Use of a-bisabolol in the preparation of a serratia liquefaciens quorum sensing quencher

Abstract

This invention discloses the application of α-bisabolol in the preparation of a quorum sensing quencher for Serratia liquefaction. It belongs to the field of pharmaceutical technology. The purpose of this invention is to provide an application in the preparation of a quencher for inhibiting the quorum sensing system of Serratia liquefaction, thereby reducing the toxicity of pathogenic bacteria without inhibiting their growth. This invention provides an application of bisabolol in the preparation of a quencher for inhibiting the quorum sensing system of Serratia liquefaction. It offers a novel solution for developing efficient and safe food preservatives.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of α-bisabolol in the preparation of liquefied Serratia marcescens quorum sensing quencher. Background Technology

[0002] Serratia liquefaction is a common foodborne spoilage bacterium that frequently causes spoilage of meat and seafood. Therefore, antimicrobial research in the food industry is most concentrated there. With the widespread clinical use of antibiotics, bacterial resistance has gradually increased. This not only reduces the sensitivity of bacteria to antibiotics but may also alter the physiological and biochemical characteristics of bacteria, causing them to develop features they did not originally possess, such as impaired drug penetration, the production of drug-inactivating enzymes, and the formation of biofilms, thereby reducing the effectiveness of antibiotics. Currently, antibiotic resistance has become a serious global public health problem.

[0003] In recent years, bacterial quorum sensing (QS) systems have become important targets for researching novel drugs against drug-resistant bacteria. QS is a mode of intracellular or intercellular signal transduction in bacteria. By monitoring the concentration of certain signaling molecules (also known as autoinducible molecules) such as acyl-homoserine lactone (AHL), QS controls and coordinates the behavior of the entire bacterial community, enabling them to respond collectively to environmental stimuli and greatly enhancing the survival ability of the entire bacterial community.

[0004] The pathogenicity of pathogens is regulated by the QS system, which achieves pathogenicity by mediating the expression of pathogenic genes. When the density of pathogenic bacteria reaches a certain level, the pathogens synthesize and release certain signaling molecules that can initiate the expression of related genes and regulate various biological behaviors of the pathogens, such as bioluminescence, toxin production, biofilm formation, and antibiotic production.

[0005] Therefore, finding drugs to inhibit bacterial quorum sensing systems has become an urgent problem for researchers in the field. Summary of the Invention

[0006] The purpose of this invention is to prepare an inhibitor for the bacterial quorum sensing system of Serratia liquefaction, thereby providing an inhibitor for the bacterial quorum sensing system, which reduces the toxicity of pathogens without inhibiting their growth.

[0007] This invention provides the application of α-bisabolol in the preparation of a quencher that inhibits the quorum sensing system of Serratia liquefaction.

[0008] This invention provides the application of α-bisabolol in the preparation of a quencher that inhibits the growth of Serratia liquefiedis, reduces the release of volatile basic nitrogen, and alleviates pH rise.

[0009] This invention provides the application of α-bisabolol in the preparation of a quencher that inhibits the formation of extracellular polysaccharides in Serratia liquefiedifolia.

[0010] This invention provides the application of α-bisabolol in the preparation of a quencher that inhibits the activity of extracellular proteases in Serratia liquefaction.

[0011] This invention provides the application of α-bisabolol in the preparation of a quencher that inhibits the activity of extracellular lipase in Serratia liquefaction.

[0012] This invention provides the application of α-bisabolol in the preparation of a quencher that inhibits the production of biofilms from Serratia liquefiedis cells.

[0013] This invention provides the application of α-bisabolol in the preparation of a quencher that inhibits the aggregation, motility, or biofilm morphology of Serratia liquefiedis.

[0014] Further specifying, the dosage form of the quencher can be any one of tablets, capsules, granules, powders, or liquid preparations.

[0015] Further specifying, the concentration of α-bisabolol is 500 μg / mL.

[0016] This invention provides an application of α-bisabolol in the preparation of food preservatives, with the target being the LuxR protein of Serratia liquefaction quorum sensing.

[0017] Beneficial Effects: This invention confirms that the screened quorum sensing quencher, α-bisabolol, can significantly interfere with the key spoilage phenotypes of *Serratia liquefaction* at the sub-minimum inhibitory concentration (MIC). Specifically, it exhibits a 26.39% inhibition rate in biofilm formation, a reduction of over 40% in extracellular protease and lipase activity, a 60% decrease in bacterial motility, and effective blockade of siderophore synthesis. This effect is achieved through targeted inhibition of the quorum sensing signaling pathway, effectively delaying product spoilage and preventing bacterial adhesion and spread on surfaces without producing bactericidal effects, thus avoiding the risk of drug resistance. This provides a novel solution for developing highly efficient and safe food preservatives. Attached Figure Description

[0018] Figure 1 To test the QS inhibitory activity of the screened small molecules of traditional Chinese medicine using the reporter bacterium C. violaceum CV026; Figure 2 The effects of different concentrations of bisabolol on the growth curve (A), extracellular polysaccharide (B), extracellular lipase (C), extracellular protease (D), aggregation (E), and migration (F) of Serratia liquefaction. Figure 3The effect of bisabolol on biofilm production of Serratia liquefiedi; Figure 4 Field emission scanning electron microscopy results of Serratia marcescens liquefied after bisabolol treatment; Figure 5 The figure shows the effects of bisabolol on bacterial count (A), pH (B), and TVB-N (C) of fresh chicken breast during storage. Detailed Implementation

[0019] Bisabolol was purchased from Yuanfeng Chemical, 20250807003.

[0020] The accession number for the LuxR protein is UniProt ID: A0A379Y5B3.

[0021] Example 1: Screening for Quorum Sensing Quorum Quenchers Using Targeted Liquefaction of Serratia marcescens Quorum Sensing Proteins Molecular docking virtual screening: Small molecules of traditional Chinese medicine with a molecular weight of less than 600 were downloaded from the database of natural Chinese medicine ingredients. Molecular docking technology was used to screen for LuxR, the quorum sensing protein of Serratia liquefaction, and small molecules of traditional Chinese medicine with docking results lower than -9 kcal / mol were selected.

[0022] Minimum inhibitory concentration (MIC) determination was performed on the high-scoring small-molecule traditional Chinese medicines: The small-molecule medicines were first dissolved in 60% dimethyl sulfoxide (DMSO) to prepare stock solutions of corresponding concentrations, ensuring a final concentration of 2 mg / mL in the first well of a 96-well plate (ensuring the final DMSO volume ratio was less than 1.5%). A two-fold dilution method was used to establish concentration gradients (a total of 10 gradients). *Serratia liquefaction* was cultured to the second-generation logarithmic growth phase, and bacterial suspensions were prepared and adjusted to a concentration of 10⁻¹⁰ using sterile culture medium. 7 CFU / mL, ensuring an inoculum density of approximately 10 CFU / mL per well. 4 CFU / mL. The 96-well plates were then incubated in a biochemical incubator for 16–24 h. LB medium without the compound was used as a negative control, and 60% DMSO as a solvent control. The lowest concentration of the compound at which no bacterial growth was observed was the MIC of that substance.

[0023] Quorum quenching activity validation: The quorum quenching activity of the screened small molecules from traditional Chinese medicine was validated using the reporter strain C. violaceum CV026. The reporter strain was cultured to OD200. 600The concentration was 1.0. The culture was added to LB agar medium cooled to approximately 50 °C at a ratio of 1:50, along with a certain amount of C4-HSL. After thorough mixing, the mixture was poured into plates (90 mm in diameter) and wells were punched using Oxford cups. Then, 150 μL of the sub-inhibitory concentration of the small molecule substance was added to the wells of the plates, and the plates were incubated for 24 h (30 °C) and the color changes were observed.

[0024] Result: From Figure 1 It can be seen that the purple halo of the α-bisabolol treatment group is the smallest, indicating that its quorum quenching effect is better than that of the other five substances.

[0025] Implementation 2: Study on the inhibitory effect of α-bisabolol on the putrefactive phenotype of Serratia liquefaction Overnight cultures of liquefied Serratia marcescens were inoculated into LB broth at a rate of 1% (v / v). Bisabolol was added to the broth at final concentrations of 0, 1 / 16 MIC, 1 / 8 MIC, 1 / 4 MIC, and 1 / 2 MIC (MIC is 250 μg / mL; concentrations are 0, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, and 250 μg / mL, respectively). None of the selected sub-MIC concentrations showed direct antibacterial activity; the aim was to specifically evaluate their regulatory effect on quorum sensing-mediated virulence phenotypes. All treatments were incubated at 37 °C for 24 h, after which growth curves, motility, biofilm formation, extracellular polysaccharide production, extracellular protease activity, and extracellular lipase activity were measured.

[0026] Growth curve: Measured every 2 hours (0-24 h) using visible spectrophotometry and recorded the OD value. 600 value.

[0027] Extracellular polysaccharides: The phenol-sulfuric acid method was used to determine the extracellular polysaccharides. A series of glucose standard solutions were prepared with concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. 1 mL of each standard solution was taken, and 1 mL of 6% (w / v) phenol solution and 5 mL of concentrated sulfuric acid were added sequentially. After thorough vortexing and mixing, the mixture was allowed to stand at room temperature for 30 min. After cooling, the absorbance (OD) at 490 nm was measured using a microplate reader. 490 Plotting glucose concentration on the x-axis and corresponding OD... 490 The standard curve is plotted with the values ​​on the ordinate, and the regression equation is obtained by fitting the curve with linear regression.

[0028] The extraction procedure for bacterial extracellular polysaccharides (EPS) is as follows: Take 1 mL of bacterial culture, centrifuge at 10000 r / min for 15 min, collect the bacterial precipitate and resuspend it in 0.5 mL of sterile physiological saline, and centrifuge again under the same conditions for 30 min. Take 1 mL of supernatant, add 3 mL of pre-cooled ethanol to precipitate the polysaccharides, vortex for 1 min, and let stand at room temperature for 10 min until a white flocculent precipitate appears; after centrifugation at 8000 r / min for 10 min, take 1 mL of supernatant and perform colorimetric determination according to the same operating steps as the phenol-sulfuric acid method described above. Finally, substitute the values ​​into the regression equation fitted by the standard curve to calculate the content of extracellular polysaccharides (EPS) in the sample.

[0029] Extracellular proteases: First, 10 mL of 10% (w / v) skim milk and 90 mL of 1.5% (w / v) agar solution were sterilized at 121 °C for 15 min. After the agar cooled to 55 °C, the skim milk and agar were thoroughly mixed and poured into plates. The bacterial culture was centrifuged at 10,000 × g for 10 min at 4 °C, and 150 μL of the supernatant was added to the wells of a solidified skim milk agar plate perforated using an autoclaved Oxford cup. After incubation at 37 °C for 24 h, the diameter of the clear hydrolysis zone was measured.

[0030] Extracellular lipase: The supernatant of bacterial culture was used as the enzyme solution, with 50 mM p-nitrophenol palmitate (p-NPP) as the substrate (dissolved in isopropanol containing 0.1% gum arabic and 0.4% Triton X-100). The specific reaction system was as follows: 0.1 mL of substrate solution was incubated with 0.5 mL of 50 mM Tris-HCl buffer (pH 8.0) at 37 ℃ for 5 min. 50 μL of enzyme solution was added, and the volume was brought to a final volume of 3 mL with the same buffer. The reaction was then carried out at 37 ℃ for 10 min, and immediately terminated with 3 mL of anhydrous ethanol. The absorbance was measured at 410 nm, and the amount of p-NP generated was calculated based on the standard curve. The standard curve used p-nitrophenol (-NP) as a standard, with concentration gradient solutions prepared. OD was measured under the same conditions after terminating the reaction as described above. 410 The concentration-absorbance relationship was established through linear regression.

[0031] Motility: The solid culture medium for migratory motility consisted of 1 g tryptone, 0.5 g sodium chloride, 0.3 g agar, and 100 mL distilled water. The solid culture medium for gregarious motility consisted of 1 g peptone, 0.5 g sodium chloride, 0.3 g agar, 0.5 g D-fructose, and 100 mL distilled water. After preparation, the solid culture media were sterilized at 121 °C for 15 min, incubated at 55 °C, and then the plates were poured. After the agar cooled, 2 μL of culture was spotted in the center of the plate. Finally, the plates were incubated at 37 °C for 24 h, and the changes on the plates were observed and recorded.

[0032] Biofilm: Overnight cultures of liquefied Serratia marcescens were inoculated into 24-well plates at a 1% (v / v) inoculation ratio, with 1 mL of LB broth added to each well. α-Bisabolol was then added at final concentrations of 0, 1 / 16 MIC, 1 / 8 MIC, 1 / 4 MIC, and 1 / 2 MIC (MIC is 250 μg / mL; concentrations are 0, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, and 250 μg / mL, respectively), and the plates were incubated at 37°C for 24 h. After incubation, the bacterial culture in the wells was aspirated with a pipette, and the plates were gently rinsed 3-4 times with deionized water to remove any unadhered airborne bacteria. Then, 1 mL of 0.1% (w / v) crystal violet staining solution was added to each well, and staining was performed at room temperature for 10 min. After staining, rinse repeatedly with deionized water until the eluent is colorless. After the plate is allowed to air dry, add 0.5 mL of 33% glacial acetic acid solution to each well to dissolve the stained biofilm. After thorough mixing, measure the absorbance (OD) of the solution at a wavelength of 595 nm. 595 The amount of biofilm formed is characterized by absorbance values.

[0033] Scanning electron microscopy: Cell slides were placed in 24-well plates containing bacterial culture medium and incubated at 37 °C for 24 h. After incubation, the slides were removed, rinsed three times with 0.1 M PBS buffer (pH 7.2), and air-dried under sterile conditions. The slides were then immersed in 2.5% glutaraldehyde fixative and fixed at 4 °C for at least 4 h. After fixation, the slides were rinsed twice with PBS buffer of the same concentration, and then subjected to a gradient dehydration process using 50%, 70%, 80%, 90%, and 100% ethanol solutions. After dehydration, the slides were first replaced once with a 1:1 volume ratio of 100% ethanol and tert-butanol, and then twice with pure tert-butanol. After drying, the slides were sputter-coated with gold. Finally, the microstructure of the biofilm was observed and recorded using field emission scanning electron microscopy.

[0034] Result: From Figure 2As can be seen from A, Serratia liquefiedis could still grow normally after sub-MIC treatment, while the growth of bacteria in the MIC-treated group was restricted. The quorum sensing system may not directly regulate the basal metabolism or growth rate of Serratia liquefiedis.

[0035] Result: From Figure 2 As shown in B, the amount of extracellular polysaccharide formation in the experimental group was reduced by 26.39% compared with that in the control group.

[0036] Result: From Figure 2 As can be seen from D, the extracellular protease activity in the experimental group was reduced by 41.91% compared with that in the control group.

[0037] Result: From Figure 2 As can be seen from C, the extracellular lipase activity in the experimental group was reduced by 79.91% compared with that in the control group.

[0038] Result: From Figure 2 E shows that the clustering behavior of the experimental group was reduced by 65.98% compared with the control group.

[0039] Result: From Figure 2 As can be seen from F, the experimental group's mobility was reduced by 69.18% compared to the control group.

[0040] Result: From Figure 3 It can be seen that the biofilm production in the experimental group was reduced by 26.39% compared with that in the control group.

[0041] Result: From Figure 4 It can be seen that the biomembrane morphology of the experimental group was looser and smaller than that of the control group, and the extracellular metabolites were reduced.

[0042] Example 3. Study on the inhibitory effect of bisabolol on the putrefactive phenotype of Serratia liquefaction 25 g of chicken breast was taken and sterilized with ultraviolet light for 30 min. The samples were divided into the following four treatment groups: ① Control group (C) (blank control); ② Inoculated with Serratia liquefaction bacteria group (1×10⁻⁶). 5 ③ Inoculation with bisabolol (500 μg / mL) (SL); ④ Inoculation with liquefied Serratia marcescens (1×10⁻⁶ CFU / mL) (BIS); 5 Smoked chicken legs were packaged in trays and stored at 4 °C. Samples were collected on days 0, 3, 6 and 9 to determine total viable count, pH and volatile basic nitrogen (TVB-N).

[0043] Colony counting: The colony counting method described in GB / T 4789.2-2016 was used. Plate counting agar was used, and inoculation was performed by pouring the agar plates. Two to three appropriate dilutions were selected based on different fermentation days, and colony counting was performed after incubation at 37 ℃ for 48 h. Each sample was tested in triplicate, and the average value was taken as the final result.

[0044] pH: Take 10.0 g of meat sample, add 90.0 mL of deionized water and shake to mix. After standing for 30 min, filter and measure the pH value of the filtrate. Take 3 parallel replicates for each sample and take the average value as the final result.

[0045] Volatile basic nitrogen: Determined by semi-micro nitrogen determination method in GB 5009.228—2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food".

[0046] Result: From Figure 5 As can be seen from A, the colony count in the BIS group was lower than that in the C group, and the colony count in the SL+BIS group was lower than that in the SL group. The addition of bisabolol alleviated the increase in colony count during product storage.

[0047] Result: From Figure 5 As can be seen from B, the overall pH increase trend in the BIS group was smaller than that in the C group, and the pH in the SL+BIS group was smaller than that in the SL group. The addition of bisabolol effectively alleviated the increase in pH of the product during storage.

[0048] Result: From Figure 5 As can be seen from C, the TVB-N in the BIS group was less than that in the C group, and the TVB-N in the SL+BIS group was less than that in the SL group, which effectively alleviated the release of volatile basic nitrogen during product storage.

Claims

1. Application of α-bisabolol in the preparation of quenchers that inhibit the quorum sensing system of Serratia liquefaction.

2. Application of α-bisabolol in the preparation of quenchers that inhibit the growth of Serratia liquefiedis, reduce the release of volatile basic nitrogen, and alleviate pH rise.

3. Application of α-bisabolol in the preparation of quenchers that inhibit the formation of extracellular polysaccharides in Serratia liquefiedifolia.

4. Application of α-bisabolol in the preparation of quenchers that inhibit the activity of extracellular proteases in Serratia liquefaction.

5. Application of α-bisabolol in the preparation of quenchers that inhibit the activity of extracellular lipase in Serratia liquefaction.

6. Application of α-bisabolol in the preparation of quenchers that inhibit biofilm production in Serratia liquefiedis cells.

7. Application of α-bisabolol in the preparation of quenchers that inhibit the aggregation, motility or biofilm morphology of Serratia liquefiedis.

8. The application according to any one of claims 1-7, characterized in that, The quenching agent can be in any of the following dosage forms: tablets, capsules, granules, powders, or liquid preparations.

9. The application according to any one of claims 1-7, characterized in that, The concentration of α-bisabolol was 500 μg / mL.

10. The application of bisabolol in the preparation of food preservatives, characterized in that, The target is the LuxR protein of Serratia liquefaction quorum sensing.

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