Application of lactobacillus plantarum cell-free supernatant in inhibiting pseudomonas fluorescens and its biofilm
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-18
- Publication Date
- 2026-08-11
AI Technical Summary
[0005]本发明旨在提供一种植物乳杆菌无细胞上清液的新应用，以解决现有技术中控制荧光假单胞菌及其生物膜时存在的化学残留、易产生耐药性、物理清除不彻底、能耗高等问题
[0025]本发明揭示了植物乳杆菌无细胞上清液能够同时抑制荧光假单胞菌浮游菌生长和生物膜形成，为食品工业提供了一种兼具抗菌和抗生物膜双重功能的天然抑菌剂。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, specifically to the application of cell-free supernatant of Lactobacillus plantarum in inhibiting Pseudomonas fluorescens and its biofilm. Background Technology
[0002] *Pseudomonas fluorescens* is a Gram-negative bacillus that produces a yellow-green fluorescein and possesses lipolytic, proteolytic, and lecithinase activities. Furthermore, its lipases promote the breakdown of dietary fats, releasing unsaturated glycerol and fatty acids. These unsaturated fatty acids can produce unpleasant flavors, including rancid or bitter compounds, significantly impacting dairy and seafood products. *Pseudomonas fluorescens* exhibits strong environmental adaptability and complex regulatory mechanisms, enabling it to contaminate food during packaging, posing a significant challenge to cold chain transportation. Simultaneously, *Pseudomonas fluorescens* can adhere to various biological and abiotic surfaces, forming thick and dense biofilms. Biofilms are a form of microbial community that attaches to solid surfaces and constructs a three-dimensional structure using extracellular polymeric matrices secreted by the microorganisms themselves. In the food industry, biofilms formed by *Pseudomonas* become a persistent source of contamination for food and its contact surfaces, leading to spoilage, shortened shelf life, and severe economic losses. Furthermore, when pathogens are present in the environment, certain Pseudomonas species can colonize food contact surfaces more effectively and act as a protective agent within mixed bacterial biofilms, shielding them from disinfectant damage. Currently, methods for controlling Pseudomonas fluorescens and its biofilm formation mainly rely on chemical disinfectants and physical removal methods. However, chemical disinfectants are prone to leaving residues, affecting food flavor and safety, and long-term use may induce antibiotic resistance in bacteria; physical removal methods (such as scrubbing and high-temperature treatment) are damaging to equipment, energy-intensive, and difficult to completely remove existing biofilms. Therefore, developing green, safe, and highly efficient natural antibacterial agents that can both inhibit the growth of planktonic bacteria and disrupt biofilm formation has become an urgent problem to be solved in the food industry.
[0003] Lactobacillus plantarum, as a probiotic strain, has been widely used in the fermentation process of various foods. It not only improves the flavor and texture of food but also acts as an antibacterial agent to inhibit pathogenic bacteria. The U.S. Food and Drug Administration has recognized Lactobacillus plantarum as a Generally Recognized As Safe (GRAS) microorganism, and the use of Lactobacillus plantarum and its metabolites as natural antibacterial agents has attracted widespread attention. Various metabolites in the cell-free supernatant of Lactobacillus plantarum, including secretory proteins, enzymes, organic acids, peptides, and extracellular vesicles, have shown antibacterial potential. Furthermore, compared with probiotic strains or other single components, the cell-free supernatant exhibits higher biological activity and stability. In recent years, Lactobacillus plantarum and its metabolites have been widely used in food preservation, feed additives, and clinical adjuvant therapy. Due to its high safety, low risk of drug resistance, and environmental friendliness, it is considered an ideal alternative to chemical preservatives. However, research on the dual effects of inhibiting planktonic bacterial growth and controlling biofilm in the control of Pseudomonas fluorescens has not yet been reported.
[0004] However, there are currently no reports on using cell-free supernatant of *Lactobacillus plantarum* to simultaneously inhibit the planktonic growth and biofilm formation of *Pseudomonas fluorescens*. Existing control methods relying on chemical disinfectants and physical removal suffer from drawbacks such as chemical residues, induced drug resistance, high energy consumption, and difficulty in completely removing biofilms. Therefore, how to provide a green, safe, and efficient natural antibacterial agent that can simultaneously inhibit the planktonic growth and biofilm formation of *Pseudomonas fluorescens* is a pressing technical problem to be solved in this field. Summary of the Invention
[0005] This invention aims to provide a novel application of cell-free supernatant from *Lactobacillus plantarum* to address the problems of chemical residues, drug resistance, incomplete physical removal, and high energy consumption in existing technologies for controlling *Pseudomonas fluorescens* and its biofilm. Specifically, this invention provides a green, safe, and highly efficient natural antibacterial agent that can simultaneously inhibit the growth of *Pseudomonas fluorescens* planktonic bacteria and biofilm formation.
[0006] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides the application of cell-free supernatant of Lactobacillus plantarum in inhibiting Pseudomonas fluorescens and its biofilm.
[0007] Furthermore, the inhibition includes inhibiting the growth of Pseudomonas fluorescens planktonic bacteria and / or inhibiting the formation of Pseudomonas fluorescens biofilms.
[0008] Furthermore, the inhibition of *Pseudomonas fluorescens* and its biofilm is achieved through at least one of the following mechanisms:
[0009] Disruption of Pseudomonas fluorescens cell membrane permeability;
[0010] Increased leakage of nucleic acids and / or proteins from the contents of *Pseudomonas fluorescens*;
[0011] Increase the reactive oxygen species level in *Pseudomonas fluorescens*;
[0012] Reduces the activity of Pseudomonas fluorescens;
[0013] Reduces the metabolic activity of cells within the biofilm of *Pseudomonas fluorescens*;
[0014] Interfering with the microstructure of Pseudomonas fluorescens biofilm.
[0015] Furthermore, the cell-free supernatant of *Lactobacillus plantarum* is obtained by centrifuging *Lactobacillus plantarum* to remove the bacterial cells, and then filtering the supernatant through a sterile filter membrane.
[0016] Furthermore, the *Lactobacillus plantarum* is either *Lactobacillus plantarum* CICC22810 or *Lactobacillus plantarum* CICC21805.
[0017] Furthermore, the volume concentration of the cell-free supernatant of *Lactobacillus plantarum* is 12.5% to 100%.
[0018] Furthermore, the volume concentration of the cell-free supernatant of *Lactobacillus plantarum* is 25%, 50%, or 100%.
[0019] Furthermore, the inhibition of Pseudomonas fluorescens biofilm formation includes:
[0020] Reduce biofilm formation;
[0021] Reduce the content of extracellular polysaccharides, extracellular proteins and / or extracellular DNA in biological membranes;
[0022] At least one of the following can cause the biomembrane structure to change from dense to loose.
[0023] Furthermore, the present invention also provides the use of Lactobacillus plantarum cell-free supernatant in the preparation of antibacterial agents or biofilm scavengers for inhibiting Pseudomonas fluorescens and its biofilm.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] This invention reveals that cell-free supernatant of Lactobacillus plantarum can simultaneously inhibit the growth of Pseudomonas fluorescens planktonic bacteria and biofilm formation, providing the food industry with a natural antibacterial agent that has both antibacterial and anti-biofilm functions.
[0026] This invention demonstrates through experiments that the cell-free supernatant of *Lactobacillus plantarum* can exert its effects through multiple pathways:
[0027] Disruption of the cell membrane integrity of Pseudomonas fluorescens leads to a large leakage of intracellular nucleic acids and proteins (as shown in Example 4, 100% CFS treatment increased protein leakage by more than 93%).
[0028] It induces oxidative stress, which significantly increases the level of reactive oxygen species (as shown in Example 5, 100% CFS treatment increases ROS by more than 155%).
[0029] The activity of *Pseudomonas fluorescens* was destroyed (as shown in Example 6, after 100% CFS treatment, the activity of *Pseudomonas fluorescens* was significantly reduced, and most of the cells stained red with PI, indicating that the cell membrane was severely damaged).
[0030] Inhibit biofilm formation (as shown in Example 7, 100% CFS treatment reduced biofilm formation by more than 85%).
[0031] Reduced metabolic activity of cells within biomembranes (as shown in Example 8, 100% CFS treatment reduced metabolic activity by more than 76%).
[0032] Disrupt the microstructure of the biomembrane, making it change from dense to porous (as shown in the scanning electron microscopy results of Example 9).
[0033] Lactobacillus plantarum is a Generally Recognized As Safe (GRAS) probiotic. Its cell-free supernatant contains no live bacteria, leaves no chemical residues, is unlikely to induce bacterial resistance, and is environmentally friendly, making it an ideal alternative to chemical preservatives. This invention can be applied to the processing and storage of cold chain foods such as dairy products, aquatic products, and food contact surfaces, effectively controlling Pseudomonas fluorescens contamination, extending food shelf life, and reducing economic losses. It exhibits significant inhibitory effects within a concentration range of 12.5% to 100% (v / v), with the best effect observed at high concentrations (100%). In practical applications, the concentration can be flexibly adjusted as needed. Attached Figure Description
[0034] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments:
[0035] Figure 1 The graph shows the effect of cell-free supernatant from *Lactobacillus plantarum* on the growth of *Pseudomonas fluorescens*. Figure 1 A represents the results of processing cell-free supernatant from Lactobacillus plantarum CICC22810. Figure 1 B represents the results of processing cell-free supernatant from Lactobacillus plantarum CICC21805.
[0036] Figure 2 The figure shows the effect of cell-free supernatant from *Lactobacillus plantarum* on leakage of contents from *Pseudomonas fluorescens*. Figure 2 A and Figure 2B represents the leakage of nucleic acids and proteins after treatment with cell-free supernatant of Lactobacillus plantarum CICC22810. Figure 2 C and Figure 2 D represents the leakage of nucleic acids and proteins after treatment with cell-free supernatant of Lactobacillus plantarum CICC21805.
[0037] Figure 3 The figure shows the effect of cell-free supernatant from *Lactobacillus plantarum* on the level of reactive oxygen species (ROS) in *Pseudomonas fluorescens*; among them, Figure 3 A represents the results of processing cell-free supernatant from Lactobacillus plantarum CICC22810. Figure 3 B represents the results of processing cell-free supernatant from Lactobacillus plantarum CICC21805.
[0038] Figure 4 A fluorescence micrograph of Pseudomonas fluorescens PI staining on cell-free supernatant of Lactobacillus plantarum;
[0039] Figure 5 The figure shows the effect of cell-free supernatant from *Lactobacillus plantarum* on the biofilm-forming ability of *Pseudomonas fluorescens*. Figure 5 A represents the results of processing cell-free supernatant from Lactobacillus plantarum CICC22810. Figure 5 B represents the results of processing cell-free supernatant from Lactobacillus plantarum CICC21805.
[0040] Figure 6 The figure shows the effect of cell-free supernatant from *Lactobacillus plantarum* on the metabolic capacity of *Pseudomonas fluorescens* biofilm; among which, Figure 6 A represents the results of processing cell-free supernatant from Lactobacillus plantarum CICC22810. Figure 6 B represents the results of processing cell-free supernatant from Lactobacillus plantarum CICC21805.
[0041] Figure 7 Scanning electron microscopy (SEM) image of a *Pseudomonas fluorescens* biofilm after treatment with cell-free supernatant of *Lactobacillus plantarum*. Detailed Implementation
[0042] To better understand this invention, specific embodiments are described in further detail below. It should be understood that these embodiments are for illustrative purposes only and do not constitute any limitation on the scope of protection of this invention. Any modifications, substitutions, or improvements made based on the principles of this invention are within the scope of protection of this invention.
[0043] The strains used in this invention are as follows:
[0044] Lactobacillus plantarum: strains CICC22810 and CICC21805, both purchased from the China Center of Industrial Culture Collection (CICC).
[0045] Pseudomonas fluorescens: strain ATCC13525, preserved in the laboratory of the applicant of this invention.
[0046] Culture media and reagents:
[0047] LB agar medium, LB broth medium, MRS liquid medium.
[0048] Phosphate-buffered saline (PBS, pH 7.2-7.4).
[0049] Crystal violet staining solution, glacial acetic acid, glutaraldehyde (2.5%, v / v), and ethanol gradient series.
[0050] Reactive oxygen species detection kit (DCFH-DA method), XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolyl-5-carboxyaniline), menadione, propidium iodide (PI).
[0051] All the above reagents are of analytical grade or biochemical grade.
[0052] Main instruments: Multifunctional microplate reader (for measuring OD value and fluorescence intensity). Fluorescence microscope. Scanning electron microscope. Growth curve analyzer. Low-temperature centrifuge (5000g, 4℃). 0.22 μm sterile filter membrane and filter.
[0053] Example 1: Resuscitation and Culture of *Pseudomonas fluorescens*
[0054] The *Pseudomonas fluorescens* ATCC13525 strain, stored at -80℃, was retrieved and streaked onto LB agar medium. It was then incubated at 30℃ for 24 h to activate the bacteria. Single colonies were picked and inoculated into LB broth medium, and cultured with shaking at 30℃ and 180 rpm. After incubation, the culture was centrifuged at 5000 g and 4℃ for 5 min. The supernatant was discarded, and the bacterial cells were washed twice with sterile PBS buffer. The cells were resuspended in PBS, and the bacterial suspension concentration was adjusted to OD500. 600nm = 0.5 (approximately 1 × 10) 8 (CFU / mL), for later use.
[0055] Example 2: Preparation of cell-free supernatant from Lactobacillus plantarum
[0056] Lactobacillus plantarum CICC22810 and CICC21805 were inoculated into MRS liquid medium and incubated statically at 37°C for 48 h. After incubation, the culture was centrifuged at 5000 g at 4°C for 15 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm sterile filter to remove residual bacteria and particulate matter, yielding cell-free supernatant (CFS) of Lactobacillus plantarum. CFS was aliquoted and stored at -80°C for later use. When needed, it should be diluted to the specified concentration (v / v).
[0057] Example 3: Effect of cell-free supernatant of Lactobacillus plantarum on the growth of Pseudomonas fluorescens planktonic bacteria
[0058] The *Pseudomonas fluorescens* bacterial suspension prepared in Example 1 was added to LB broth medium at an inoculum volume of 1% (v / v). Different volume concentrations of *Lactobacillus plantarum* cell-free supernatant (0%, 25%, 50%, 75%, 100%, v / v) were added, mixed thoroughly, and then added to 96-well plates, 200 μL per well. The plates were continuously incubated at 30°C in a growth curve analyzer for 44 h, and OD was measured every 2 h. 600nm Values are used to plot growth curves.
[0059] The results are as follows Figure 1 As shown. Compared with the control group (0% CFS), the addition of Lactobacillus plantarum CICC22810 ( Figure 1 A) or CICC21805 ( Figure 1 B) In the cell-free supernatant experimental group, the growth rate of *Pseudomonas fluorescens* decreased significantly, and the higher the CFS concentration, the stronger the inhibitory effect. This indicates that *Lactobacillus plantarum* cell-free supernatant can effectively inhibit the growth of planktonic *Pseudomonas fluorescens*.
[0060] Example 4: Effect of cell-free supernatant of Lactobacillus plantarum on leakage of contents from Pseudomonas fluorescens.
[0061] The fluorescent Pseudomonas suspension prepared in Example 1 was mixed with different volume concentrations (12.5%, 25%, 50%, 100%, v / v) of cell-free supernatant of Lactobacillus plantarum, with the CFS-free group serving as a control (0%). The mixture was incubated at 30°C and 180 rpm for 24 h. After incubation, the mixture was centrifuged at 5000 g and 4°C for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter. The absorbance of the supernatant at 260 nm (nucleic acid) and 280 nm (protein) was measured.
[0062] The results are as follows Figure 2As shown, cell-free supernatants of *Lactobacillus plantarum* CICC22810 (12.5%, 25%, 50%, and 100%) increased nucleic acid leakage by 26.41%, 57.65%, 71.97%, and 80.84%, respectively. Figure 2 A), which increased protein leakage by 34.01%, 66.78%, 82.80%, and 93.41% ( Figure 2 B). Cell-free supernatants of *Lactobacillus plantarum* CICC21805 (12.5%, 25%, 50%, 100%) increased nucleic acid leakage by 28.26%, 33.82%, 37.38%, and 54.59%, respectively. Figure 2 C), which increased protein leakage by 25.37%, 46.07%, 71.03%, and 77.89% (C). Figure 2 D). The above results indicate that cell-free supernatant of *Lactobacillus plantarum* can significantly disrupt the cell membrane integrity of *Pseudomonas fluorescens*, leading to a large amount of intracellular nucleic acid and protein leakage in a concentration-dependent manner.
[0063] Example 5: Effect of cell-free supernatant of Lactobacillus plantarum on reactive oxygen species levels in Pseudomonas fluorescens
[0064] Reactive oxygen species (ROS) levels were determined using a DCFH-DA-based reactive oxygen species (ROS) detection kit (fluorescent probe method). The fluorescent Pseudomonas aeruginosa suspension prepared in Example 1 was mixed with different volume concentrations (12.5%, 25%, 50%, 100%, v / v) of cell-free Lactobacillus plantarum supernatant and incubated at 37°C for 2 h. Then, 10 μM DCFH-DA was added, and incubation was continued for 20 min in the dark. The bacterial cells were collected by centrifugation, washed twice with PBS, resuspended, and the fluorescence intensity was measured using a multi-mode microplate reader (excitation wavelength 485 nm, emission wavelength 535 nm). The relative ROS level was calculated using the untreated group as a control (100%).
[0065] The results are as follows Figure 3 As shown. Cell-free supernatants of *Lactobacillus plantarum* CICC22810 (12.5%, 25%, 50%, and 100%) increased the ROS level of *Pseudomonas fluorescens* to 18.46%, 107.42%, 118.62%, and 155.36% of the control group, respectively. Figure 3 A). Cell-free supernatants of *Lactobacillus plantarum* CICC21805 (12.5%, 25%, 50%, 100%) increased ROS levels by 24.45%, 83.89%, 104.59%, and 132.67%, respectively. Figure 3 B). This indicates that cell-free supernatant of Lactobacillus plantarum can induce oxidative stress in Pseudomonas fluorescens, and the ROS level is significantly increased at high concentrations.
[0066] Example 6: Effect of cell-free supernatant of Lactobacillus plantarum on the activity of Pseudomonas fluorescens
[0067] The fluorescent Pseudomonas bacterial suspension prepared in Example 1 was taken and added to Lactobacillus plantarum cell-free supernatant at final concentrations of 0%, 12.5%, and 100% (v / v), respectively, and incubated at 30°C. The cells were collected by centrifugation, washed twice with PBS, and resuspended in PBS. Propidium iodide (PI, final concentration 5 μg / mL) was added, and the mixture was incubated in the dark for 20 min. 5 μL of the bacterial suspension was dropped onto a glass slide and observed and photographed under a fluorescence microscope (PI excitation wavelength 535 nm, emission wavelength 615 nm; red fluorescence represents dead bacteria with damaged membranes).
[0068] The results are as follows Figure 4 As shown, the control group (0% CFS) had fewer red fluorescent bacteria, indicating that most bacterial cell membranes remained intact. With increasing concentration of the cell-free supernatant of *Lactobacillus plantarum*, the number of red fluorescent bacteria significantly increased, with almost all bacteria in the 100% CFS treatment group exhibiting red fluorescence. This indicates that the cell-free supernatant of *Lactobacillus plantarum* can significantly reduce the activity of *Pseudomonas fluorescens*.
[0069] Example 7: Effect of cell-free supernatant of Lactobacillus plantarum on biofilm formation ability of Pseudomonas fluorescens
[0070] The biofilm formation amount was determined using crystal violet staining. A suitable amount of the *Pseudomonas fluorescens* suspension prepared in Example 1 was transferred to a 96-well plate containing cell-free supernatant of *Lactobacillus plantarum* prepared in Example 2 at different volume concentrations (0%, 25%, 50%, 75%, 100%, v / v), with a final volume of 200 μL per well. After static incubation at 30°C for 48 h, the plate was gently rinsed twice with PBS buffer to remove airborne bacteria and allowed to air dry. 200 μL of 0.1% (w / v) crystal violet solution was added to each well, and staining was performed at room temperature for 20 min. The plate was then rinsed twice with sterile distilled water to remove unbound crystal violet and dried. 200 μL of 33% (v / v) glacial acetic acid was added to each well, and the crystal violet was dissolved at room temperature for 20 min. The absorbance was measured at 570 nm using a microplate reader to evaluate the biofilm formation amount.
[0071] The results are as follows Figure 5 As shown, cell-free supernatants of *Lactobacillus plantarum* CICC22810 (12.5%, 25%, 50%, and 100%) reduced the biofilm-forming ability of *Pseudomonas fluorescens* by 76.13%, 79.34%, 81.05%, and 87.04%, respectively. Figure 5 A). Cell-free supernatants of *Lactobacillus plantarum* CICC21805 (12.5%, 25%, 50%, 100%) reduced biofilm formation capacity by 68.59%, 75.36%, 77.74%, and 85.68%, respectively. Figure 5 B). This indicates that the cell-free supernatant of *Lactobacillus plantarum* can effectively inhibit the formation of *Pseudomonas fluorescens* biofilm in a concentration-dependent manner.
[0072] Example 8: Effect of cell-free supernatant of Lactobacillus plantarum on the metabolic capacity of Pseudomonas fluorescens biofilm
[0073] Prepare XTT (1 mg / mL) and menadione (1 mM) working solutions at a volume ratio of 12.5:1. Take the *Pseudomonas fluorescens* suspension prepared in Example 1 and add an appropriate amount of the suspension to a 96-well plate containing cell-free supernatant of *Lactobacillus plantarum* prepared in Example 2 at different volume concentrations (0%, 25%, 50%, 75%, 100%, v / v), with a final volume of 200 μL per well. After incubating at 30°C for 48 h, rinse the plate twice with sterile distilled water to remove airborne bacteria. Add PBS buffer and the above XTT-menadione working solution to each well, incubate in the dark for an appropriate time, and then measure the OD. 490nm value.
[0074] The results are as follows Figure 6 As shown. Cell-free supernatants of *Lactobacillus plantarum* CICC22810 (12.5%, 25%, 50%, and 100%) reduced the metabolic capacity of *Pseudomonas fluorescens* biofilm by 48.16%, 60.68%, 65.30%, and 78.30%, respectively. Figure 6 A). Cell-free supernatants of *Lactobacillus plantarum* CICC21805 (12.5%, 25%, 50%, 100%) reduced metabolic capacity by 35.50%, 49.39%, 68.18%, and 74.02%, respectively. Figure 6 B). This indicates that cell-free supernatant from *Lactobacillus plantarum* can significantly reduce the metabolic activity of cells within biomembranes.
[0075] Example 9: Effects of cell-free supernatant from *Lactobacillus plantarum* on the microstructure of *Pseudomonas fluorescens* biofilm
[0076] The ultrastructure of the biofilm was observed using scanning electron microscopy. Fluorescent Pseudomonas suspension prepared in Example 1 was pipetted into 24-well plates containing different volume concentrations (0%, 12.5%, 100%, v / v) of cell-free supernatant of Lactobacillus plantarum prepared in Example 2. Sterile circular cell smears (approximately 8 mm in diameter) were placed in each well beforehand. After static incubation at 30°C for 48 h, the cell smears were removed, gently washed three times with sterile PBS buffer, and fixed overnight at 4°C in 2.5% (v / v) glutaraldehyde. After three washes with PBS buffer, the biofilm was dehydrated using a gradient of ethanol (30%, 50%, 70%, 80%, 90%, 100%) for 15 min each time. After critical point drying, a gold sputtering coating was deposited under vacuum. The microscopic surface of the biofilm was observed and photographed using scanning electron microscopy.
[0077] The results are as follows Figure 7 As shown, the control group formed a complex and tightly packed biofilm with numerous extracellular polymers visible between cells. After treatment with 12.5% *Lactobacillus plantarum* cell-free supernatant, the biofilm structure became looser, and cell aggregation was significantly reduced. In the 100% *Lactobacillus plantarum* cell-free supernatant treatment group, cell aggregation was minimal, and almost no complete biofilm structure was observed. Microscopic analysis indicates that *Lactobacillus plantarum* cell-free supernatant can inhibit the biofilm formation ability of *Pseudomonas fluorescens*.
[0078] In summary, this invention reveals for the first time the dual inhibitory effect of Lactobacillus plantarum cell-free supernatant on Pseudomonas fluorescens: it can both inhibit the growth of planktonic bacteria and effectively inhibit biofilm formation. This invention exerts its effects through multiple pathways, including disrupting cell membrane permeability (increasing nucleic acid and protein leakage), inducing oxidative stress (increasing ROS levels), reducing bacterial activity, inhibiting cellular metabolic activity within biofilms, and disrupting the microstructure of biofilms.
[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Those skilled in the art can make various adjustments and modifications without departing from the principles of the present invention, and these adjustments and modifications should also be considered within the scope of protection of the present invention.
Claims
1. Application of cell-free supernatant of Lactobacillus plantarum in inhibiting Pseudomonas fluorescens and its biofilm.
2. Use according to claim 1, characterized in that, The inhibition includes inhibiting the growth of Pseudomonas fluorescens planktonic bacteria and / or inhibiting the formation of Pseudomonas fluorescens biofilms.
3. Use according to claim 1 or 2, characterized in that, The inhibition of *Pseudomonas fluorescens* and its biofilm includes at least one of the following mechanisms of action: Disruption of Pseudomonas fluorescens cell membrane permeability; Increased leakage of nucleic acids and / or proteins from the contents of *Pseudomonas fluorescens*; Increase the reactive oxygen species level in *Pseudomonas fluorescens*; Reduces the activity of Pseudomonas fluorescens; Reduces the metabolic activity of cells within the biofilm of *Pseudomonas fluorescens*; Interfering with the microstructure of Pseudomonas fluorescens biofilm.
4. The application according to claim 1, characterized in that, The cell-free supernatant of *Lactobacillus plantarum* is obtained by centrifuging *Lactobacillus plantarum* to remove the bacterial cells, and then filtering the supernatant through a sterile filter membrane.
5. The application according to claim 4, characterized in that, The Lactobacillus plantarum is Lactobacillus plantarum CICC22810 or Lactobacillus plantarum CICC21805.
6. The application according to claim 1, characterized in that, The volume concentration of the cell-free supernatant of *Lactobacillus plantarum* is 12.5% to 100%.
7. The application according to claim 6, characterized in that, The volume concentration of the cell-free supernatant of *Lactobacillus plantarum* is 25%, 50%, or 100%.
8. The application according to claim 1, characterized in that, The inhibition of Pseudomonas fluorescens biofilm formation includes: Reduce biofilm formation; Reduce the content of extracellular polysaccharides, extracellular proteins and / or extracellular DNA in biological membranes; This causes the biological membrane structure to change from dense to loose.
9. Application of cell-free supernatant of Lactobacillus plantarum in the preparation of antibacterial agents or biofilm scavengers for inhibiting Pseudomonas fluorescens and its biofilm.