Bacterial qs inhibitor and use thereof

Abstract

The application discloses a bacterial QS inhibitor and application thereof, and the active ingredient of the bacterial QS inhibitor is apigenin, and the use concentration is a sub-inhibitory concentration of target bacteria. The application first discovers and systematically proves that apigenin has a wide and significant inhibitory effect on a variety of bacterial QS systems and a spoilage phenotype regulated by the QS systems. Experiments show that under a sub-inhibitory concentration, the inhibition rate of apigenin on biofilm formation of pectobacterium is up to 96.33%, the inhibition rate of apigenin on synthesis of a signal molecule is up to 88.42%, and the inhibition rate of apigenin on a key virulence enzyme is more than 70%; the fresh-keeping effect of apigenin on food is significant, and the application of apigenin in the field of food fresh-keeping is expanded; and apigenin is a natural dietary ingredient and has high safety.

Description

Technical Field

[0001] This invention relates to a bacterial QS inhibitor, and more particularly to a bacterial QS inhibitor that can prevent food spoilage and its application. Background Technology

[0002] *Pectinobacterium carotensis* is a typical Gram-negative, motile plant soft rot pathogen that primarily infects cruciferous vegetables such as potatoes, taro, carrots, potatoes, cabbage, lettuce, and *Dendrobium officinale*. Its pathogenic mechanism involves the secretion of pectin-lysing enzymes and polygalacturonases to decompose plant cell walls, leading to tissue disintegration and soft rot. These virulence factors are mainly regulated by the bacterial signaling mechanism of quorum sensing (QS). QS is a regulatory mechanism by which bacteria communicate and coordinate collective behavior based on changes in cell density. It controls the expression of various virulence factors in foodborne spoilage bacteria and pathogens, such as biofilm formation, extracellular enzyme secretion, and motility. Traditional food preservation methods often rely on chemical preservatives for direct sterilization, which easily leads to bacterial resistance and poses safety risks. In *Pectinobacter carotensis*, a class of homoserine lactones (AHLs) signaling molecules regulate collective behavior. When the concentration of AHLs reaches a certain threshold, AHLs bind to receptor molecules such as LuxR to form the AHL-LuxR complex. This complex regulates the expression and secretion of a series of virulence factors according to the external environment.

[0003] Plant-derived natural products have become an important source of QS inhibitors due to their safety and diverse biological activities. For *Pectinobacterium*, existing technical literature reports methyl cinnamate and benzothiazole compounds as quorum sensing inhibitors. Apigenin, widely found in various fruits and vegetables, is a common dietary flavonoid with antioxidant and anti-inflammatory physiological functions. CN120053424A reports that apigenin can inhibit the flagellar motility of *Salmonella*, but flagellar motility is an individual bacterial movement behavior, not quorum sensing. Whether apigenin possesses QS inhibitory activity, particularly against *Pectinobacterium*, a key bacterium causing soft rot in fruits and vegetables, and its potential application as a food preservative, has not been publicly reported or thoroughly studied. Summary of the Invention

[0004] The first technical problem to be solved by the present invention is to provide a bacterial QS inhibitor.

[0005] The second technical problem to be solved by the present invention is to provide an application of the bacterial QS inhibitor in inhibiting bacterial QS-related phenotypes.

[0006] The third technical problem to be solved by the present invention is to provide an application of the bacterial QS inhibitor in food preservation.

[0007] The fourth technical problem to be solved by the present invention is to provide a food preservative.

[0008] The technical solution adopted by the present invention to solve its first technical problem is a bacterial QS inhibitor, characterized in that its active ingredient is apigenin.

[0009] In some specific embodiments, the concentration of apigenin used is a sub-inhibitory concentration against the target bacteria.

[0010] In some specific embodiments, the bacteria are pectinobacterium or violaceum.

[0011] The technical solution adopted by the present invention to solve its second technical problem is the application of the bacterial QS inhibitor in inhibiting bacterial QS-related phenotypes, wherein the application includes inhibiting one or more of the following: bacterial signaling molecule (N-acylhomoserine lactone) synthesis, motility, biofilm formation, extracellular polysaccharide synthesis, or virulent extracellular enzyme activity.

[0012] In some specific embodiments, the virulent extracellular enzymes include pectin lyase, cellulase, protease, and galacturonase.

[0013] The technical solution adopted by the present invention to solve its third technical problem is the application of the bacterial QS inhibitor in food preservation.

[0014] The technical solution adopted by this invention to solve its fourth technical problem is a food preservative comprising the aforementioned bacterial QS inhibitor, and a food-grade acceptable carrier or solvent.

[0015] For example, the carrier can be guar gum, and the solvent can be an aqueous solution of ethanol.

[0016] In some specific embodiments, the concentration of apigenin in the food preservative ranges from 39 to 1250 μg / mL, the concentration of guar gum ranges from 1250 to 3000 μg / mL, and the concentration of ethanol ranges from 10 to 20 mg / mL.

[0017] In some specific embodiments, the food is a fruit or vegetable.

[0018] The present invention has the following advantages:

[0019] This invention is the first to discover and systematically demonstrate that apigenin has a broad and significant inhibitory effect on the QS system and its regulated putrefactive phenotypes of various bacteria. Experiments show that at sub-inhibitory concentrations, apigenin can inhibit the formation of biofilms of Pectinobacterium pectinum by up to 94.62%, the synthesis of signaling molecules by up to 69.23%, and the inhibition rate of key virulence enzymes by over 70%. The preservation effect is significant, expanding the new application of apigenin in the field of food preservation. Apigenin is a natural dietary component with high safety.

[0020] The QS inhibitor of this invention is specifically targeted at Pectinobacterium, which causes soft rot in fruits and vegetables. By inhibiting the expression of key genes in its QS system, it weakens the pathogenicity at the source, providing a precise solution for post-harvest preservation of fruits and vegetables.

[0021] The food preservative of this invention employs an "antiviral" strategy, interfering with bacterial QS communication at sub-inhibitory concentrations to inhibit key spoilage behaviors such as biofilm formation, motility, and virulent enzyme secretion, rather than directly killing bacteria, thus helping to reduce the risk of drug resistance. The active ingredient, apigenin, has no toxic side effects on humans or fruits and vegetables at effective concentrations, is easily degraded without pollution, and aligns with the green and sustainable development direction of the food industry. Attached Figure Description

[0022] Figure 1 Photographs showing the effect of different concentrations of apigenin on the quorum sensing inhibition of Vibrio CV026;

[0023] Among them, A, the quorum sensing inhibitory activity of apigenin against *Porphyromonas violaceus* CV026 was determined by α-furanone C-30 (156 μL / mL), β-apigenin (156 μL / mL), apigenin (78 μL / mL), d-apigenin (39 μL / mL), and o-DMSO; B, the inhibitory activity of apigenin against *Porphyromonas violaceus* CV026 producing violetin was determined by α-furanone C-30 (156 μL / mL), apigenin (156 μL / mL), apigenin (78 μL / mL), and apigenin (39 μL / mL).

[0024] Figure 2 Photograph showing the effect of 1 / 2 MIC apigenin on the motility inhibition of pectinobacteria;

[0025] Among them, A, the effect of apigenin on the motility inhibition of Pectinobacterium on day 1, DMSO (a, b, c), apigenin (d, e, f); B, the effect of apigenin on the motility inhibition of Pectinobacterium on day 2, DMSO (a, b, c), apigenin (d, e, f); C, the effect of apigenin on the motility inhibition of Pectinobacterium on day 3, DMSO (a, b, c), apigenin (d, e, f); D, the trend of apigenin's inhibitory effect on the motility of Pectinobacterium during the 1-3 day culture period.

[0026] Figure 3 Image showing the effect of apigenin on the inhibitory effect of pectinobacter signaling molecules;

[0027] Among them, A, LC / MS chromatograms of the inhibitory effect of different concentrations of apigenin on AHLs of Pectinobacterium, where a represents 3-oxo-C6-HSL standard; b represents DMSO; c represents 1 / 16 MIC apigenin; d represents 1 / 8 MIC apigenin; e represents 1 / 4 MIC apigenin; and f represents 1 / 2 MIC apigenin; B, UPLC-MS / MS chromatogram of AHLs produced by Pectinobacterium; and C, the inhibition rate of apigenin on AHLs production by Pectinobacterium.

[0028] Figure 4 Photographs showing the effect of apigenin on the inhibitory effect of pectinobacter biofilm formation.

[0029] Among them, A) the inhibitory effect of apigenin on biofilm formation of *Pectinobacterium tumefaciens* observed under different microscopes, the inhibitory effect of apigenin on biofilm formation of *Pectinobacterium tumefaciens* observed under fluorescence microscopy (ae), where a represents DMSO, b represents 1 / 16 MIC, c represents 1 / 8 MIC, d represents 1 / 4 MIC, and e represents 1 / 2 MIC; the inhibitory effect of apigenin on biofilm formation of *Pectinobacterium tumefaciens* observed under scanning electron microscopy (fj), where f represents DMSO, g represents 1 / 16 MIC, h represents 1 / 8 MIC, i represents 1 / 4 MIC, and j represents 1 / 2 MIC; and the inhibitory effect of apigenin on biofilm formation of *Pectinobacterium tumefaciens* observed under laser confocal microscopy (ko), where k represents DMSO, l represents 1 / 16 MIC, m represents 1 / 8 MIC, n represents 1 / 4 MIC, and o represents 1 / 2 MIC; B) the inhibition rate of apigenin on biofilm formation of *Pectinobacterium tumefaciens*; and C) the inhibition rate of apigenin on extracellular polysaccharide production by *Pectinobacterium tumefaciens*.

[0030] Figure 5 Photographs showing the effect of apigenin on the inhibitory effect of extracellular enzymes on the virulence of pectinobacter;

[0031] Among them, A. the inhibitory effect of apigenin on pectinobacterial protease, B. the inhibitory effect of apigenin on pectinobacterial cellulase, C. the inhibitory effect of apigenin on pectin lyase, and D. the inhibitory effect of apigenin on pectinobacterial galacturonase.

[0032] Figure 6 Figure showing the effect of 1 / 2 MIC apigenin on the expression levels of QS and related pathogenic genes in Pectinobacterium praecoxib;

[0033] Where A represents the expression levels of apigenin on the two-component system regulatory genes (qseB / qseC) and flagellar regulation (flhC / flhD) of *Pectinobacterium*, and B represents the expression levels of apigenin on the pectin lyase synthesis gene (pelL), galacturonase synthesis gene (pehA), signal molecule synthesis gene (carI), and lipopolysaccharide synthesis gene (lptE) of *Pectinobacterium*; "**" indicates P < 0.05, and "***" indicates P < 0.01.

[0034] Figure 7 Photographs showing the inhibitory effect of celery garlic on the rotting of Chinese cabbage inoculated with P. carotovorum;

[0035] Wherein, a is the blank control group; b is the treatment with apigenin concentration of 1 / 2 MIC (313 μL / mL); c is the treatment with apigenin concentration of 1 / 4 MIC (156 μL / mL); d is the treatment with apigenin concentration of 1 / 8 MIC (78 μL / mL); and e is the treatment with apigenin concentration of 1 / 16 MIC (39 μL / mL). Detailed Implementation

[0036] The preferred embodiments of the present invention will be further described below with reference to the examples and accompanying drawings, but the scope of protection of the present invention is not limited thereto. The *Pectobacterium carotovorum* strain RC5297 chromosome (hereinafter referred to as *Pectobacterium*) used in the examples was purchased from the China Industrial Microbial Culture Collection Center; *Vibrio vulgaris* CV026 was donated by Professor Jia Aiqun's research group at Nanjing University of Science and Technology; calcium chloride, ethyl acetate, acetic acid, nucleic acid, dimethyl sulfoxide (DMSO), sodium dihydrogen phosphate, disodium hydrogen phosphate, acetone, sodium acetate, concentrated sulfuric acid, phenol, glucose, trichloroacetic acid, sodium hydroxide, and hydrochloric acid were all analytical grade and purchased from Sinopharm Group; methanol was chromatographic grade and purchased from TEDIA, USA; 95% anhydrous ethanol, 0.2% crystal violet solution, and Tris-HCl were also used. Buffer solution, 3,5-dinitrosalicylic acid, and physiological saline were purchased from Shanghai Bioengineering Co., Ltd.; signal molecules: N-hexanoyl-L-homoserine lactone (C6-HSL); N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL), with a purity ≥96%, were purchased from Sigma.

[0037] The apigenin (98%) and guar gum (95%) used in the examples were purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.

[0038] Example 1: Preliminary verification of the QS inhibitory activity of apigenin

[0039] The inhibitory activity of apigenin QS was detected using the reporter strain of Vibrio CV026.

[0040] Quorum sensing activity assay: A single colony of *Vibrio violaceus* CV026 was inoculated into 100 mL of LB liquid medium and cultured at 28℃ and 180 rpm for 24 h. 100 μL of the bacterial culture was then transferred to 100 mL of melted LB solid medium (at approximately 50℃), at which point the bacterial concentration in the medium was 1 × 10⁻⁶. 6 Add CFU / mL, then add 200 μL of the signal molecule C6-HSL and 100 μL of kanamycin (final concentration 20 μg / mL), shake to mix thoroughly, and pour evenly into 6 culture dishes. After solidification, place in Oxford cups, and add 30 μL of apigenin (one of four concentrations below the MIC, 1 / 4 MIC, 1 / 8 MIC, 1 / 16 MIC) to the Oxford cups. Use an equal volume of DMSO as a negative control. The experiment was conducted in triplicate, with 28 days of repeated readings. o Observe the experimental results after culturing in C for 24 hours.

[0041] The results showed that at sub-inhibitory concentrations, all apigenin-treated groups produced distinct colorless inhibition zones on agar plates. Quantitative analysis indicated that apigenin at concentrations of 156 μg / mL, 78 μg / mL, and 39 μg / mL inhibited the production of violacein by 98.21%, 61.13%, and 45.89%, respectively, comparable to the positive control furanone C-30, confirming that apigenin possesses clear QS inhibitory activity.

[0042] Example 2: Inhibition of pectin motility on pectinobacteria

[0043] The effect of apigenin at 1 / 2 MIC concentration on the motility of pectinobacteria was determined.

[0044] Motility assay: A single colony of *Pectinobacterium* was inoculated into 100 mL of NB liquid medium and incubated at 28°C and 180 rpm for 24 h. The OD of the bacterial culture was then measured. 600 All values ​​were adjusted to 0.05, at which point the bacterial concentration was approximately 1-5 × 10⁻⁵. 5 CFU / mL was used as seed culture for later use; 90 mL of swimming medium (NB liquid medium + 0.3% agar), 90 mL of gregarious movement medium (NB liquid medium + 0.5% agar), and 90 mL of rubbing movement medium (NB liquid medium + 1.0% agar) were prepared and sterilized at 121℃ for 30 min; 1000 μL of apigenin was added to each of the above solid media (around 50℃) to make the final concentration of apigenin in the medium 1 / 2 MIC, with an equal volume of DMSO as a negative control. After mixing, the three solid media were poured into 6 plates (90 mm in diameter). After the plates were solidified, 2 μL of the above seed culture was inoculated into the swimming, gregarious, and rubbing movement culture plates respectively; for swimming movement (0.3% agar), 2 μL of bacterial solution was dropped directly onto the surface (center) of the agar to determine the swimming ability of Pectinobacterium in semi-solid agar; for gregarious movement (0.5% agar), 2 μL of bacterial solution was dropped directly onto the surface (center) of the agar to determine the swimming ability of Pectinobacterium in semi-solid agar; for gregarious movement (0.5% agar), 2 μL of bacterial solution was dropped into the surface (center) of the agar. μL of bacterial suspension was directly dropped onto the agar surface (center) to determine the aggregation of Pectinobacteria on the agar surface; for scour activity (1% agar), 2 μL of bacterial suspension was injected deep into the agar using a sterile pipette tip to determine the scour activity of Pectinobacteria inside the agar. After incubation at 28°C for 24 hours, the diameters of swimming, aggregation, and scour activity were measured.

[0045] The results are as follows Figure 2 As shown, on the second day of culture, the apigenin-treated group inhibited the scouring, swimming, and swarming movements of Pectinobacterium by 92.83%, 94.85%, and 86.33%, respectively, indicating that apigenin can significantly weaken the migration and colonization abilities of bacteria.

[0046] Example 3: Inhibition of pectin on signal molecule synthesis in pectinobacteria

[0047] Signal molecule assay: A single colony of *Pectinobacterium* was inoculated into 100 mL of NB liquid medium and cultured at 28°C and 180 rpm for 24 h. The OD of the bacterial culture was then measured. 600 All values ​​were adjusted to 0.05, at which point the bacterial concentration was approximately 1.5 × 10⁻⁵. 5 CFU / mL was used as seed culture. 48 mL of the above seed culture was added to 2 mL of four different concentrations of apigenin (to final concentrations of 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, and 1 / 16 MIC, respectively). An equal volume of DMSO was used as a negative control. The culture was incubated at 28℃ and 180 rpm for 24 h. The cultured bacterial culture was centrifuged at 4℃ and 10000 r / min for 15 min to remove the bacterial cells. The supernatant was extracted twice with an equal volume of acidified ethyl acetate (50 mL, 0.5% formic acid). The extracts were combined and dried under reduced pressure at 43℃. 5 mL of methanol was added and shaken to dissolve the bacteria. The methanol solution was filtered through a 0.22 μm filter and stored at -20℃ for later use. 3-oxo-C6-HSL standard was dissolved in methanol to prepare a standard solution of 50–800 µg / mL for determining the standard curve. 1 mL of the above extract was added to 2 mL of apigenin. The effects of different concentrations of apigenin on the content of the signal molecule 3-oxo-C6-HSL produced by Erwinia were analyzed using LC-MS / MS in mL autosampler vials. The detection of the signal molecule was based on the treatment time and ion fragments of the standard. The characteristic ion fragment peak was m / z 102. The experimental results were integrated based on the peak area of ​​the signal molecule to calculate the relative content.

[0048] Chromatographic conditions: Waters Sunfire C18 column (2.1 mm × 100 mm 2.0 µm), flow rate 0.3 mL / min, injection volume 10 µL, column temperature 35℃, mobile phase gradient conditions: A is methanol, B is water; gradient elution program is shown in Table 1.

[0049] Table 1 Gradient elution process

[0050]

[0051] The results are as follows Figure 3 As shown, apigenin can dose-dependently inhibit the synthesis of 3-oxo-C6-HSL, a characteristic signaling molecule of Pectinobacterium. The inhibition rates at 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, and 1 / 16 MIC concentrations were 88.42%, 79.67%, 63.28%, and 44.65%, respectively, indicating that its target site is located upstream of the QS signaling pathway.

[0052] Example 4: Inhibition of biofilm and extracellular polysaccharide formation by apigenin in Pectinobacterium

[0053] Extracellular polysaccharide assay: A single colony of *Pectinobacterium* was inoculated into 100 mL of NB liquid medium and cultured at 28°C and 180 rpm for 24 h. The OD600 value of the bacterial solution was then adjusted to 0.05, at which point the bacterial concentration was approximately 1.5 × 10⁻⁶. 5 CFU / mL was used as seed culture. 1900 μL of the seed culture was added to a 24-well microplate, along with 100 μL of four different concentrations of apigenin (final concentrations of 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, and 1 / 16 MIC). An equal volume of DMSO served as a negative control. The experiment was conducted in triplicate. The culture was incubated at 28°C for 24 h. Two mL of the cultured bacterial solution was centrifuged at 6000 r / min for 20 min. The supernatant was filtered through a 0.22 μm filter membrane, and three volumes of anhydrous ethanol (pre-cooled) were added. The mixture was allowed to precipitate at 4°C for 24 h. After centrifugation at 6000 r / min for 20 min at 2°C, the supernatant was removed, and the precipitate was dissolved in 1 mL of distilled water and stored at -20°C for later use. The total carbohydrate content in EPS was quantified using the phenol-sulfuric acid method with glucose as the standard.

[0054] The inhibition rate is calculated as follows: Inhibition rate (%) = 100% × (total carbohydrate content of control - total carbohydrate content of sample) / total carbohydrate content of control.

[0055] Microscopic experiment: A single colony of *Pectinobacterium* was inoculated into 100 mL of NB liquid medium and incubated at 28°C and 180 rpm for 24 h. The OD of the bacterial culture was then measured. 600 All values ​​were adjusted to 0.05, at which point the bacterial concentration was approximately 1.5 × 10⁻⁵. 5 CFU / mL was used as seed culture. 1900 μL of the above seed culture was added to a 24-well microplate with a round coverslip at the bottom. At the same time, 100 μL of four different concentrations of apigenin were added to make the final concentrations 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC and 1 / 16 MIC, respectively. An equal volume of DMSO was used as a negative control. The experiment was conducted in triplicate. The culture was carried out at 28℃ for 24 h. After the culture, the upper culture medium was aspirated and the round coverslip at the bottom was removed. The plate was washed three times with PBS solution to remove airborne bacteria.

[0056] Fluorescence microscopy: The film on the coverslip was stained with 0.01% acridine orange for 15 min. It was washed three times with PBS solution to remove excess staining agent, and then observed under a fluorescence microscope after air drying.

[0057] Scanning electron microscopy (SEM): The film on the coverslip was fixed with 2.5% glutaraldehyde for 12 h, then dehydrated in a gradient manner with 50%, 70%, 80%, 90%, and 100% (v / v) ethanol, and then freeze-dried for 6 h. After sputtering with gold, it was observed under a SEM microscope.

[0058] Laser confocal scanning electron microscope (CLSM): The film on the coverslip was stained with AO / EB (1:1, v / v) for 5 min, excess dye was washed away with PBS solution, and the coverslip was placed under the CLSM lens for observation.

[0059] like Figure 4 As shown, apigenin strongly disrupts biomembrane structure and reduces biomass. At a concentration of 1 / 2 MIC, the biomembrane inhibition rate reached 94.62%, and almost no intact biomembrane structure was observed. Simultaneously, at this concentration, the inhibition rate of extracellular polysaccharide synthesis reached 68.49%. The key matrix for biomembrane formation was effectively inhibited.

[0060] Example 5: Inhibition of extracellular enzyme activity of virulent pectin by apigenin

[0061] Determination of pectin lyase: A single colony of *Pectinobacterium* was inoculated into 100 mL of NB liquid medium and incubated at 28°C and 180 rpm for 24 h. The OD of the bacterial culture was then measured. 600 All values ​​were adjusted to 0.05, at which point the bacterial concentration was approximately 1.5 × 10⁻⁵. 5 CFU / mL was used as seed culture. 9.5 mL of the seed culture was taken, and 500 µL of four different concentrations of apigenin (to final concentrations of 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, and 1 / 16 MIC) were added respectively. The mixture was thoroughly mixed, with DMSO used as a negative control. The culture was incubated at 28℃ and 180 rpm for 24 h. The cultured bacterial solution was centrifuged at 10000 r / min for 15 min at 4℃ to remove the bacterial cells. 500 μL of the supernatant was added to 9.5 mL of reaction mixture [100 mM Tris.HCl buffer, 50 mM calcium chloride, 0.5% polygalacturonic acid, pH=8.5] and reacted in a 50℃ water bath for 30 min. After centrifugation at 10000 r / min for 5 min, the absorbance was measured at 235 nm using a UV spectrophotometer. 235 The activity calculation formula is ΔA. 235 / (h・mL). NB liquid medium was used as a blank control.

[0062] Cellulase assay: A single colony of *Pectinobacterium* was inoculated into 100 mL of NB liquid medium and incubated at 28°C and 180 rpm for 24 h. The OD of the bacterial culture was then measured. 600All values ​​were adjusted to 0.05, at which point the bacterial concentration was approximately 1.5 × 10⁻⁵. 5 CFU / mL was used as seed culture. 9.5 mL of the seed culture was added to 500 µL of four different concentrations of apigenin (to final concentrations of 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, and 1 / 16 MIC), mixed thoroughly, and DMSO was used as a negative control. The culture was incubated at 28℃ and 180 rpm for 24 h. The cultured bacterial solution was centrifuged at 10000 r / min for 15 min at 4℃ to remove the bacterial cells. 500 μL of the supernatant was mixed with 2 mL of the reaction mixture [2.5 mg / mL carboxymethyl cellulose, 0.2 M phosphate buffer, pH=7] and incubated in a 42℃ water bath for 1 h. The reaction was stopped with 7.5 mL of ethanol-acetone (2:1 v / v) solution. After centrifugation at 10000 r / min for 5 min, the absorbance was measured at 550 nm using a UV spectrophotometer. 550 The activity calculation formula is ΔA. 550 / (h・mL). NB liquid medium was used as a blank control.

[0063] Assay for polygalacturonase: A single colony of *Pectinobacterium* was inoculated into 100 mL of NB liquid medium and incubated at 28°C and 180 rpm for 24 h. The OD of the bacterial culture was then measured. 600 All values ​​were adjusted to 0.05, at which point the bacterial concentration was approximately 1-5 × 10⁻⁵. 5 CFU / mL was used as seed culture for later use; 9.5 mL of seed culture was taken and 500 µL of four different concentrations of apigenin were added respectively (to make the final concentrations 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC and 1 / 16 MIC respectively), mixed well, and DMSO was used as a negative control. The mixture was incubated at 28℃ and 180 rpm for 24 h. After culturing, the bacterial culture was centrifuged at 10,000 r / min for 15 min at 4℃ to remove the bacterial cells. 500 μL of the supernatant was mixed with 2.5 mL of reaction mixture [0.05 M sodium acetate buffer, 0.5% polygalacturonic acid, pH 5.5] and reacted in a water bath at 28℃ for 30 min. 2.5 mL of distilled water was added to a final volume of 5 mL, followed by 5 mL of 3,5-dinitrosalicylic acid. The mixture was thoroughly mixed and placed in a boiling water bath (100℃) for 5 min. After centrifugation at 10,000 r / min for 5 min, the absorbance was measured at 540 nm using a UV spectrophotometer. 540 The activity calculation formula is ΔA. 540 / (h・mL). NB liquid medium was used as a blank control.

[0064] Protease assay: A single colony of *Pectinobacterium* was inoculated into 100 mL of NB liquid medium and incubated at 28°C and 180 rpm for 24 h. The OD of the bacterial culture was then measured. 600 All values ​​were adjusted to 0.05, at which point the bacterial concentration was approximately 1-5 × 10⁻⁵. 5 CFU / mL was used as seed culture. 9.5 mL of the seed culture was added to 500 µL of four different concentrations of apigenin (to final concentrations of 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, and 1 / 16 MIC, respectively), mixed thoroughly, and DMSO was used as a negative control. The culture was incubated at 28℃ and 180 rpm for 24 h. The cultured bacterial solution was centrifuged at 10000 r / min for 15 min at 4℃ to remove the bacterial cells. 500 μL of the supernatant was mixed with 5.5 mL of 1% azocasein and 1 M Tris-HCl buffer (pH=8.5), and then incubated at 43℃ for 1 h. The reaction was terminated by adding 4 mL of 10% trichloroacetic acid solution. After centrifugation at 10000 r / min for 5 min, 5 mL of the supernatant was mixed with an equal volume of 1 M sodium hydroxide solution, and the absorbance at 436 nm was measured using a UV spectrophotometer (A). 436 The activity calculation formula is ΔA. 436 / (h・mL). NB liquid medium was used as a blank control.

[0065] like Figure 5 As shown, apigenin significantly inhibited the activities of pectin lyase, cellulase, protease, and galacturonase. At a 1 / 2 MIC concentration, the inhibition rates were 87.16%, 84.47%, 78.19%, and 82.33%, respectively, indicating that apigenin can significantly reduce the ability of bacteria to infect and damage plant tissues.

[0066] Example 6: Regulation of QS-related gene expression in Pectinobacterium by apigenin

[0067] A single colony of *Pectinobacterium* was inoculated into 100 mL of NB liquid medium and cultured at 28°C and 180 rpm for 24 h. The OD600 value of the bacterial solution was then adjusted to 0.05, at which point the bacterial concentration was approximately 1.5 × 10⁻⁶. 5CFU / mL was used as seed culture. 100 μL of the above seed culture was added to 100 mL of fresh NB liquid medium containing a final concentration of 1 / 2 MIC apigenin. DMSO was used as a blank control. The culture was carried out at 28℃ and 180 rpm for 12 h. After centrifugation at 12000 rpm for 10 min, the bacterial cells were collected, washed three times with pre-cooled PBS water, and centrifuged again at 12000 rpm for 10 min. The bacterial cells were then placed in liquid nitrogen for later use. Total RNA was extracted using an RNA kit produced by Beijing Adley Biotechnology Co., Ltd. The primer sequences are shown in Table 2. The ribosomal gene yqgF was used as an internal reference gene. Primers were synthesized by Wuhan Qingke Biotechnology Co., Ltd. A qRT-PCR reaction system was constructed according to the instructions of the EASYspin Plus bacterial RNA rapid extraction kit (produced by Beijing Adley Biotechnology Co., Ltd.), and the experiment was performed using an Applied Biosystems 7300 Real-time PCR System.

[0068] Table 2 Primer sequences for qRT-PCR

[0069]

[0070] like Figure 6 As shown, 1 / 2 MIC apigenin significantly downregulated the expression of multiple pathogenicity-related genes in *Pectinobacterium*, including signal molecule synthesis genes (carI), QS regulatory genes (qseB / qseC), flagella assembly genes (flhC / flhD), biofilm component-related genes (lptE), and virulence enzyme genes (pelL, pehA). This molecularly confirms that apigenin exerts its antiviral effect by interfering with the core QS pathway.

[0071] Example 7: Application Experiment of Apigenin in Simulated Fruit and Vegetable Preservation

[0072] Apigenin, a quorum sensing inhibitor, is used in vegetable preservation. This study aimed to determine whether apigenin could effectively inhibit the infection of lettuce leaves by *P. carotovorum*. Single colonies of *P. carotovorum* were inoculated into 100 mL of NB liquid medium and cultured at 28°C and 180 rpm for 24 h. The OD values ​​of the bacterial culture were then calculated. 600 All values ​​were adjusted to 0.1, at which point the bacterial concentration was approximately 1×10⁻⁶. 6CFU / mL was used as the seed culture for later use. Lettuce leaves were purchased from a local market in Changsha, thoroughly washed with tap water, and air-dried at room temperature. Damaged outer leaves were removed, and fresh leaves free from disease, pests, and mechanical damage were selected and cut into 4 cm × 4 cm square slices using sterile scissors. The sliced ​​leaves were then immersed in 1% (w / v) sodium hypochlorite solution and 75% (v / v) ethanol for 30 s each, rinsed with sterile water, and air-dried for 30 min. A small hole was then made in the center of each slice using a sterile needle, and 5 µL of the above seed culture was inoculated into the hole. The inoculated samples were placed at 25 °C for 1 h to allow them to dry completely so that the bacteria could adhere effectively. Subsequently, 10 µL of apigenin containing different concentrations (1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, 1 / 16 MIC) was added to the hole and the surrounding area. DMSO was used as a negative control. The treated samples were stored in sterile petri dishes and incubated at 28°C and 90% humidity for 5 days to prevent excessive drying, and damage was checked during the incubation period. The experiment was performed independently three times, with each experiment repeated three times.

[0073] Using lettuce leaves as a model, after inoculating with *Pectinobacterium*, four different concentrations of apigenin solution were applied. Figure 7 As shown, compared with the control group, apigenin treatment can significantly delay the expansion of lesions and reduce the area of ​​tissue decay, proving its potential application value in the actual preservation of fruits and vegetables.

[0074] This invention reveals for the first time that apigenin, as a highly efficient natural QS inhibitor, can effectively weaken the spoilage ability of putrefactive bacteria such as Pectinobacterium without affecting bacterial growth, through multiple pathways including inhibiting signal molecule synthesis, disrupting biofilms, and reducing motility and toxic enzyme activity. This provides a solid experimental basis and a novel technical approach for developing novel, safe, and efficient food preservatives based on apigenin, especially fruit and vegetable coating preservatives.

Claims

1. A bacterial QS inhibitor, characterized in that, Its active ingredient is apigenin.

2. The bacterial QS inhibitor according to claim 1, characterized in that, The concentration of apigenin used is a sub-inhibitory concentration against the target bacteria.

3. The bacterial QS inhibitor according to claim 1 or 2, characterized in that, The bacteria are pectinobacterium or violaceum.

4. The use of the bacterial QS inhibitor according to any one of claims 1-3 in inhibiting bacterial QS-related phenotypes.

5. The application according to claim 4, characterized in that, The applications include inhibiting one or more of the following: bacterial signaling molecule synthesis, motility, biofilm formation, extracellular polysaccharide synthesis, or virulent extracellular enzyme activity.

6. The application according to claim 5, characterized in that, The virulent extracellular enzymes include pectin lyase, cellulase, protease, and galacturonase.

7. The application of the bacterial QS inhibitor according to any one of claims 1-3 in food preservation.

8. A food preservative, characterized in that, It includes the bacterial QS inhibitor according to any one of claims 1-3.

9. The food preservative according to claim 8, characterized in that, The food mentioned is fruit or vegetables.

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