nitrite nitrogen was less than 0.1 mg / L.

[0057] Three days after drug administration, the bacteria were challenged with the virus. Based on preliminary experiments, the concentration of the challenge bacterial solution was determined to be 5 x 10⁻⁶. 6 CFU / mL (mortality rate approximately 80%–100%). Each shrimp in the test drug group, control drug group, and challenge control group was intramuscularly injected with 0.1 mL of Vibrio parahaemolyticus PBS resuspension. The blank control group received an equal volume of PBS per shrimp. Shrimp were observed continuously for 7 days after challenge, and mortality was recorded. The relative protection rate (RPS) was calculated using the following formula: RPS = (1 - mortality rate of the test group / mortality rate of the challenge control group) x 100%.

[0058] Giant freshwater prawns were challenged with Vibrio parahaemolyticus after being fed a mixture of garlic E and a control drug (enrofloxacin). The results showed that within 7 days of challenge, the survival rates of prawns in the garlic E groups (1000 mg / kg, 500 mg / kg, and 250 mg / kg) were 80%, 50%, and 10%, respectively, while the survival rate in the 1000 mg / kg enrofloxacin control group was 70%. These results indicate that feeding with garlic E effectively reduces mortality in giant freshwater prawns caused by Vibrio parahaemolyticus, and at the same dosage, its effect is superior to that of the control compound enrofloxacin.

[0059] discuss

[0060] The experimental results show that:

[0061] (1) Garlic E has good in vitro antibacterial activity against Vibrio parahaemolyticus, with MIC and MBC values ​​of 7.8 mg / L and 31.25 mg / L, respectively.

[0062] (2) Garlic E can destroy the cell structure of Vibrio parahaemolyticus, increase cell membrane permeability, and cause leakage of macromolecules such as β-D-galactosase and proteins.

[0063] (3) Garlic E can effectively reduce the mortality rate of giant freshwater prawns infected with Vibrio parahaemolyticus.

[0064] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A use of garlic E, characterized in that, Feed composition for preparing in macrobrachium rosenbergii inhibiting vibrio parahaemolyticus; wherein, the allicin E has the following formula:

2. Use according to claim 1, characterized in that, The feed composition is used to treat or improve diseases or symptoms caused by Vibrio parahaemolyticus infection in giant freshwater prawns.

3. Use according to claim 1, characterized in that, In the feed composition, the amount of garlic E is 100-2000 mg / kg of feed.

4. The use according to claim 1, characterized in that, In the feed composition, the amount of garlic E is 900-1100 mg / kg of feed.

5. The use according to claim 1, characterized in that, The feed composition described herein is used to increase bacterial cell membrane permeability.

6. The use according to claim 1, characterized in that, The feed composition described herein is used to inhibit the formation of Vibrio parahaemolyticus biofilm.

7. The use as described in claim 1, characterized in that, The feed composition described herein is used to reduce the mortality rate of giant freshwater prawns infected with Vibrio parahaemolyticus.

8. A feed composition for Macrobrachium rosenbergii, characterized in that, The feed composition includes garlic E.

9. The composition according to claim 8, characterized in that, The content of allicin E in the feed composition is 100-2000 mg / kg feed.

10. The composition of claim 8, wherein In the feed composition, the amount of garlic E is 900-1100 mg / kg of feed.

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