Application of perillaldehyde in inhibiting formation of shewanella putrefaciens biofilm

Abstract

The invention discloses application of perillaldehyde in inhibiting formation of a shewanella putrefaciens biofilm, and belongs to the technical field of microorganisms. Perillaldehyde not only can inhibit the formation of a shewanella putrefaciens biofilm in the culture medium, but also can effectively inhibit the formation of the shewanella putrefaciens biofilm on the surface, the contact surface or the interior of the aquatic product. The perillaldehyde is safe in component and free of fragrance, can be used as a food additive, and has a wide application prospect in food antibiosis.

Description

Technical Field

[0001] The invention relates to application of perillaldehyde in inhibiting biofilm formation of Shewanella putrefaciens, and belongs to the technical field of microorganisms. Background Art

[0002] Shewanella putrefaciens is a Gram-negative bacterium and a common food spoilage bacterium that can reduce trimethylamine oxide to trimethylamine and produce volatile gases such as putrescine and hydrogen sulfide, which can cause the deterioration of aquatic products and emit an unpleasant odor. It is also associated with various important clinical infections and diseases (such as bacteremia, skin tissue and soft tissue infections), especially in the summer in tropical and temperate zones.

[0003] Bacterial biofilm refers to the microbial community attached to the surface by bacteria that can embed and grow in the extracellular matrix such as exopolysaccharides and proteins secreted by themselves. It is also a way of life for most microorganisms to adapt to changes in the external environment. Shewanella putrefaciens can adhere to the surfaces of organisms and non-organisms to form biofilms. The biofilm formation process can be divided into the following four stages: First, it is the migration stage of microorganisms to the attachment surface. In this stage, bacteria will use active or passive transport mechanisms to migrate to possible attachment surfaces. Secondly, it enters the reversible attachment stage. At this time, bacteria will use a variety of physical and chemical effects to initially attach to the surface, which is a process of dynamic balance between attachment and shedding. Subsequently, it is the irreversible attachment stage. This stage usually involves sticky metabolites secreted by microorganisms, such as exopolysaccharides, which can strengthen the binding of microorganisms to the attachment surface and improve their ability to resist water erosion. Finally, bacteria build a relatively stable microenvironment on the attachment surface, that is, form a biofilm. In this environment, bacteria can grow and reproduce stably and may spread further.

[0004] The biofilm growth mode is the preferred lifestyle of the microbial world because it promotes growth and survival by providing nutrients and protection from predators and antimicrobial compounds. Once formed, biofilms are difficult to remove and become a continuous source of recontamination. Biofilms formed on food processing equipment can lead to energy loss during processing, reduce heat transfer efficiency, affect production efficiency, and cause corrosion of processing equipment. The formation of bacterial biofilms facilitates the attachment of other microorganisms, causing multiple cross-contamination. Even if the biofilm spreads, the cells in the biofilm will recolonize the surface and reform the biofilm in the appropriate environment. Food safety problems caused by biofilms may still occur even after rigorous cleaning and disinfection procedures. Shewanella putrefaciens can even aggravate food spoilage after forming biofilms on the surface of food and food processing media in contact with it, causing food safety problems and threatening human health.

[0005] In aquatic product processing facilities, physical methods for removing biofilms are not ideal for many aquatic products. The use of chemical reagents may affect the flavor and quality of food and involve potential safety hazards. The economic cost and effectiveness of using biological methods must be considered. Therefore, there is a need for an environmentally friendly biofilm removal method suitable for industrial production. Patent CN113142291A discloses a method for inhibiting the formation of biofilms of Shewanella putrefaciens in aquatic products using cactus fruit extract. However, cactus fruit extract, as a mixture, has a complex composition and its safety is not yet clear, and it is not suitable for use as a food additive. The prior art lacks a natural source aquatic product anti-biofilm agent that is safe, does not affect the flavor of the product, and can be used as a food additive. Summary of the invention

[0006] In view of the above-mentioned defects, the present invention aims to provide an anti-biofilm agent for aquatic products which is safe, does not affect the flavor of the product and can be used as a food additive.

[0007] The invention provides a method for inhibiting biofilm formation of Shewanella putrefaciens, wherein perillaldehyde is added into an environment containing Shewanella putrefaciens.

[0008] In one embodiment, in the environment, the added amount of perillaldehyde is 0.05-0.2 mg / mL.

[0009] In one embodiment, perilla is a short-day annual plant and a popular leafy vegetable used for therapeutic and food preparation in Asian countries, particularly in China, Korea, and Japan. Perillaldehyde is extracted from perilla and has potent biological activities such as antioxidant, vasodilator, anti-inflammatory, antifungal, and anticancer properties. As a widely used spice, perillaldehyde is often added to sauces, puddings, salad dressings, beverages, and bark foods to obtain a stronger spicy flavor and woody, citrus flavors.

[0010] In one embodiment, perillaldehyde can inhibit the formation of biofilm without inhibiting bacterial growth, thereby providing a theoretical basis for expanding the use of perillaldehyde as an anti-biofilm inhibitor in the food industry.

[0011] The present invention also provides the use of perillaldehyde in preparing a product for inhibiting the biofilm formation of Shewanella putrefaciens.

[0012] In one embodiment, the dosage form of the product includes a fluid, semi-fluid or solid preparation.

[0013] In one embodiment, the product is applied to a food surface or a food contact surface.

[0014] In one embodiment, the inhibiting of biofilm formation of Shewanella putrefaciens comprises: reducing the biofilm formation ability of Shewanella putrefaciens on food surfaces (shrimp shells and razor clam shells) and food contact surfaces (glass, stainless steel and silica gel), and weakening the biofilm formation ability of Shewanella putrefaciens in laboratory broth (LB broth) and food nutrient broth (shrimp, sea bass and oyster juice).

[0015] The invention also provides the use of perillaldehyde in preparing the biofilm inhibitor of Shewanella putrefaciens.

[0016] In one embodiment, the biofilm inhibitor includes an antimicrobial film preparation for food and an antimicrobial film preparation for aquaculture.

[0017] In one embodiment, the inhibitor has at least one of the following functions:

[0018] (1) Reduce the metabolic activity of cells in the biofilm of Shewanella putrefaciens;

[0019] (2) disturbing the microstructure of Shewanella putrefaciens biofilm;

[0020] (3) weaken the activity of cells in the biofilm of Shewanella putrefaciens;

[0021] (4) inhibit the swimming and clustering ability of Shewanella putrefaciens;

[0022] (5) reduce the secretion of extracellular polymers (exopolysaccharides, extracellular proteins and extracellular DNA) of Shewanella putrefaciens;

[0023] (6) downregulating pathways enriched in the two-component system / quorum sensing system;

[0024] (7) Inhibit the expression of some genes involved in biofilm formation ability.

[0025] The invention also provides the use of perillaldehyde in preparing an antimicrobial agent for Shewanella putrefaciens, and the use concentration of perillaldehyde is not less than 0.2 mg / mL.

[0026] The invention also provides the use of perillaldehyde in preparing aquatic product preservatives.

[0027] In the present invention, perillaldehyde has the ability to significantly inhibit the formation of biofilm of Shewanella putrefaciens, and does not inhibit the growth of Shewanella putrefaciens. At sub-inhibitory concentrations, perillaldehyde significantly reduces biofilm components (exopolysaccharides, extracellular proteins and extracellular DNA), which targets the related mechanism of biofilm generation by bacteria themselves. Scanning electron microscopy results confirmed that the biofilm of the control group was dense and viscous, and perillaldehyde at sub-inhibitory concentrations made the dense biofilm loose, showing less aggregation of extracellular polymers, and decreased in a concentration-dependent manner. The reduction of extracellular polymers in the biofilm also makes it more difficult for it to adhere to and colonize on the surface. This is also related to the results of motility. Perillaldehyde significantly weakens the swimming and clustering ability of Shewanella putrefaciens, indicating that it can reduce the colonization ability of Shewanella putrefaciens on the surface of objects. In summary, perillaldehyde disrupts the adhesion and colonization ability of Shewanella putrefaciens and reduces the risk of infection by Shewanella putrefaciens by inhibiting the formation of biofilm. Perillaldehyde also reduces the metabolic activity and vitality of biofilm. Transcriptomics was further applied to analyze the transcriptional levels of differentially expressed genes and the regulation of enriched pathways after treatment with perillaldehyde at the molecular level. The transcriptomic results showed that perillaldehyde downregulated the amino acid biosynthesis and metabolic pathways of Shewanella putrefaciens, and downregulated the quorum sensing and two-component system enrichment pathways. In addition, perillaldehyde at sub-inhibitory concentrations also reduced the amount of biofilm formed by its adhesion to food and food contact surfaces.

[0028] Beneficial effects:

[0029] The present invention provides an application of perillaldehyde in inhibiting the formation of biofilm of Shewanella putrefaciens. Perillaldehyde can not only inhibit the formation of biofilm of Shewanella putrefaciens in culture medium, but also effectively inhibit the formation of biofilm of Shewanella putrefaciens on the surface and contact surface of aquatic products, which is specifically embodied in:

[0030] (1) Perillaldehyde reduces the film-forming ability of Shewanella putrefaciens in laboratory broth (LB): Under 15°C culture, different concentrations of perillaldehyde (0.05, 0.10 and 0.20 mg / mL) reduced the film-forming ability by 28.38%, 41.62% and 61.89%, respectively; under 30°C culture, the film-forming ability was reduced by 17.91%, 30.35% and 39.30%, respectively.

[0031] (2) Perillaldehyde reduces the biofilm-forming ability of Shewanella putrefaciens on the surface of aquatic products: Under 15°C incubation, perillaldehyde (0.1 mg / mL) reduced the biofilm-forming ability of Shewanella putrefaciens in shrimp, sea bass and oyster juice by 39.20%, 37.98% and 35.36%, respectively; under 30°C incubation, it reduced the biofilm-forming ability of Shewanella putrefaciens in shrimp, sea bass and oyster juice by 23.94%, 44.94% and 30.25%, respectively.

[0032] (3) Perillaldehyde reduces the metabolic activity of Shewanella putrefaciens biofilm: After treatment with perillaldehyde (0.05, 0.10 and 0.20 mg / mL), the metabolic activity of cells in the biofilm of Shewanella putrefaciens cultured in laboratory broth was reduced by 18.77%, 30.65% and 39.08%, respectively.

[0033] (4) Perillaldehyde reduces the swimming ability and clustering ability of Shewanella putrefaciens: After treatment with perillaldehyde (0.05, 0.10 and 0.20 mg / mL), the swimming diameter of Shewanella putrefaciens decreased by 34.68%, 50.58% and 60.05%, and the clustering diameter decreased by 35.27%, 45.98% and 48.21%, respectively.

[0034] (5) Perillaldehyde significantly reduced the secretion of extracellular polymers in the biofilm of Shewanella putrefaciens. The degree of influence was extracellular polysaccharides > extracellular proteins > extracellular DNA. Sub-inhibitory concentrations of perillaldehyde (0.05, 0.10 and 0.20 mg / mL) reduced the production of extracellular polysaccharides by 21.95%, 27.72% and 45.33%, reduced the secretion of extracellular proteins by 15.14%, 16.44% and 19.92%, and reduced the content of extracellular DNA by 12.29%, 17.62% and 18.35%, respectively. We speculate that affecting the secretion of extracellular polymers and thus affecting the adhesion of bacteria may be one of the important reasons why perillaldehyde inhibits biofilm formation.

[0035] (6) Perillaldehyde significantly reduced the biofilm formation ability of Shewanella putrefaciens on the surfaces of stainless steel, silica gel, glass, shrimp shells, and razor clam shells. When the treatment concentration reached 0.2 mg / mL, the amount of biofilm formed on these surfaces was reduced by 14.20%, 19.32%, 16.76%, 19.32%, and 23.97%, respectively.

[0036] (7) Transcriptome analysis showed that perillaldehyde downregulated the differential gene expression in pathways such as the two-component system, quorum sensing system, and amino acid biosynthesis and metabolism. These factors may be the key mechanism by which perillaldehyde inhibits the biofilm formation of Shewanella putrefaciens. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a graph showing the effect of perillaldehyde on the growth of Shewanella putrefaciens;

[0038] Figure 2 It is the effect of perillaldehyde on the biofilm formation ability of Shewanella putrefaciens in LB broth after being cultured at 15℃ and 30℃;

[0039] Figure 3 is a graph showing the inhibition of metabolic activity of cells in Shewanella putrefaciens biofilm by perillaldehyde;

[0040] Figure 4This is a graph showing the inhibition of biofilm formation by perillaldehyde on Shewanella putrefaciens in food broth (A: oyster, B: sea bass, C: shrimp);

[0041] Figure 5 This is a scanning electron microscope observation of the microstructure of the biofilm of Shewanella putrefaciens after treatment with perillaldehyde;

[0042] Figure 6 This is a fluorescence microscope observation of the biofilm activity of Shewanella putrefaciens after treatment with perillaldehyde;

[0043] Figure 7 It is the inhibition graph of perillaldehyde on the motility (A, C) and clustering (B, D) of Shewanella putrefaciens;

[0044] Figure 8 This is the inhibition graph of perillaldehyde on the content of extracellular polysaccharide (A), extracellular protein (B) and extracellular DNA (C) in the biofilm of Shewanella putrefaciens;

[0045] Fig. 9 is the volcano plot of differentially expressed genes in Shewanella putrefaciens treated with perillaldehyde;

[0046] Fig.10 is the enrichment pathway diagram of up-regulated GO (A) and down-regulated GO (B);

[0047] Fig.11 is the enriched pathway diagram of up-regulated COG (A) and down-regulated COG (B);

[0048] Fig.12 is the enriched pathway diagram of up-regulated KEGG (A) and down-regulated KEGG (B);

[0049] Fig.13 This is a graph showing the effect of perillaldehyde on the ability of Shewanella putrefaciens to adhere to food surfaces (A) and form biofilms on food contact surfaces (B). DETAILED DESCRIPTION

[0050] The perillaldehyde used in the experiment of the present invention was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; and Shewanella putrefaciens CICC22940 was purchased from the China Industrial Microbiological Culture Collection Management Center.

[0051] The "broth" or "broth medium" described in the following examples refers to broth LB medium.

[0052] In order to better understand the essence of the new use of the present invention, further description will be given below in conjunction with embodiments.

[0053] Example 1: Recovery and cultivation of Shewanella putrefaciens

[0054] First, the Shewanella putrefaciens stored in a -80°C ultra-low temperature refrigerator was streaked on LB agar medium for activation. Incubated overnight in a 30°C incubator, the colonies were selected and inoculated in LB broth, incubated overnight at 30°C and 160 rpm, centrifuged at 5000g and 4°C for 5 minutes, the supernatant was discarded, washed and resuspended, and the suspension was adjusted to OD 600nm =0.5(10 8 CFU / mL).

[0055] Example 2: Effect of perillaldehyde on the growth of Shewanella putrefaciens

[0056] Experimental method: two-fold broth dilution method.

[0057] Adjust the density of bacterial suspension (10 6 CFU / mL), 100 μL of bacterial solution was mixed with 100 μL of perillaldehyde solution to make the final concentration of perillaldehyde reach 12-0.05 mg / mL. The growth of Shewanella putrefaciens was monitored using a fully automatic microbial growth curve analysis system at an interval of 1 hour. The concentration of perillaldehyde that had no effect on the growth of Shewanella putrefaciens was selected as the sub-inhibitory concentration.

[0058] The results are as follows Figure 1 As shown, the growth of Shewanella putrefaciens was completely inhibited at a concentration of perillaldehyde above 12 mg / mL. The growth was basically not affected below 0.20 mg / mL. Therefore, it can be determined that perillaldehyde below 0.20 mg / mL is the sub-inhibitory concentration (SICs) of perillaldehyde for Shewanella putrefaciens. In this experiment, 0.20, 0.10, and 0.05 mg / mL were selected as the concentrations for subsequent experiments.

[0059] Example 3: Effect of perillaldehyde on the biofilm formation ability of Shewanella putrefaciens cultured in LB broth

[0060] Experimental method: Crystal violet staining

[0061] 200 μL of broth containing Shewanella putrefaciens (final concentration 10 6 CFU / mL) and perillaldehyde (final concentrations were 0.05, 0.10 and 0.20 mg / mL, respectively). Place the well plate in a constant temperature incubator at 15°C and 30°C, respectively. After culturing for 48 hours, aspirate the culture medium and wash each well twice with PBS solution to remove floating bacteria. After drying for 30 minutes, add 200μL of 0.1% crystal violet, stain at room temperature for 20 minutes, and then aspirate the crystal violet solution. Then wash three times with PBS solution and dry for 30 minutes. Then add 200μL of 33% glacial acetic acid to each well, let it stand for 20 minutes, and use an enzyme reader to measure the OD 630nm .

[0062] The results are as follows Figure 2 As shown in the results, perillaldehyde (0.05, 0.10 and 0.20 mg / mL) significantly inhibited the growth of biofilm of Shewanella putrefaciens, and the biofilm formation ability was strongest at 15°C. Compared with Shewanella putrefaciens cultured at 30°C (the biofilm inhibition effect was 17.91%, 30.35% and 39.30%, respectively), the inhibition effect at 15°C was more obvious, reaching 28.38%, 41.62% and 61.89%. Therefore, 15°C was determined to be the optimal film-forming temperature for Shewanella putrefaciens, and subsequent experiments were carried out at this temperature.

[0063] Example 4: Effect of perillaldehyde on the metabolic activity of cells in Shewanella putrefaciens biofilm

[0064] Experimental method: XTT reduction method

[0065] XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolyl-5-carboxanilide) (1 mg / mL) menadione (1mM) was prepared in a ratio of 12.5:1. Biofilm was formed at 15 ° C for 48 h as described in Example 3. 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 .

[0066] The results are as follows Figure 3 As shown, the effect of perillaldehyde on the metabolic activity of bacteria in the biofilm of Shewanella putrefaciens was determined by the XTT method. Compared with the control group, perillaldehyde significantly inhibited the metabolic activity of the biofilm of Shewanella putrefaciens in a dose-dependent manner. After treatment with perillaldehyde (0.05, 0.10 and 0.20 mg / mL), the metabolic activity of cells in the biofilm of Shewanella putrefaciens decreased by 18.77%, 30.65% and 39.08%, respectively.

[0067] Example 5: Effect of Perillaldehyde on the Biofilm Formation Ability of Shewanella putrefaciens in Food Matrix

[0068] Take shrimp, oysters, and sea bass as different food matrices. Take the meat of shrimp, oysters, and sea bass and put them into a homogenizer bag, and add an equal proportion of PBS solution. Put them in a homogenizer and homogenize until the meat is broken. Then filter with gauze, pour the liquid into a 500mL beaker, put it in a water bath and boil it vertically, remove the upper foam, filter the membrane, and put it into a blue-capped bottle. Dilute the suspension of Shewanella putrefaciens with the homogenized food matrix and add perillaldehyde to make the final concentrations of 0.05, 0.10, and 0.20 mg / mL, respectively, and the number of viable bacteria of Shewanella putrefaciens is 10 6 CFU / mL, the biofilm formation procedure described in Example 3 was followed to determine OD 630nm . Degree of reduction in biofilm formation ability:

[0069] The results are as follows Figure 4 As shown, perillaldehyde significantly inhibited the biofilm formation ability of Shewanella putrefaciens in shrimp, oyster and sea bass juice as culture medium in a concentration-dependent manner. Under 15℃ culture, perillaldehyde (0.1mg / mL) reduced the biofilm formation ability of Shewanella putrefaciens in shrimp, sea bass and oyster juice by 39.20%, 37.98% and 35.36%, respectively; under 30℃ culture, it reduced the biofilm formation ability of Shewanella putrefaciens in shrimp, sea bass and oyster juice by 23.94%, 44.94% and 30.25%, respectively.

[0070] Example 6: Determination of the effect of perillaldehyde on the microstructure of Shewanella putrefaciens biofilm

[0071] Biofilm formation of Shewanella putrefaciens in sterile round cell slides (Φ=8 mm) in 24-well plates. Perillaldehyde solution was added at the same time (final concentrations were 0.05, 0.10 and 0.20 mg / mL, respectively). After 48 h of incubation at 15°C, the cells were washed with PBS solution, fixed with 2.5% glutaraldehyde overnight, and washed with PBS. Dehydrated with a series of ethanol gradients (30%, 50%, 70%, 80%, 90% and 100%). The samples were attached to the electron microscope stage, gold-plated under vacuum conditions, and observed using a scanning electron microscope.

[0072] The results are as follows Figure 5 As shown in the figure, the control group formed a complex and compact biofilm, while the addition of perillaldehyde solution significantly inhibited the biofilm formation of Shewanella putrefaciens. In the perillaldehyde solution at a concentration of 0.20 mg / mL, the cells aggregated the least, and the microscopic structure showed that the perillaldehyde solution could inhibit the formation of Shewanella putrefaciens.

[0073] Example 7: Determination of bacterial activity in biofilms of Shewanella putrefaciens using fluorescence microscopy

[0074] The green fluorescent dye SYTO and the red fluorescent dye PI were used for staining. Biofilm formation was as described in Example 6, the well plate was placed in an incubator at 15°C for 48 hours, washed with PBS, then 5 μM SYTO9 dye and 30 μM PI dye were added, stained in the dark for 20 minutes, washed with PBS to remove background dye, and then observed using a fluorescence microscope.

[0075] The results are as follows Figure 6As shown in the figure, after being stained with PI / SYTO 9 dye, dead cells are red and live cells are green. SYTO can penetrate into the intact cell membrane and bind to nucleic acids to show green fluorescence, while PI can only penetrate into cells with damaged cell membranes to show red fluorescence. When the two dyes exist at the same time, PI will compete with SYTO to cause the weakening of SYTO staining fluorescence. The control group without the addition of perillaldehyde solution was almost all green, and as the concentration of perillaldehyde increased, the green fluorescence gradually decreased and the red fluorescence gradually increased. Therefore, it can be known that perillaldehyde can interfere with the activity of cells in the biofilm and affect the film-forming ability of Shewanella putrefaciens.

[0076] Example 8: Effect of perillaldehyde on putrefactive Schwann motility

[0077] For swimming medium, weigh 25g of LB broth, 3g of agar powder, add 1L of deionized water, dissolve and heat to boil, and then sterilize at 121℃ for 20min. For cluster medium, weigh 25g of LB broth, 5g of agar powder, add 1L of deionized water, dissolve and heat to boil, and then sterilize at 121℃ for 20min. Add perillaldehyde solution to the medium to make its concentration reach 0.05, 0.10 and 0.20mg / mL. Inoculate 5μL of Shewanella putrefaciens into the medium (final concentration is 10 6 CFU / mL), cultured in a 30°C incubator, and the movement area of ​​bacteria on the agar plate was captured and the diameter of the activity area was measured using ImageJ software.

[0078] The results are as follows Figure 7 As shown, the range of motion of the control group was the largest, and the range of motion of the addition of 0.20 mg / mL perillaldehyde was the smallest. 0.05, 0.10 and 0.20 mg / mL perillaldehyde reduced the swimming ability of Shewanella putrefaciens by 34.68%, 50.58% and 60.05%, respectively, and the clustering ability by 35.27%, 45.98% and 48.21%, respectively. Compared with the control group, the swimming and clustering pictures also showed a significant decrease in the range of motion. It can be concluded that perillaldehyde can significantly reduce the motility of Shewanella putrefaciens, and perillaldehyde can regulate the formation of biofilm by controlling the motility of Shewanella putrefaciens.

[0079] Example 9: Biofilm Formation

[0080] The biofilm formation procedure of Shewanella putrefaciens was as described in Example 6. After culturing at 15°C for 48 h, the cell slide containing the biofilm was taken out, the biofilm was washed and resuspended in 1 mL of 0.01 mol / L KCl solution. The biofilm cells were stored on ice and ultrasonically disrupted. The ultrasonic suspension was centrifuged at 5,000×g, 4°C for 5 min, and the supernatant was filtered with a 0.22-μm membrane. The filtered supernatant was used to determine the content of extracellular polysaccharides, extracellular proteins and extracellular DNA using the phenol-sulfuric acid method, BCA protein assay and M5 Pic5 kit, respectively.

[0081] The results are as follows Figure 8 As shown in the figure, perillaldehyde significantly inhibited the content of extracellular polymers in the biofilm of Shewanella putrefaciens, with the degree of influence being extracellular polysaccharides>extracellular proteins>extracellular DNA. After perillaldehyde treatment, the content of extracellular polysaccharides in the biofilm of Shewanella putrefaciens was reduced by 23.26%, 27.91% and 46.51%, respectively, the content of extracellular proteins was reduced by 15.14%, 16.44% and 19.92%, and the content of extracellular DNA was reduced by 12.29%, 17.62% and 18.35%, respectively. Influencing the secretion of extracellular polysaccharides and thus affecting the adhesion of bacteria may be one of the important reasons why perillaldehyde inhibits biofilm formation.

[0082] Example 10: Effect of perillaldehyde on the transcriptome of Shewanella putrefaciens

[0083] RNA was extracted from the control group and the Shewanella putrefaciens treated with perillaldehyde (0.05 mg / mL). The quality of the extracted total RNA was evaluated using agarose detection and Nanodrop measurement methods. Agarose detection was used to observe whether there was contamination by impurities such as sugar, protein or DNA in the sample, and to preliminarily evaluate the purity and integrity of the RNA sample. Nanodrop measurement was used to determine the concentration of the RNA sample. By comprehensively judging the results of agarose detection and Nanodrop measurement, it can be ensured that the quality of the RNA sample meets the requirements of sequencing library construction to improve the reliability and accuracy of subsequent sequencing analysis. Subsequently, a cDNA library and sequencing were established (Sangon Biotech, Shanghai). After screening out the differentially expressed genes, in order to further explore the differences in gene function between the samples treated with perillaldehyde and the control group, the differentially expressed genes were enriched, their functions in different classification databases were annotated, and their distribution was statistically analyzed. The GO database, COG database and KEGG database were used for enrichment.

[0084] Fig. 9 As shown in the figure, compared with the control group, 553 differentially expressed genes were identified after perillaldehyde treatment, of which 254 were down-regulated and 299 were up-regulated. KEGG pathway analysis, GO and COG functional enrichment were used to mine differentially expressed genes to further explore the molecular regulatory mechanism of perillaldehyde on Shewanella putrefaciens.

[0085] like Fig.10 As shown in A and B, the top 30 up-regulated and down-regulated GO functional enrichment pathways, 29 down-regulated differentially expressed genes belong to the cellular amino acid biosynthesis process, and 31 down-regulated genes belong to the cellular amino acid metabolism process. 26 up-regulated genes are involved in ribosome function enrichment, and 85 up-regulated genes are involved in the cellular nitrogen compound biosynthesis process. Fig.11 A and B show COG functional enrichment pathways, and the up-regulated differentially expressed genes are mainly enriched in energy generation and conversion, nucleotide transport and metabolism, translation, ribosome structure and biogenesis. The down-regulated differentially expressed genes are mainly involved in amino acid transport and metabolism, biosynthesis, transport and catabolism of secondary metabolites, lipid transport and metabolism, and inorganic ion transport and metabolism. Fig.12 The top 30 up-regulated and down-regulated KEGG pathways were observed in A and B, respectively. A total of 25 up-regulated genes were attributed to ribosome, 19 up-regulated genes were related to purine metabolism, and 14 up-regulated genes were attributed to methane metabolism pathway. The main down-regulated genes were enriched in valine, leucine and isoleucine degradation (10 genes), two-component system (13 genes) and valine, leucine and isoleucine biosynthesis (7 genes). In conclusion, perillaldehyde regulates the formation of biofilm of Shewanella putrefaciens by regulating multiple GO, COG and KEGG pathways.

[0086] Example 11: Effect of perillaldehyde on the ability of Shewanella putrefaciens to adhere to food surfaces and form biofilms on food contact surfaces

[0087] Shrimp shells and razor clam shells were selected as different food shells. After peeling and removing the meat of the South American white shrimp (purchased in Qianhe Market, Ganjingzi District, Dalian) with a body length of about 15 cm, the shrimp shells were cut into 1.5cm×1.5cm rectangles, and the razor clam shells were cut into 1.5cm×7.5cm after removing the meat, and soaked in sodium hypochlorite solution for 30 minutes. After washing three times with sterile water, they were placed under ultraviolet light and irradiated on both sides for 1 hour each to remove the bacteria. The experiment selected 304 stainless steel sheets, glass 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. Then take out the above materials and operate in a biosafety cabinet. Put the above experimental materials into a sterile centrifuge tube, add bacterial suspension and perillaldehyde solution to make the concentration 0.05, 0.10, 0.20 mg / mL, and culture in a 15℃ incubator for 48 hours before taking out and operating in a biosafety cabinet. Pour out the liquid in the centrifuge tube, wash and resuspend with PBS solution, and add high-temperature sterilized glass beads and vortex for 1 minute. Then use the serial dilution coating method to count the number of colonies.

[0088] like Fig.13As shown in A, compared with the control group, perillaldehyde significantly reduced the biofilm formed by Shewanella putrefaciens adhering to the surface of shrimp shells and razor clam shells. Among them, Shewanella putrefaciens had the strongest ability to form biofilm on the surface of razor clam shells, and the inhibitory effect decreased in a perillaldehyde concentration-dependent manner. The results showed that perillaldehyde can significantly reduce the risk of Shewanella putrefaciens biofilm infection on food shells. Fig.13 B, C and D show that perillaldehyde reduces the biofilm formation ability of Shewanella putrefaciens on stainless steel glass and silica gel surfaces to varying degrees. Among them, perillaldehyde has the strongest biofilm formation ability on the silica gel surface. In summary, perillaldehyde solution can significantly reduce the infection risk of Shewanella putrefaciens on food contact surfaces.

[0089] In summary, this study showed that perillaldehyde has effective anti-biofilm properties against Shewanella putrefaciens at sub-inhibitory concentrations. We demonstrated that perillaldehyde inhibited the formation of biofilms of Shewanella putrefaciens grown in laboratory culture media and food culture media. At the same time, it reduced the amount of biofilm formation on different foods and food contact surfaces, inhibited the motility of Shewanella putrefaciens, reduced the production of extracellular polymers, and inhibited the metabolic activity of biofilm cells. Transcriptome analysis confirmed that multiple biofilm-related pathways and genes were regulated by perillaldehyde. This study explored the anti-biofilm properties of perillaldehyde in Shewanella putrefaciens at the molecular level, providing a theoretical basis for expanding the application of perillaldehyde in the food industry.

[0090] Preventing the formation of bacterial biofilms is one of the important ways to ensure the safety and quality of seafood. This study showed that perillaldehyde had a significant inhibitory effect on biofilm formation of Shewanella putrefaciens at subinhibitory concentrations, which could reduce the content of extracellular polymers, inhibit motility, interfere with the microstructure of biofilms, and reduce the number of biofilm cells attached to different surfaces. In addition, transcriptional analysis revealed the anti-biofilm mechanism of perillaldehyde against Shewanella putrefaciens. A large number of differentially expressed genes were found to be components of molecular pathways, such as ribosomes, two-component systems, quorum sensing systems, ABC transporters, amino acid metabolism, biosynthesis, and degradation. Therefore, understanding the mechanisms of expressing these advantageous properties will help to develop intervention strategies for controlling Shewanella putrefaciens biofilms in the future.

[0091] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for inhibiting biofilm formation of Shewanella putrefaciens, characterized in that: Perillaldehyde was added to an environment containing Shewanella putrefaciens.

2. The method according to claim 1, characterized in that: In the environment, the added amount of perillaldehyde is 0.05-0.2 mg / mL.

3. Use of perillaldehyde in the preparation of a product for inhibiting biofilm formation of Shewanella putrefaciens.

4. The use according to claim 3, characterized in that: The dosage form of the product includes fluid, semi-fluid or solid preparations.

5. The use according to claim 4, characterized in that: The product is applied to food surfaces or food contact surfaces.

6. The use of perillaldehyde in the preparation of a biofilm inhibitor of Shewanella putrefaciens, characterized in that: The biofilm inhibitors include antimicrobial film preparations for food and antimicrobial film preparations for aquaculture.

7. The use according to claim 6, characterized in that The biofilm inhibitor has at least one of the following functions: (1) Reduce the metabolic activity of cells in the biofilm of Shewanella putrefaciens; (2) disturbing the microstructure of Shewanella putrefaciens biofilm; (3) weaken the activity of cells in the biofilm of Shewanella putrefaciens; (4) inhibit the swimming and clustering ability of Shewanella putrefaciens; (5) reduce the secretion of extracellular polymers of Shewanella putrefaciens; (6) downregulating pathways enriched in the two-component system / quorum sensing system; (7) Inhibit the expression of genes related to biofilm formation.

8. The use of perillaldehyde in the preparation of a Shewanella putrefaciens antibacterial agent, characterized in that: The usage concentration of perillaldehyde is not less than 0.2 mg / mL.

9. Application of perillaldehyde in the preparation of aquatic product preservatives.

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