Application of fumaric acid in inhibition of formation of vibrio parahaemolyticus biofilm and inhibition method

Abstract

The invention discloses application of fumaric acid for inhibiting formation of a vibrio parahaemolyticus biological membrane and an inhibition method, and belongs to the field of biological membranes. The invention provides an application of fumaric acid in inhibition of formation of a vibrio parahaemolyticus biological membrane, and proves that the fumaric acid can significantly reduce the ability of vibrio parahaemolyticus to adhere to different foods and the ability of vibrio parahaemolyticus to form a biological membrane on food contact surfaces, weaken the biological membrane quantity of vibrio parahaemolyticus in food nutrition gravy, interfere the activity of bacteria in the biological membrane and inhibit the formation of the vibrio parahaemolyticus biological membrane. The transcription level of related genes of the vibrio parahaemolyticus biological membrane is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biofilms, and particularly relates to an application of fumaric acid in inhibiting the formation of Vibrio parahaemolyticus biofilm and an inhibition method. Background Art

[0002] Due to its many potential health benefits, seafood has become an increasingly important part of the human diet worldwide. Currently, one of the main obstacles hindering the development of the seafood industry is pathogen contamination. Vibrio parahaemolyticus is a Gram-negative pathogen widely found in the marine environment, which can cause gastroenteritis, sepsis and wound infections, and can also cause serious quality and economic losses to seafood. The adaptation of Vibrio species to different environmental conditions is essential for their survival and colonization. The formation of biofilms is a key factor in the survival and spread of bacteria in various environments. Vibrio parahaemolyticus can adhere to the surfaces of living and non-living organisms to form a complex microbial community with a spatial three-dimensional structure composed of microbial cells and extracellular polymeric substances, called biofilms. The process from planktonic bacteria to biofilm formation is a complex dynamic process, and these stages are the bacterial planktonic stage, adhesion stage, microcolony formation stage, biofilm maturation and diffusion stage. First, free bacteria adhere to the surface of living or non-living objects, and discrete cell clusters gather together to form small colonies (which can occur either through clonal growth of attached cells or through active translocation of cells on the surface); small colonies grow in size and combine to form large colonies composed of membrane structures of varying thicknesses. The extracellular polysaccharide matrix is ​​retained within the large colonies to form a biofilm; finally, the large colonies can dissolve, releasing cells from the biofilm, and the detached individuals attach again.

[0003] Compared with planktonic bacteria, the biofilm survival mode enhances the bacteria's resistance to antibiotics and adverse environments (low pH, low temperature and high salt), prolongs the survival time of bacteria in the food industry, causing persistent pollution that is difficult to remove, causing great economic losses to the food industry, posing food safety risks and endangering human health.

[0004] The use of antibiotics is a common method to combat biofilm contamination, but excessive use of antibiotics not only makes bacteria resistant, but also leads to the accumulation of toxic substances in the edible tissues of farmed fish and shellfish, causing long-term adverse effects on the health of humans and other animals.

[0005] There are existing reports on inhibiting biofilm formation and resisting biofilm pollution, such as Chinese patent document CN114917239A, which discloses the use of pomegranate polyphenols to inhibit biofilm formation of Vibrio parahaemolyticus. However, the pomegranate polyphenols used in this method further inhibit biofilm formation by acting as an antibacterial agent to exert a bactericidal effect, and the pomegranate polyphenols themselves are derived from biological extraction, which is relatively expensive.

[0006] Therefore, it is crucial to find a safe, effective, and low-cost inhibitor against biofilm formation of V. parahaemolyticus. Summary of the invention

[0007] The invention aims to provide application of fumaric acid in inhibiting biofilm formation of Vibrio parahaemolyticus.

[0008] Fumaric acid, as a low-cost food additive, is widely used in condiments, beverages, juices, jams, bread, cakes, candies, chewing gum and other products in the food industry. Fumaric acid can effectively inhibit the growth of bacteria and molds in food. Adding fumaric acid to perishable foods such as meat products can extend their shelf life and ensure the safety and quality of the products. Fumaric acid has a strong sour and salty taste. Adding it to foods such as fish products can improve the taste and appetite. In addition, fumaric acid can be used as a fermentation seasoning to make pickled vegetables and cheese products to increase the flavor of the products. At present, the effect of fumaric acid on the biofilm formation of Vibrio parahaemolyticus has not been reported.

[0009] The present invention discovers for the first time that fumaric acid significantly reduces the ability of Vibrio parahaemolyticus to adhere to different foods and form biofilms on food contact surfaces, weakens the amount of biofilm in food nutrient gravy, interferes with bacterial activity in the biofilm, and reduces the transcription level of Vibrio parahaemolyticus biofilm-related genes.

[0010] The specific contents of the plan are as follows:

[0011] The invention provides an application of fumaric acid in inhibiting the formation of biofilm of Vibrio parahaemolyticus.

[0012] The invention provides application of fumaric acid in reducing the secretion of extracellular polymers by biofilm of Vibrio parahaemolyticus.

[0013] In one embodiment of the present invention, the extracellular polymers include extracellular polysaccharides, extracellular proteins, and extracellular DNA.

[0014] The invention provides application of fumaric acid in preparing a product for resisting biofilm formation of Vibrio parahaemolyticus.

[0015] In one embodiment of the present invention, fumaric acid can be used as an antimicrobial film preparation to control Vibrio parahaemolyticus biofilm contamination in the food industry, providing a theoretical basis for the application of fumaric acid in the food industry.

[0016] In one embodiment of the present invention, the products include: drugs for treating biofilm interference of Vibrio parahaemolyticus, antimicrobial film preparations for food, and antimicrobial film preparations for aquaculture.

[0017] In one embodiment of the present invention, the product further contains any substance that can synergistically inhibit biofilm formation with fumaric acid.

[0018] In one embodiment of the present invention, the dosage form of the product can be a fluid, a semi-fluid or a solid powder.

[0019] In one embodiment of the present invention, the strain number of the Vibrio parahaemolyticus is ATCC17802.

[0020] The invention provides application of fumaric acid in preparing a Vibrio parahaemolyticus biofilm formation inhibitor.

[0021] The invention provides application of fumaric acid in preparing a biofilm inhibitor for food contact surfaces.

[0022] The present invention also provides a method for inhibiting the formation of Vibrio parahaemolyticus biofilm on non-human organisms or non-biological surfaces, liquid surfaces and interfaces, which uses fumaric acid as a Vibrio parahaemolyticus biofilm formation inhibitor, and the concentration of fumaric acid used does not exceed 250 μg / mL.

[0023] The present invention also provides a method for inhibiting the formation of Vibrio parahaemolyticus biofilm on the surface of food or food processing medium, which uses fumaric acid as a Vibrio parahaemolyticus biofilm formation inhibitor, and the concentration of the fumaric acid used does not exceed 250 μg / mL.

[0024] The present invention also provides a method for inhibiting the formation of Vibrio parahaemolyticus biofilm in an aquaculture environment, which uses fumaric acid as a Vibrio parahaemolyticus biofilm formation inhibitor, and the concentration of the fumaric acid used does not exceed 250 μg / mL.

[0025] In one embodiment of the present invention, the concentration of fumaric acid is further 62.5-250 μg / mL, and specifically 62.5, 125 and 250 μg / mL can be selected.

[0026] In one embodiment of the present invention, the non-human organisms may be marine organisms, including fish, shrimps, crabs, shellfish, etc.

[0027] In one embodiment of the present invention, the food processing medium comprises stainless steel, glass, food grade silicone and the like.

[0028] In one embodiment of the present invention, the food may be a nutrient gravy, such as shrimp, oyster, or sea bass gravy.

[0029] Preferably, the method for inhibiting biofilm formation of Vibrio parahaemolyticus comprises at least one of the following:

[0030] (1) Inhibit the motility of Vibrio parahaemolyticus;

[0031] (2) reduce the metabolic activity of cells within the biofilm of V. parahaemolyticus;

[0032] (3) Reduce the secretion of extracellular polymers (exopolysaccharides, extracellular proteins and extracellular DNA) of Vibrio parahaemolyticus;

[0033] (4) affecting the microstructure of Vibrio parahaemolyticus biofilm;

[0034] (5) Down-regulate the expression of biofilm-related genes of Vibrio parahaemolyticus.

[0035] In one embodiment of the present invention, fumaric acid can reduce the amount of biofilm formed by Vibrio parahaemolyticus adhering to the surface of food and food processing media (stainless steel, glass and food-grade silica gel), and weaken the biofilm forming ability of Vibrio parahaemolyticus in food nutrient gravy (shrimp, oysters, sea bass).

[0036] Compared with the prior art, the present invention has the following inventive principles and beneficial effects:

[0037] The present invention does not use an antibacterial agent with antibacterial activity, and only needs to add a small amount of fumaric acid to achieve the effect of significantly inhibiting the formation of biofilm of Vibrio parahaemolyticus.

[0038] In the present invention, fumaric acid has the ability to significantly inhibit the biofilm formation of Vibrio parahaemolyticus, and does not inhibit the growth of Vibrio parahaemolyticus. At sub-inhibitory concentrations, fumaric acid significantly reduces the amount of biofilm formation of Vibrio parahaemolyticus in laboratory broth (3% NaCl LB broth) and food nutrient gravy (shrimp juice, sea bass juice and oyster juice), and also reduces the biofilm formation ability of Vibrio parahaemolyticus adhering to food (shrimp shell and clam shell) and food contact surface (silica gel and stainless steel). Fumaric acid significantly reduces the motility of Vibrio parahaemolyticus. And it can reduce the colonization ability of Vibrio parahaemolyticus on the surface of objects. Fumaric acid significantly reduces biofilm components (exopolysaccharides, extracellular proteins and extracellular DNA), which targets the related mechanism of biofilm generation by bacteria themselves. Scanning electron microscopy further confirmed this result. We observed that the biofilm of the control group was dense and viscous, and fumaric acid at sub-inhibitory concentrations made the dense biofilm loose, showing less aggregation of extracellular polymers, and reduced in a concentration-dependent manner. The reduction of extracellular polymers in biofilms also makes it more difficult for them to adhere to and colonize the surface. In summary, fumaric acid inhibits the formation of biofilms, disrupts the adhesion and colonization ability of Vibrio parahaemolyticus, and reduces the risk of infection with Vibrio parahaemolyticus. Laser confocal microscopy observed that fumaric acid reduced the viability of cells in biofilms. The metabolic activity of cells in biofilms was also significantly weakened under fumaric acid treatment. Real-time fluorescence quantitative PCR was further used to analyze the effect of fumaric acid treatment on the transcription level of Vibrio parahaemolyticus biofilm-related genes at the molecular level.

[0039] Specific:

[0040] (1) The film-forming ability of Vibrio parahaemolyticus at 3h, 12h, 96h and 120h was lower than that at 24h, 48h and 72h. The film-forming ability of Vibrio parahaemolyticus reached the highest level at 48h, and the inhibitory effect of fumaric acid on it was also the most obvious. Therefore, the subsequent experiments were conducted first to form a film at 48h, and then to test the subsequent indicators.

[0041] (2) The biofilm formation ability of Vibrio parahaemolyticus treated with fumaric acid at sub-inhibitory concentrations (62.5, 125 and 250 μg / mL) was reduced by 32.31%, 56.41% and 70.26% in oyster juice culture medium, by 27.09%, 39.44% and 55.59% in seabass juice culture medium, and by 22.63%, 28.83% and 52.92% in shrimp juice culture medium, respectively.

[0042] (3) Fumaric acid inhibits the secretion of extracellular polymers in Vibrio parahaemolyticus biofilms. The degree of influence is polysaccharides > proteins > extracellular DNA (eDNA). Influencing the secretion of extracellular polysaccharides and thus affecting bacterial adhesion may be one of the important reasons why fumaric acid inhibits biofilm formation.

[0043] (4) The swimming diameter of Vibrio parahaemolyticus decreased by 24.63%, 32.75% and 49.78% after treatment with fumaric acid (62.5, 125 and 250 μg / mL).

[0044] (5) Fumaric acid (62.5, 125 and 250 μg / mL) reduced the metabolic activity of Vibrio parahaemolyticus biofilm in laboratory broth by 30.43%, 57.61% and 74.46%, respectively; in oyster broth medium, the metabolic activity of biofilm cells was reduced by 25.21%, 40.82% and 56.12%, respectively; in seabass broth medium, the metabolic activity of biofilm cells was reduced by 23.40%, 28.72% and 46.81%, respectively.

[0045] (6) After treatment with fumaric acid (62.5, 125 and 250 μg / mL), the biofilm formation ability of Vibrio parahaemolyticus on food surfaces (shrimp shells) decreased by 9.84%, 16.59% and 24.60%, and the biofilm formation ability on razor clam shells decreased by 10.94%, 15.88% and 29.56%. The biofilm formation ability on food processing media surfaces (such as stainless steel and silica gel) was also reduced to varying degrees.

[0046] (7) Real-time fluorescence quantitative PCR analysis showed that fumaric acid downregulated the expression levels of biofilm-related genes in Vibrio parahaemolyticus. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a graph showing the effect of different concentrations of fumaric acid on the growth of Vibrio parahaemolyticus;

[0048] Figure 2 This is a graph showing the effect of fumaric acid treatment on the biofilm formation ability of Vibrio parahaemolyticus at different culture times in laboratory broth (LB broth containing 3% NaCl);

[0049] Figure 3 This is a graph showing the effect of fumaric acid treatment on the biofilm-forming ability of Vibrio parahaemolyticus in food broth (A: shrimp, B: oyster, C: sea bass);

[0050] Figure 4 The effect of fumaric acid on the metabolism of cells in biofilms of Vibrio parahaemolyticus cultured in laboratory broth (A) and food broth (B: shrimp, C: oyster, D: sea bass) after 48 hours of cultivation.

[0051] Figure 5 This is a scanning electron microscope observation of the microscopic structure of the biofilm of Vibrio parahaemolyticus after treatment with fumaric acid;

[0052] Figure 6 This is the effect of fumaric acid on the swimming ability of Vibrio parahaemolyticus;

[0053] Figure 7 This is a graph showing the effect of fumaric acid on the contents of extracellular polysaccharides (A), extracellular proteins (B) and eDNA (C) in biofilms of Vibrio parahaemolyticus;

[0054] Figure 8 This is a graph showing the effect of fumaric acid on the expression of biofilm-related genes in Vibrio parahaemolyticus;

[0055] Fig. 9 This is a graph showing the effect of fumaric acid on the biofilm-forming ability of Vibrio parahaemolyticus adhering to the surfaces of shrimp (A), razor clams (B), silica gel (C) and stainless steel (D). DETAILED DESCRIPTION

[0056] The food-grade fumaric acid used in the experiments of the present invention was purchased from Anhui Xuelang Biotechnology Co., Ltd.; Vibrio parahaemolyticus ATCC 17802 was purchased from the American Type Strains Collection Center.

[0057] In order to better understand the essence of the new use of the present invention, the following will be further described in conjunction with the embodiments:

[0058] Example 1: Effect of fumaric acid on the growth of Vibrio parahaemolyticus

[0059] Experimental method: two-fold broth dilution method.

[0060] A single colony of Vibrio parahaemolyticus was picked from the TCBS agar plate and inoculated into 3% NaCl LB broth (laboratory broth) for overnight culture. The OD of the bacterial solution was adjusted to 600nm =0.5(10 8CFU / mL) and then adjust the bacterial suspension density (10 6 CFU / mL) was mixed with 100 μL of bacterial solution and different concentrations of fumaric acid solution (final concentration reached 1000-62.5 μg / mL) into the well plate. The growth of Vibrio parahaemolyticus was monitored using a fully automatic microbial growth curve analysis system, and the bacterial growth was tested once every hour. The concentration of fumaric acid that had no effect on the growth of Vibrio parahaemolyticus was selected as the sub-inhibitory concentration.

[0061] The results are as follows Figure 1 As shown, fumaric acid at a concentration not exceeding 250 μg / mL (such as 250 μg / mL, 125 μg / mL and 62.5 μg / mL) has minimal or no inhibitory effect on the growth of Vibrio parahaemolyticus.

[0062] Example 2: Effect of fumaric acid on biofilm formation ability of Vibrio parahaemolyticus cultured in 3% NaCl LB broth

[0063] Experimental method: Crystal violet staining

[0064] 200 μL of bacterial suspension and fumaric acid (250 μg / mL, 125 μg / mL and 62.5 μg / mL) were mixed into a 96-well plate and cultured in a constant temperature incubator for 3 h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h. After culturing at the corresponding time points, the culture medium was aspirated, and each well was washed twice with PBS solution to remove floating bacteria. After drying for 30 min, 200 μL of 0.1% crystal violet was added to each well, and the crystal violet solution was aspirated after staining at room temperature for 20 min. Then, it was washed three times with PBS solution and dried for 30 min. Then, 200 μL of 33% glacial acetic acid was added to each well, and after standing for 20 min, OD 570 was measured using an enzyme reader. nm .

[0065] The results are as follows Figure 2 As shown in the results, fumaric acid significantly inhibited the growth of biofilm of Vibrio parahaemolyticus, and the biofilm formation ability was strongest at 48h. Moreover, after 48h of culture, the inhibitory effect of fumaric acid was more obvious than that at other time periods. Therefore, 48h was determined to be the optimal culture time for Vibrio parahaemolyticus, and the subsequent biofilm formation experiment was carried out at 48h.

[0066] Example 3: Effect of fumaric acid on biofilm formation ability of Vibrio parahaemolyticus in food broth

[0067] Take oysters, sea bass and shrimp as different food matrices. Take the meat of oysters, sea bass and shrimp and put them into a homogenizing bag, and add an equal proportion of PBS solution. Put them into a homogenizer and homogenize until the meat is broken. Then filter with gauze, pour the liquid into a 500mL beaker, put it into a water bath and boil it vertically, remove the upper foam, filter the membrane, and put it into a blue-capped bottle. Dilute the bacterial suspension with food broth and add fumaric acid solution to make the final concentrations of 250, 125 and 62.5μg / mL. According to the biofilm formation procedure described in Example 2, measure the OD 570nm .

[0068] The results are as follows Figure 3 As shown, fumaric acid significantly inhibited the biofilm formation ability of Vibrio parahaemolyticus in shrimp, oyster and sea bass juice as culture medium and the inhibition was concentration-dependent.

[0069] Example 4: Effect of fumaric acid on the metabolic activity of cells in Vibrio parahaemolyticus biofilm

[0070] Experimental method: XTT reduction method

[0071] XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolyl-5-carboxanilide) (1 mg / mL) menadione (1mM) was prepared in a working solution at a ratio of 12.5:1. Biofilms were formed as described in Example 2 for 48 h. After washing with PBS, 200 μL PBS and 25 μL working solution were added to each well and incubated in the dark for 4 h. OD was measured. 490nm .

[0072] The results are as follows Figure 4 As shown in the figure, XTT can reduce mitochondria in living cells to orange substances. The darker the orange color, the stronger the cell metabolic activity. And compared with the control group, after fumaric acid treatment, the metabolic activity of Vibrio parahaemolyticus biofilm decreased in a dose-dependent manner, whether in laboratory culture medium or food culture medium.

[0073] Example 5: Scanning electron microscopy observation of the effect of fumaric acid on the microstructure of Vibrio parahaemolyticus biofilm

[0074] Vibrio parahaemolyticus was formed into a biofilm on a sterile round cover glass (Φ=8 mm) in a 24-well plate. Fumaric acid was added to the wells, and after 48 hours of incubation, the wells were washed twice with PBS solution, fixed with 2.5% glutaraldehyde overnight, and washed with PBS. Finally, ethanol (30%, 50%, 70%, 80%, 90% and 100%) was used for continuous dehydration. The sample was attached to the electron microscope stage, gold-plated under vacuum conditions, and observed using a scanning electron microscope.

[0075] The results are as follows Figure 5As shown, untreated V. parahaemolyticus formed a dense biofilm with a complex three-dimensional, multicellular, self-assembled structure, and fumaric acid treatment significantly inhibited the development of the biofilm structure. In the presence of fumaric acid, V. parahaemolyticus secreted less extracellular polymers, and fewer cell aggregates and multicellular structures were observed.

[0076] Example 6: Determination of the swimming ability of Vibrio parahaemolyticus

[0077] Resuspend the bacterial suspension in broth and adjust the OD 600nm =0.5. When the swimming (0.3% agar) medium is cooled to about 55°C, add fumaric acid solutions with final concentrations of 250, 125 and 62.5 μg / mL, then pour the plate and let the plate stand. When the plate is in a semi-solid state, inoculate 5 μL of the bacterial suspension into the middle of the swimming agar plate surface. Incubate the plate at 37°C. Use an automatic colony counter to capture the movement area of ​​bacteria on the agar plate, and use ImageJ software to measure the diameter of the active area.

[0078] The results are as follows Figure 6 As shown, fumaric acid significantly reduced the swimming diameter of Vibrio parahaemolyticus in a concentration-dependent manner. Surface fumaric acid weakened the motility of Vibrio parahaemolyticus, thereby reducing its adhesion.

[0079] Example 7: Determination of the content of main components of extracellular polymers in biofilm

[0080] The biofilm formation of Vibrio parahaemolyticus was as described in Example 5. After cultivation, the cell slides were placed in a potassium chloride solution (1 mL 0.01 mol / L) and frozen at -20°C for 2 h. Ultrasonic disruption was performed, followed by centrifugation, and the supernatant was filtered through a 0.22 μM filter membrane. The supernatant was filtered using the phenol-sulfuric acid method, BCA protein assay, and M5 Pic5 kit to determine the contents of extracellular polysaccharides, proteins, and eDNA, respectively.

[0081] The results are as follows Figure 7 As shown in the results, fumaric acid reduced the content of extracellular polysaccharides, extracellular proteins and eDNA in biofilm of Vibrio parahaemolyticus. Influencing the secretion of extracellular polymers and thus affecting the adhesion of bacteria may be one of the important reasons why fumaric acid inhibits biofilm formation.

[0082] Example 8: Real-time fluorescence quantitative PCR

[0083] RNA was extracted from the control group and the fumaric acid (125 μg / mL and 250 μg / mL) treated group using an RNA extraction kit. RNA degradation and contamination were analyzed using agarose gel electrophoresis. RNA was then adjusted to a consistent concentration and reverse transcribed into cDNA. The SYBR Premix was configured into a 20 μL system for reaction, with 16s rRNA as the internal reference. The results were 2 -△△Ct Gene expression levels were calculated. The specific primer sequences used are shown in Table 1 below.

[0084] Table 1 Primer sequences

[0085]

[0086]

[0087] The test results are as follows Figure 8 As shown, fumaric acid treatment significantly reduced the expression levels of biofilm-related genes of Vibrio parahaemolyticus.

[0088] Example 9: Fumaric acid inhibits the biofilm formation ability of Vibrio parahaemolyticus adhering to shrimp shells, razor clam shells, silica gel and stainless steel surfaces

[0089] This experiment selected 304 stainless steel sheets and food-grade silicone sheets with a size of 1cm×1cm as experimental materials. First, put the above materials into a beaker, wash with 75% ethanol solution, and sterilize at 121℃ for 20 minutes. Cut the shrimp shells into 1.5cm×1.5cm, and the razor clam shells into rectangles of 1.5cm×7.5cm, and soak them in sodium hypochlorite solution for 30 minutes. After washing twice with deionized water and wiping the moisture with kitchen paper, take out the above materials and irradiate them with ultraviolet light on both sides in the biological safety cabinet for 1 hour. Put the above experimental materials into a sterile centrifuge tube, add Vibrio parahaemolyticus suspension (10 6 CFU / mL) and fumaric acid solution (to make the final concentration reach 250, 125 and 62.5μg / mL). After culturing at 30℃ for 48h, take out. Operate in a biological safety cabinet, pour out the liquid in the centrifuge tube, wash with PBS solution, add glass beads sterilized by high temperature and high pressure, vortex and oscillate for 1min, and count by serial dilution coating method.

[0090] The results are as follows Fig. 9 As shown, fumaric acid significantly reduced the ability of Vibrio parahaemolyticus to adhere to food and form biofilm on food contact surfaces, and the inhibitory effect decreased in a dose-dependent manner.

[0091] In summary, fumaric acid can reduce the adhesion ability of Vibrio parahaemolyticus on the surfaces of various objects, thereby reducing the infection ability of biofilms formed by Vibrio parahaemolyticus on the surfaces of food and food processing media in the food processing field.

[0092] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of fumaric acid in inhibiting biofilm formation of Vibrio parahaemolyticus.

2. Application of fumaric acid in the preparation of products for inhibiting biofilm formation of Vibrio parahaemolyticus.

3. The use according to claim 2, characterized in that: The products include: drugs for treating biofilm interference caused by Vibrio parahaemolyticus, antimicrobial film preparations for food, and antimicrobial film preparations for aquaculture.

4. The use according to claim 2, characterized in that: The product may also contain any substance that can synergize with fumaric acid to inhibit biofilm formation.

5. The use according to claim 2, characterized in that: The dosage form of the product is fluid, semi-fluid or solid powder.

6. Application of fumaric acid in reducing the secretion of extracellular polymers by Vibrio parahaemolyticus biofilm.

7. The use according to any one of claims 1 to 6, characterized in that: The concentration of fumaric acid used should not exceed 250 μg / mL.

8. A method for inhibiting the formation of Vibrio parahaemolyticus biofilm on non-human organisms or non-biological surfaces, liquid surfaces and interfaces, comprising using fumaric acid as a Vibrio parahaemolyticus biofilm formation inhibitor, wherein the concentration of fumaric acid used does not exceed 250 μg / mL.

9. A method for inhibiting the formation of Vibrio parahaemolyticus biofilm on the surface of food or food processing medium, which uses fumaric acid as a Vibrio parahaemolyticus biofilm formation inhibitor, and the concentration of fumaric acid used does not exceed 250 μg / mL.

10. A method for inhibiting the formation of Vibrio parahaemolyticus biofilm in an aquaculture environment, comprising using fumaric acid as a Vibrio parahaemolyticus biofilm formation inhibitor, wherein the concentration of the fumaric acid used does not exceed 250 μg / mL.

Images