Application of food preservative in inhibition of aeromonas hydrophila

Abstract

The invention discloses application of a food preservative in inhibition of aeromonas hydrophila, and belongs to the field of aquatic bacteriostatic agents. The invention provides application of nisin in inhibition of aeromonas hydrophila, and experimental results show that the nisin has an obvious antibacterial effect on aeromonas hydrophila when the concentration of the nisin is 7.5 mg / mL, and has bactericidal activity when the concentration of the nisin is 50 mg / mL; moreover, the nisin can effectively reduce the hemolytic activity of the aeromonas hydrophila and the hydrolytic activity of extracellular protease, and the nisin can destroy the outer membrane and the inner membrane of the aeromonas hydrophila, so that the toxicity of the aeromonas hydrophila is finally reduced, and in a word, the nisin shows comprehensive inhibition capability and potential on the aeromonas hydrophila. The invention provides a new strategy for preventing and / or treating infection caused by aeromonas hydrophila in aquaculture, and has great economic benefits and wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of aquatic antibacterial agents, and in particular to application of a food preservative in inhibiting Aeromonas hydrophila. Background Art

[0002] Aeromonas hydrophila is a common, Gram-negative, short bacillus belonging to the Vibrio family. It possesses motility in liquid culture due to its polar flagella. It lacks spores and a capsule, and its optimal growth temperature is 25-30°C. It is one of the most common pathogens of aquatic animals, particularly fish, and can cause epidemic outbreaks during the warm summer months. Studies have shown that Aeromonas hydrophila produces highly toxic exotoxins, including hemolysins, tissue toxins, necrotizing toxins, and enterotoxins. The interaction of these multiple virulence factors contributes to its potent and widespread pathogenicity. Aeromonas hydrophila is ubiquitous in freshwater, sewage, and soil, and is a typical pathogen of humans, animals, and fish. It harms fish, shrimp, crabs, farmed frogs, livestock, and humans, causing localized infections such as sepsis and skin ulcers, as well as acute gastroenteritis. Outbreaks often cause devastating economic losses to the aquaculture industry. Currently, antibiotics are commonly used for prevention and treatment during production. However, their overuse has led to the development of multidrug resistance and the proliferation of resistance genes. Aeromonas hydrophila is resistant to multiple antibiotics, with the prevalence reaching as high as 76%, and exhibits broad-spectrum resistance. While the use of green and effective natural products as alternatives to antibiotics is becoming a growing trend, research remains limited.

[0003] Nisin is a naturally occurring bioactive antimicrobial peptide composed of 34 amino acids. It exhibits high thermal stability, retaining over 90% of its bioactivity after heating at 100°C for 10-15 minutes. It is also stable in solutions at pH 2.0, while it is inactivated at pH 7.0. Nisin can be used as a non-toxic food preservative that is digestible by digestive enzymes and highly storage-stable. As an antimicrobial peptide, nisin is highly effective and safe, and has found initial application in the food and pharmaceutical industries. Studies have shown that nisin, due to its presence of multiple dehydrated or lanthioamino acid residues, exhibits potent bactericidal activity against Gram-positive bacteria such as Staphylococcus, Enterococcus, Pediococcus, Leuconostoc, and Listeria, particularly spore-forming bacteria such as Bacillus and Clostridium. However, it has no inhibitory effect on Gram-negative bacteria, yeasts, and molds. Nisin can inhibit the synthesis of substances such as peptidoglycan, thereby hindering the synthesis of cell membranes and phospholipid compounds, leading to the leakage of intracellular substances and cell lysis. Summary of the Invention

[0004] The present invention aims to provide a use of a food preservative in inhibiting Aeromonas hydrophila to address the problems of the prior art. Nisin can inhibit the growth of Aeromonas hydrophila and exhibits comprehensive inhibitory ability and potential against Aeromonas hydrophila. The present invention provides a new strategy for preventing and / or treating infections caused by Aeromonas hydrophila in aquaculture, and has significant economic benefits and broad application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of nisin in inhibiting the proliferation of aquatic pathogens in vitro.

[0007] Preferably, the aquatic pathogen is Aeromonas hydrophila.

[0008] The present invention also provides an application of nisin in preparing an Aeromonas hydrophila antibacterial agent.

[0009] The present invention also provides an Aeromonas hydrophila antibacterial agent, which takes nisin as a main active ingredient.

[0010] Preferably, the bacteriostatic agent further comprises other bacteriostatic active substances.

[0011] The present invention also provides an application of nisin in preparing a reagent for reducing the toxicity of Aeromonas hydrophila.

[0012] The present invention also provides a reagent for reducing the toxicity of Aeromonas hydrophila, which contains nisin as a main active ingredient.

[0013] The present invention also provides a use of nisin in preparing a medicine for preventing and / or treating Aeromonas hydrophila infection.

[0014] The present invention also provides a medicine for preventing and / or treating Aeromonas hydrophila infection, which contains nisin as a main active ingredient.

[0015] Preferably, the drug further comprises other antibacterial active ingredients.

[0016] The present invention discloses the following technical effects:

[0017] The present invention provides the use of nisin in inhibiting Aeromonas hydrophila. Experimental results show that nisin has a significant antibacterial effect on Aeromonas hydrophila at 7.5 mg / mL and bactericidal activity at 50 mg / mL. Furthermore, nisin can effectively reduce the hemolytic activity and hydrolytic activity of extracellular proteases of Aeromonas hydrophila. Furthermore, nisin can destroy the outer and inner membranes of Aeromonas hydrophila, ultimately reducing its virulence. In summary, nisin demonstrates comprehensive inhibitory ability and potential against Aeromonas hydrophila. The present invention provides a new strategy for preventing and / or treating infections caused by Aeromonas hydrophila in aquaculture, with significant economic benefits and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 The figure shows the results of the MIC determination experiment of nisin against Aeromonas hydrophila; wherein - is the negative control and + is the positive control;

[0020] Figure 2 This is a line graph of the inhibition rate of nisin (starting at 30 mg / mL) against Aeromonas hydrophila;

[0021] Figure 3 This is a line graph of the inhibition rate of nisin (starting at 40 mg / mL) against Aeromonas hydrophila;

[0022] Figure 4 The growth observation diagram of Aeromonas hydrophila after 24 hours of smear culture; (A) is the 12.5 mg / mL nisin group, (B) is the 25 mg / mL nisin group, and (C) is the 50 mg / mL nisin group;

[0023] Figure 5 Figure 2 is a graph showing the results of the inhibition zone test of nisin against Aeromonas hydrophila; (A) shows the results of nisin treatment at concentrations of 30, 40, and 50 mg / mL; (B) shows the results of nisin treatment at concentrations of 25, 15, and 7.5 mg / mL;

[0024] Figure 6 Statistical graph of the hemolytic activity of nisin against Aeromonas hydrophila; * indicates P < 0.05;

[0025] Figure 7This is the result of the extracellular protease hydrolysis activity experiment of Aeromonas hydrophila cultured for 24 hours;

[0026] Figure 8 This is a statistical graph showing the effect of nisin on the AKP activity of Aeromonas hydrophila;

[0027] Figure 9 This is a statistical graph of the extracellular β-D-galactosidase content of Aeromonas hydrophila after 6 hours of nisin treatment;

[0028] Figure 10 Statistical graph of the extracellular DNA content of Aeromonas hydrophila after 6 hours of nisin treatment. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Aeromonas hydrophila was donated by the Zhejiang Freshwater Fisheries Research Institute. Nisin was purchased from Shanghai Maclean. LB broth premix powder was purchased from Guangdong Huankai. Agar powder was purchased from Sinopharm Reagent. Resazurin was purchased from Shanghai Maclean. Skim milk medium, made from skim milk powder, was purchased from Zhebei Milan Supermarket in Huzhou. BHI medium was purchased from Guangdong Huankai.

[0035] Preparation of nisin stock solution: Prepare nisin with sterile water to a concentration of 50 mg / mL, filter through a 0.45 μm filter for sterilization, and prepare immediately before use.

[0036] Preparation of LB broth liquid culture medium: Add 25 g of LB broth medium premix powder to every 1000 mL of sterile water. Sterilize the prepared culture medium by autoclaving at 121°C for 20 min before use.

[0037] Preparation of LB broth solid culture medium: Add 25 g of LB broth medium premix powder to every 1000 mL of sterile water, then add 15 g of agar powder and mix well. Autoclave at 121°C for 20 min, pour into a plate and let dry. Store in a refrigerator at 4°C until used.

[0038] Preparation of Resazurin stock solution: Prepare Resazurin working solution with sterile water to a concentration of 40 μg / mL, sterilize through a 0.22 μm filter, and store in a -20°C refrigerator away from light.

[0039] After the bacteria were activated, they were picked up with an inoculation loop and inoculated into a conical flask containing 50 mL of sterilized LB liquid medium. The culture was shaken at 28°C and 120 rpm for about 24 hours. The cell count was controlled at 10 8 CFU / mL.

[0040] Example 1

[0041] 1. Experimental Methods

[0042] 1.1 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

[0043] In columns 1-10 of a 96-well microtiter plate, 100 μL of drug-containing culture medium of varying concentrations (serial dilutions, final nisin concentration of 50-0.098 mg / mL) was added, followed by 100 μL of bacterial solution. Column 11 added 100 μL of culture medium and 100 μL of bacterial solution as a positive control, and column 12 added 200 μL of culture medium as a negative control. After incubation at 28°C for approximately 20 hours, 50 μL of resazurin working solution was added. Three replicates were set up for all experimental and control groups, and incubation continued for 30 minutes. The absorbance was measured at a wavelength of 600 nm using a microtiter plate reader, and the color of each well was observed. The inhibition rate was calculated according to the following formula. The antibiotic concentration in the wells with an inhibition rate ≥90% and no color change was defined as the minimum inhibitory concentration (MIC) of the antibiotic against Vibrio.

[0044]

[0045] Take solutions with nisin at or above the minimum inhibitory concentration, add 100 μL of bacterial solution, inoculate into LB solid culture medium, and incubate at 28°C for about 24 hours. The lowest concentration at which no colony growth is observed is the minimum bactericidal concentration (MBC).

[0046] 1.2 Determination of the diameter of the drug's inhibition zone on bacteria

[0047] The antibacterial sensitivity of Aeromonas hydrophila to nisin was determined using the Oxford cup method. -3 Place 100 μL of the 200 μL Aeromonas hydrophila suspension onto a plate and spread evenly with a spreader. Then, use tweezers to gently place a sterilized Oxford cup into the Petri dish. Place the Oxford cups evenly on a flat surface, forming a triangular pattern. Pipette 0.25 mL of nisin at a specific concentration into the Oxford cup. Place the Petri dish containing nisin in a 4°C refrigerator to diffuse for 24 hours. Then, incubate at the optimal growth temperature of 28°C for 24 hours.

[0048] 1.3 Hemolytic activity assay

[0049] According to the method of Zhu Ludan et al. (Zhu Ludan, Chen Kai, Xi Bingwen, et al. Antibacterial effect of quinoline and its effect on the virulence of Aeromonas hydrophila [J]. Chinese Fisheries Science, 2019, 26(05): 984-992.), the fish red blood cells were washed with sterile PBS by continuous low-speed low-temperature centrifugation (4°C, 2000r / min) to prepare a 4% fish red blood cell solution. 100μL of nisin solution of different concentrations was taken in a 2mL centrifuge tube, 100μL of 4% fish red blood cells and 800μL of sterile saline were added, incubated at 28°C for 30min, centrifuged at 5000r / min for 10min, and 200μL of supernatant was taken to an enzyme-labeled plate. Sterile saline was used as a negative control and distilled water (hemolytic ability was 100%) was used as a positive control. The OD was measured in a full-wavelength enzyme reader. 540 value.

[0050] OD 540 The value is calculated according to the following formula:

[0051] OD 540 =(sample OD 540 -Negative control OD 540 ) / (positive control OD 540 -Negative control OD 540 ).

[0052] 1.4 Effects on extracellular protease synthesis

[0053] 1.4.1 Preparation of extracellular products

[0054] Activate Aeromonas hydrophila and plate onto skim milk medium for 24 hours. Select colonies with the largest hydrolysis zone and inoculate them onto BHI medium at 28°C, 120 rpm, and incubate for 36 hours. Harvest the culture and centrifuge at 12,000 rpm for 30 minutes at 4°C. Collect the supernatant. Sterilize the supernatant with a 0.22 μm pore size sterile filter, precipitate with 75% saturated ammonium sulfate, and refrigerate overnight at 4°C. Centrifuge at 12,000 rpm for 30 minutes at 4°C, then discard the supernatant. Resuspend the precipitate in a small amount of 0.02 mol / L Tris-HCl (pH 7.5) buffer. Transfer the resuspended solution into a dialysis bag with a cutoff of 8,000-14,000 Da and dialyze extensively against the same buffer to obtain crude ESPase. Store at -20°C until use.

[0055] 1.4.2 Protease activity detection

[0056] Dilute nisin to an appropriate concentration and filter-sterilize (50, 25, 7.5 mg / mL). Place four Oxford cups on a skim milk plate. Add a mixture (100 μL exoenzyme + 100 μL nisin) that has been incubated at 28°C for 2 h to the three experimental wells. Add 100 μL exoenzyme + 100 μL Tris-HCL (pH 7.5) to the control wells and incubate in a 28°C incubator. After 15 h, terminate the reaction by submerging the agar in a saturated ammonium sulfate solution and take out the agar to observe its hydrolysis zone.

[0057] 1.5 Effects on the membrane of Aeromonas hydrophila

[0058] 1.5.1 Effects on cell walls

[0059] Aeromonas hydrophila was activated and cultured to the logarithmic phase. 50% (v / v) of the culture medium was inoculated into various concentrations of nisin, resulting in final nisin concentrations of 2MIC, MIC, and 1 / 2MIC, respectively. A bacterial suspension supplemented with 1% DMSO served as a DMSO positive control. Samples were taken every 2.5 hours until 7.5 hours. The cells were centrifuged at 3500 rpm for 10 minutes, and the supernatant was collected and the alkaline phosphatase (AKP) content was determined according to the instructions of the alkaline phosphatase (AKP) kit.

[0060] 1.5.2 Effects on cell membranes

[0061] β-D-galactosidase activity assay: Aeromonas hydrophila was inoculated into BHI medium and cultured at 28°C, 120 rpm, in a shaking incubator until the logarithmic phase. The concentration of the bacterial suspension was adjusted to 1 × 10 7 CFU / mL, and inoculated into culture medium containing different concentrations of nisin at a ratio of 50% (V / V) to make the final concentration of nisin 2MIC, MIC and 1 / 2MIC. The bacterial suspension supplemented with 1% DMSO was set as the DMSO positive control. The suspension was cultured in a shaker at 28°C and 120 r / min for 12 h. The absorbance of the bacterial suspension was determined by ONPG colorimetry. The bacterial suspension was diluted to 5×10 7 CFU / mL, take 10 μL of 30mmol / L ONPG solution and add it to 100 μL of bacterial suspension, react at 37℃ for 4 hours and measure OD 405nm The experiment was set up with 3 parallels and the results were averaged.

[0062] Determination of extracellular DNA content: Aeromonas hydrophila was inoculated into BHI medium and cultured at 28°C, 120 rpm, and shaken for 18 h. The culture was then washed three times with 0.1 mol / L PBS and resuspended to a concentration of 1 × 10 7CFU / mL, inoculated into culture medium containing different concentrations of nisin at a ratio of 50% (V / V) to make the final concentration of nisin 2MIC, MIC and 1 / 2MIC. A culture medium with 1% DMSO was set as a DMSO positive control. After incubation at 28°C, 120 rpm for 6 hours, centrifuge at 4°C, 4500 rpm for 10 minutes, and take the supernatant to measure OD 260nm The experiment was set up in 3 parallels and the results were averaged. The extracellular DNA content was calculated according to the following formula:

[0063] Extracellular DNA content (μg / mL) = OD 260nm ×50.

[0064] 1.5.3 Electron microscopy observation

[0065] In the presence of 1×10 7 To a test tube containing 100 CFU / mL of Aeromonas hydrophila bacterial liquid, nisin was added to a final concentration of 1 / 2 MBC. A control group with an equal amount of PBS was also set up and cultured at 28°C, 120 rpm for 24 hours. 1.5 mL of the bacterial liquid was taken at 12 and 24 hours, respectively, and centrifuged at 4°C, 3000 rpm for 5 minutes. The supernatant was discarded, and the precipitate was washed three times with sterile PBS. The precipitate was collected, 4% glutaraldehyde was added, and the precipitate was fixed in a 4°C refrigerator for 4 hours and centrifuged at 3000 rpm for 5 minutes. The fixative was aspirated and the cells were washed twice with PBS. The buffer solution was aspirated from the tube and dehydrated by adding ethanol in a stepwise gradient (ethanol concentration was 30%-50%-70%-80%-90%-100%), with each stage staying for 15 minutes. Finally, aspirate the ethanol and add a 1:1 (v / v) mixture of isoamyl acetate and ethanol. Soak for 10-20 minutes with appropriate shaking. Discard the mixture and add pure isoamyl acetate to soak for 10-20 minutes with appropriate shaking. Observe the bacterial structure and morphology using a scanning electron microscope.

[0066] 2. Experimental Results

[0067] 2.1 Antibacterial activity of nisin against Aeromonas hydrophila

[0068] 2.1.1 Minimum inhibitory concentration (MIC)

[0069] This example uses the resazurin microplate method to study the antibacterial activity of nisin against Aeromonas hydrophila. Figure 1 As shown in the figure, the inhibition rate curve was calculated according to the inhibition rate formula. The results are as follows Figure 2 、 Figure 3As shown in the figure, when the concentration of nisin is between 5-7.5 mg / mL, the inhibition rate exceeds 90%. When the minimum inhibitory concentration of nisin against Aeromonas hydrophila is 5.0 mg / mL, the inhibition rate corresponding to the purple well (5.0 mg / mL) is 85.4%, while the inhibition rate corresponding to the blue well (7.5 mg / mL) is 106.7%. The minimum inhibitory concentration is between 5.0-7.5 mg / mL.

[0070] 2.1.2 Minimum bactericidal concentration (MBC)

[0071] Take out 100 μL of bacteria from each well with a concentration of 12.5 mg / mL, 25 mg / mL, and 50 mg / mL and spread it on the plate. The results are as follows: Figure 4 As shown in the figure, after 24 hours of culture, the bacterial solution with a concentration of 50 mg / mL did not grow on the plate, while a few colonies appeared on the plate coated with the bacterial solution with a concentration of 25 mg / mL, indicating that the minimum bactericidal concentration of nisin against Aeromonas hydrophila is between 25 mg / mL and 50 mg / mL.

[0072] 2.1.3 Inhibition zone

[0073] Refer to the results of the size determination of the diameter of the nisin inhibition zone (Li Huanrong, Tian Liying, Cui Defeng, et al. Sensitivity test of pathogenic Aeromonas hydrophila to 31 antibacterial drugs [J]. Journal of Beijing Agricultural College, 2001(03):99-103.) to determine the sensitivity of pathogens to different drugs. Figure 5 As shown in the figure, when the concentration of nisin is 30 mg / mL and above, the inhibition zone is greater than 20 mm, which is extremely sensitive; when the concentration of nisin is 25 mg / mL, the inhibition zone is 16 mm, and between 15-20 mm, it is highly sensitive; when the concentration of nisin is 15-7.5 mg / mL, the inhibition zone size is between 10-14 mm, which is moderately sensitive.

[0074] 2.2 Regulation of virulence factors of Aeromonas hydrophila by nisin

[0075] 2.2.1 Hemolytic activity

[0076] Hemolytic activity results Figure 6 As shown, compared with the positive control, the results were not significantly different when the added concentration was MIC (7.5 mg / mL) (P ≥ 0.05), but when the added concentration was MBC (50 mg / mL), the hemolytic activity of Aeromonas hydrophila was significantly different (P < 0.05). This shows that as the concentration continues to increase, nisin inhibits the hemolytic ability of Aeromonas hydrophila to varying degrees.

[0077] 2.2.2 Extracellular protease hydrolytic activity

[0078] like Figure 7As shown in the figure, the hydrolysis zone of the extracellular product suspension in the wells with a concentration of 50 mg / mL after culture on the plate is extremely small, with a diameter of 108.5 mm, and is translucent. The hydrolysis zone of the extracellular product suspension in the wells with a nisin concentration of 25 mg / mL (122.5 mm) and 7.5 mg / mL (135.0 mm) is smaller than that in the wells without nisin addition. The hydrolysis zone of the wells without nisin addition is clear and larger (162.5 mm), indicating that nisin can destroy extracellular proteases.

[0079] 2.3 Effects on the membrane of Aeromonas hydrophila

[0080] 2.3.1 Effects on cell walls

[0081] The results are as follows Figure 8 As shown, in the DMSO positive control, there was no significant change in AKP activity in the culture medium between 0 and 5 hours. Compared with the DMSO control, there was no significant change in AKP activity in the culture medium at a nisin concentration of 1 / 2 the MIC (3.75 mg / mL) (P>0.05). However, at nisin concentrations of 7.5 mg / mL and 2 of the MIC (15 mg / mL), AKP activity in the culture medium increased significantly (P<0.05). AKP activity also increased in the culture medium over the same time period (P<0.05). Furthermore, at the same nisin concentration, AKP activity in the culture medium increased with prolonged nisin exposure (P<0.05), indicating that AKP continuously penetrates into the bacterial culture after nisin exposure.

[0082] The results showed that nisin could destroy the cell wall of Aeromonas hydrophila, allowing AKP to penetrate outside the bacterial cells. The degree of damage to the bacterial cell wall was roughly proportional to the concentration of nisin.

[0083] 2.3.2 Effects on cell membranes

[0084] The ONPG colorimetric method was used to determine the activity of β-D-galactosidase in the culture medium of Aeromonas hydrophila. Figure 9 As shown, compared with the DMSO positive control, when the concentration of nisin was ≥ MIC (7.5, 15 mg / mL), the OD 405nm The value increased significantly (P<0.05), and with the increase of nisin concentration, the OD 405nm The greater the value increases, the greater the leakage of β-D-galactosidase, indicating that nisin has a destructive effect on the cell membrane of Aeromonas hydrophila.

[0085] The effect of nisin on the extravasation of DNA from Aeromonas hydrophila was determined. Figure 10As shown in the figure, compared with the DMSO positive control, the extracellular DNA content of Aeromonas hydrophila showed an upward trend under the treatment of three concentrations of nisin, and there were significant differences (P<0.05), indicating that nisin can destroy the cell membrane of Aeromonas hydrophila and cause its DNA to extravasate.

[0086] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of nisin in inhibiting the proliferation of aquatic pathogens in vitro.

2. The use according to claim 1, characterized in that The aquatic pathogen is Aeromonas hydrophila.

3. Use of nisin in the preparation of an Aeromonas hydrophila antibacterial agent.

4. An Aeromonas hydrophila antibacterial agent, characterized in that The main active ingredient is nisin.

5. The antibacterial agent according to claim 4, characterized in that The bacteriostatic agent also includes other bacteriostatic active substances.

6. Use of nisin in the preparation of an agent for reducing the toxicity of Aeromonas hydrophila.

7. An agent for reducing the toxicity of Aeromonas hydrophila, characterized in that The main active ingredient is nisin.

8. Use of nisin in the preparation of a medicament for preventing and / or treating Aeromonas hydrophila infection.

9. A drug for preventing and / or treating Aeromonas hydrophila infection, characterized in that: The main active ingredient is nisin.

10. The drug according to claim 9, characterized in that The drug also includes other antibacterial active ingredients.

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