Application of δ-juniperene in the preparation of liquefied Serratia marcescens quorum sensing quencher

Abstract

This invention discloses the application of δ-juniperene in the preparation of quenchers that inhibit the quorum sensing system of *Serratia liquefaction*. It belongs to the field of pharmaceutical technology. The purpose of this invention is to provide a quencher for inhibiting the quorum sensing system of *Serratia liquefaction*, thereby reducing the toxicity of pathogens without inhibiting their growth. This invention provides an application of δ-juniperene in drugs that inhibit the quorum sensing system of *Serratia liquefaction*, offering a novel solution for developing highly efficient and safe food preservatives.

Description

[0001] This invention discloses the application of δ-juniperene 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 a quencher for inhibiting the quorum sensing system of *Serratia liquefaction*, thereby reducing the toxicity of pathogens without inhibiting their growth. This invention provides an application of δ-juniperene in a drug for inhibiting the quorum sensing system of *Serratia liquefaction*, offering a novel solution for developing highly efficient and safe food preservatives. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of δ-juniperene in the preparation of liquefied Serratia marcescens quorum sensing quencher. Background Technology

[0003] 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.

[0004] 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.

[0005] The pathogenicity of pathogens is regulated by the QS system, which mediates the expression of pathogenic genes to achieve pathogenicity. 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.

[0006] Therefore, finding drugs to inhibit bacterial quorum sensing systems has become an urgent problem for researchers in the field. Summary of the Invention

[0007] The purpose of this invention is to prepare a drug for inhibiting the bacterial quorum sensing system of Serratia liquefaction, thereby providing a drug for inhibiting the bacterial quorum sensing system and reducing the toxicity of pathogens without inhibiting their growth.

[0008] This invention provides the application of δ-juniperene in a quencher that inhibits the quorum sensing system of Serratia liquefaction.

[0009] This invention provides the application of δ-juniperene as a quencher for inhibiting the formation of extracellular polysaccharides in Serratia liquefiedis.

[0010] This invention provides the application of δ-juniperene as a quencher for inhibiting the activity of extracellular proteases in Serratia liquefiedis.

[0011] This invention provides the application of δ-juniperene as a quencher for inhibiting the extracellular lipase activity of Serratia liquefaction.

[0012] This invention provides the application of δ-juniperene as a quencher for inhibiting the production of biofilms by Serratia liquefiedis cells.

[0013] This invention provides the application of δ-juniperene as a quencher for inhibiting 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, the concentration of δ-juniperene was specified as 125 μg / mL.

[0016] This invention provides an application of δ-juniperene in the preparation of food preservatives.

[0017] Further, the target was identified as the LuxR protein of Serratia liquefaction quorum sensing.

[0018] Beneficial Effects: This invention verifies a novel application of δ-juniperene, a known active ingredient in traditional Chinese medicine, targeting the LuxR protein and inhibiting bacterial quorum sensing. It demonstrates that even at sub-inhibitory concentrations, it effectively inhibits biofilm formation and the activity of virulence factors (proteases and lipases, etc.) in food-grade Serratia liquefaction. These independent inhibitors based on food-drug homologous substances provide a variety of innovative and practical candidate solutions for developing safe, precise, and non-resistant food-grade antiviral agents. Attached Figure Description

[0019] Figure 1 The effect of different concentrations of δ-juniperene on the growth curves of Serratia liquefiedifolia; Figure 2The effects of different concentrations of δ-juniperene on extracellular polysaccharide (A), extracellular lipase (B), biofilm formation (C), extracellular protease (D), aggregation (E), and migration (F) of Serratia liquefiedis; Figure 3 Field emission scanning electron microscopy results of Serratia marcescens liquefied after treatment with δ-juniperene; Figure 4 The figure shows the effect of CAD-T80 on colony count and TVB-N during pork storage; Note: different uppercase letters (AD) indicate significant differences between different treatments at the same storage time (P<0.05); different lowercase letters (ad) indicate significant differences between the same treatments at different storage times (P<0.05). Detailed Implementation

[0020] δ-juniperene was purchased from Yuanfeng Chemical, PB87571.

[0021] LuxR protein accession number: UniProt ID: A0A379Y5B3.

[0022] Example 1: Determination of the minimum inhibitory concentration of δ-juniperene on Serratia liquefaction and evaluation of its growth effect Minimum inhibitory concentration (MIC) determination: δ-juniperene was first dissolved in 60% dimethyl sulfoxide (DMSO) to prepare a stock solution of the corresponding concentration, ensuring a final concentration of 2 mg / mL in the first well of a 96-well plate (ensuring the final volume fraction of DMSO was less than 1.5%). A two-fold dilution method was used to establish concentration gradients (10 gradients in total). Serratia liquefaction was cultured to the second generation logarithmic growth phase, and a bacterial suspension was prepared, 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] Growth curve determination: Overnight cultures of *Serratia marcescens* were inoculated into LB broth at a rate of 1% (v / v). δ-juniperene was added to the broth at final concentrations of 0, 1 / 16 MIC, 1 / 8 MIC, 1 / 4 MIC, and 1 / 2 MIC (MIC = 250 μg / mL; concentrations were 0, 16.125 μg / mL, 31.25 μg / mL, 62.5 μg / mL, and 125 μg / mL, respectively). All treatments were incubated at 37 °C, and OD values ​​were measured every 2 hours (0–24 h) using visible spectrophotometry and recorded. 600 value.

[0024] Result: From Figure 1 It can be seen that Serratia liquefiedis can still grow normally after sub-MIC treatment, while the growth of bacteria in the MIC treatment group is restricted. The quorum sensing system may not directly regulate the basal metabolism or growth rate of Serratia liquefiedis.

[0025] Example 2: Effects of quorum sensing quenchers on biofilm formation and virulence factor release in Serratia liquefiedifolia.

[0026] Overnight cultures of liquefied Serratia marcescens were inoculated into LB broth at a rate of 1% (v / v). Isochrysis was added to the broth at final concentrations of 0, 1 / 16 MIC, 1 / 8 MIC, 1 / 4 MIC, and 1 / 2 MIC, respectively. None of the selected sub-MIC concentrations showed direct antibacterial activity; the aim was to specifically evaluate its regulatory effect on quorum sensing-mediated virulence phenotypes. All treatment groups were incubated at 37 °C for 24 h, after which extracellular polysaccharide production, extracellular protease activity, extracellular lipase activity, motility, and biofilm formation were measured.

[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 *Serratia liquefaction* were inoculated into 24-well plates at a 1% (v / v) inoculation ratio, with each well containing 1 mL of LB broth. Isochrysistin was added at final concentrations of 0, 1 / 16 MIC, 1 / 8 MIC, 1 / 4 MIC, and 1 / 2 MIC, respectively, and the plates were incubated at 37 °C for 24 h. After incubation, the bacterial culture was aspirated from the wells, 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, the plates were repeatedly rinsed with deionized water until the eluent was colorless. After the plates were allowed to air dry, 0.5 mL of 33% glacial acetic acid solution was added to each well to dissolve the stained biofilm. After thorough mixing, the absorbance (OD) of the solution was measured at 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 2 As can be seen from A, the amount of extracellular polysaccharide formation in the experimental group was reduced by 21.09% and 25.02% compared with the control group, respectively.

[0035] Result: From Figure 2 As shown in B, the extracellular lipase activity in the experimental group was reduced by 67.29% and 60.71% compared with the control group, respectively.

[0036] Result: From Figure 2 As can be seen from C, the amount of biofilm generated in the experimental group was reduced by 21.09% and 25.02% compared with the control group, respectively.

[0037] Result: From Figure 2 As can be seen from D, the protease activity in the experimental group was reduced by 41.32% and 36.89% compared with that in the control group, respectively.

[0038] Result: From Figure 2 E shows that the clustering of the experimental group was reduced by 53.99% and 51.46% compared with the control group, respectively.

[0039] Result: From Figure 2 As can be seen from F, the swimming mobility of the experimental group was reduced by 60.60% and 64.56% compared with that of the control group, respectively.

[0040] Result: From Figure 3 It can be seen that the experimental group showed a looser and smaller structure and a reduction in extracellular metabolites compared to the control group.

[0041] Example 3: Preparation and application of δ-juniperene nanoemulsion Preparation of nanoemulsion: First, accurately weigh 1 g of Tween 80 and dissolve it in 50 g of deionized water. Stir magnetically (200 rpm) for 15 min at room temperature until completely dissolved. Then, under magnetic stirring, slowly add 0.5 g of δ-juniperene dropwise into the aqueous phase using a syringe, and continue stirring for 30 min to form a homogeneous crude emulsion. Place the obtained crude emulsion in an ultrasonic cell disruptor for ultrasonic emulsification. The ultrasonic power is 450 W, the working mode is 2 s ultrasonication followed by 2 s intermittent sonication, and the total ultrasonic time is 10 min under ice-water bath conditions to obtain δ-juniperene nanoemulsion (CAD-T80).

[0042] Application of δ-juniperene nanoemulsion in fresh pork: δ-juniperene nanoemulsion (125 μg / mL) was applied to the surface of fresh pork and stored on pallets at 4 °C for 6 days. Its actual impact on product quality was evaluated by measuring its microbial characteristics and volatile basic nitrogen (TVB-N) during storage.

[0043] 50 g of pork samples were sterilized with ultraviolet light for 30 min. The samples were divided into four treatment groups: ① Control group (C) (blank control); ② δ-juniperene (125 μg / mL) treatment group (CAD); ③ CAD-T80 treatment group (125 μg / mL) (CAD-T80). The pork was stored in trays at 4 ℃. Samples were collected on days 0, 2, 4, and 6 to determine the total viable bacterial count and volatile basic nitrogen (TVB-N).

[0044] 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.

[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] The results are as follows Figure 4 As shown: Result: From Figure 4 As can be seen from A, the addition of δ-juniperene can significantly reduce the total bacterial count in pork during storage, and the δ-juniperene nanoemulsion treatment group is more effective than the δ-juniperene alone treatment group.

[0047] Result: From Figure 4 As can be seen from B, the addition of δ-juniperene can significantly reduce the TVB-N content in pork during storage, and the δ-juniperene nanoemulsion treatment group has a better effect than the δ-juniperene alone treatment group.

Claims

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

2. Application of δ-juniperene in the preparation of quenchers that inhibit the formation of extracellular polysaccharides in Serratia liquefiedifolia.

3. Application of δ-juniperene in the preparation of quenchers that inhibit the activity of extracellular proteases in Serratia liquefaction.

4. Application of δ-juniperene in the preparation of quenchers that inhibit the activity of extracellular lipase in Serratia liquefaction.

5. Application of δ-juniperene in the preparation of quenchers that inhibit the production of biofilms from Serratia liquefiedis cells.

6. Application of δ-juniperene in the preparation of quenchers that inhibit the aggregation, motility or biofilm morphology of Serratia liquefiedis.

7. The application according to any one of claims 1-6, characterized in that, The quenching agent can be in any of the following dosage forms: tablets, capsules, granules, powders, or liquid preparations.

8. The application according to any one of claims 1-6, characterized in that, The concentration of δ-juniperene was 125 μg / mL.

9. Application of δ-juniperene in the preparation of food preservatives.

10. The application according to claim 9, characterized in that, The target is the LuxR protein of Serratia liquefaction quorum sensing.

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