An antibacterial composition for inhibiting bacteria in VBNC state and its application and product

Abstract

The present invention belongs to the field of food safety and poultry processing technology, and specifically relates to an antibacterial composition for inhibiting bacteria in the VBNC state, and its application and product. The present invention provides an antibacterial composition, which is composed of carvacrol, diallyl sulfide and aluminum oxide nanoparticles. The antibacterial composition significantly improves the inhibitory effect on VBNC Campylobacter jejuni through synergistic action, not only enhancing the antibacterial activity but also effectively reducing the risk of drug resistance. The present invention calculates the optimal ratio of antibacterial agents through a dosage optimization method based on a mathematical model, maximizes the antibacterial effect and ensures its stability in different processing environments. The present invention solves the problem of the inability to inhibit VBNC bacteria in the prior art and ensures food safety. In addition, the combination of natural plant ingredients and non-toxic nanoparticles is both safe and environmentally friendly, suitable for multiple fields such as poultry processing, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety and poultry processing, and particularly relates to an antibacterial composition for inhibiting bacteria in VBNC state, and an application and product thereof. Background Art

[0002] Campylobacter jejuni ( Campylobacter jejuni Campylobacter jejuni is one of the main pathogens causing foodborne gastroenteritis, with infection mostly caused by contaminated poultry products. Typical symptoms of Campylobacter jejuni infection include diarrhea, abdominal pain, nausea, vomiting, and fever. Severe cases can lead to dehydration and shock, posing a greater threat to people with weakened immune systems (such as children, the elderly, and pregnant women).

[0003] During the production and processing of poultry products, Campylobacter jejuni is transmitted through the poultry's intestines and environmental contamination, making it highly susceptible to contamination of meat products such as chicken. Even with strict hygiene measures implemented during slaughter and processing, Campylobacter jejuni can still spread through the food chain through cross-contamination. Campylobacter jejuni is highly resilient in processing environments and exhibits a degree of tolerance to many common disinfectants, making it a significant challenge in food safety prevention and control.

[0004] The viable non-culturable state (VBNC) refers to a dormant state that bacteria enter when exposed to environmental stressors (such as nutrient deprivation, temperature fluctuations, or pH shifts). In this state, while the bacteria remain viable and retain a certain level of virulence, they are unable to grow on conventional culture media. Studies have shown that Campylobacter jejuni is prone to entering the VBNC state during poultry processing, particularly under adverse environmental conditions such as low-temperature storage or sterilization. This allows the bacteria to transition to the VBNC state, thus evading conventional detection methods.

[0005] Although bacteria in the VBNC state cannot be detected using traditional culture methods, they remain pathogenic and can resuscitate and resume their pathogenic activity under suitable conditions. This characteristic makes VBNC bacteria a significant risk in food safety monitoring, particularly during food processing, storage, and transportation, where resuscitation can lead to food contamination and foodborne illness. Therefore, the control and monitoring of VBNC bacteria has become a major issue that needs to be addressed in the current food safety field.

[0006] Currently, there is a lack of effective antimicrobial methods for bacteria in the VBNC state. Most existing antimicrobial agents are designed for bacteria in their viable state and fail to fully consider the specific characteristics of VBNC bacteria. Consequently, traditional antimicrobial agents have limited effectiveness in controlling VBNC bacteria, failing to effectively inhibit their resurgence and eliminate potential food safety risks.

[0007] Furthermore, most existing methods for using antimicrobial compositions lack systematic dosage optimization. This is particularly true when targeting VBNC bacteria, where the ratio and concentration of the antimicrobial agents are not precisely adjusted to maximize their antibacterial efficacy. While there has been some research on the combined use of antimicrobial agents, many studies lack in-depth analysis of synergistic effects between antimicrobial agents and fail to quantify the combined inhibitory effects of different antimicrobial combinations against VBNC bacteria. This leads to the risk of unstable antimicrobial efficacy or bacterial resistance in practical applications.

[0008] Furthermore, the application of existing mathematical models in antimicrobial optimization is relatively limited, particularly in evaluating the effects against VBNC bacteria. Traditional models are mostly based on the growth state of conventional bacteria and lack precise quantification of the inhibitory effect under VBNC conditions. Consequently, the optimization and formulation design of antimicrobial compositions fail to fully utilize mathematical models for precise control, resulting in less than ideal results.

[0009] In summary, existing technologies have significant defects in the selection of antimicrobial agents, dosage optimization, synergistic effect evaluation and application of mathematical models, and are unable to effectively solve the problem of controlling bacteria in the VBNC state. Especially in the processing of poultry products, efficient control of Campylobacter jejuni remains a technical challenge. Summary of the Invention

[0010] In response to the above-mentioned deficiencies, the present invention provides an antibacterial composition for inhibiting bacteria in the VBNC state, and its applications and products. The present invention provides an antibacterial composition, which is composed of carvacrol, diallyl sulfide and aluminum oxide nanoparticles. The antibacterial composition significantly improves the inhibitory effect on VBNC Campylobacter jejuni through synergistic effects, not only enhancing the antibacterial activity but also effectively reducing the risk of drug resistance. The present invention calculates the optimal ratio of antibacterial agents through a dosage optimization method based on a mathematical model, maximizes the antibacterial effect and ensures its stability in different processing environments. The present invention solves the problem that the existing technology cannot inhibit VBNC bacteria, and ensures food safety. In addition, the combination of natural plant ingredients and non-toxic nanoparticles is both safe and environmentally friendly, suitable for multiple fields such as poultry processing, and has broad application prospects.

[0011] The technical solution of the present invention includes:

[0012] In a first aspect, the present invention provides an antibacterial composition for inhibiting bacteria in a VBNC state, wherein the antibacterial composition is composed of carvacrol, diallyl sulfide and aluminum oxide nanoparticles.

[0013] Specifically, the antibacterial composition consists of 0.025-0.4 mg / mL carvacrol, 0.025-0.4 mg / mL diallyl sulfide and 0.025-0.4 mg / mL aluminum oxide nanoparticles.

[0014] Preferably, the antibacterial composition consists of 0.1 mg / mL carvacrol, 0.1 mg / mL diallyl sulfide and 0.1 mg / mL aluminum oxide nanoparticles.

[0015] In a second aspect, the present invention provides a dosage optimization method for the above-mentioned antibacterial composition, which includes using the median effect theorem to evaluate the combined effect of carvacrol, diallyl sulfide and aluminum oxide nanoparticles; the median effect theorem equation is fa / fu=(D / Dm)^m.

[0016] Specifically, fa represents the proportion of inactivated VBNC Campylobacter jejuni; fu represents the proportion of remaining VBNC Campylobacter jejuni; D represents the dose of the administered antimicrobial agent, Dm represents the median effect dose, and m represents the S-shape of the dose-effect curve.

[0017] Specifically, the dosage optimization method uses the logarithmic form of the median effect theorem log[fa / (1-fa)] = m log(D) - m log(Dm) to draw a relationship graph of y=log[fa / (1-fa)] and x=log(D), thereby determining m and log(Dm); using m and Dm to calculate the CI value to determine the combined effect; the m represents the slope; the log(Dm) represents the x-axis intercept; and the CI value represents the combination index value.

[0018] Preferably, a CI value <1 indicates a synergistic effect; a CI value = 1 indicates an additive effect; and a CI value > 1 indicates an antagonistic effect.

[0019] In a third aspect, the present invention provides use of the above antibacterial composition in the preparation of antibacterial products.

[0020] Specifically, the antibacterial products include antibacterial drugs or antibacterial daily necessities.

[0021] Specifically, the antibacterial product targets bacteria in the VBNC state.

[0022] Preferably, the bacteria include one or more of Gram-negative bacteria and Gram-positive bacteria.

[0023] More preferably, the bacteria is Campylobacter jejuni.

[0024] In a fourth aspect, the present invention provides an antibacterial drug, which includes the above-mentioned antibacterial composition.

[0025] Specifically, the dosage form of the antibacterial drug includes a parenteral dosage form or a gastrointestinal dosage form.

[0026] Preferably, the dosage forms for administration via the gastrointestinal tract include but are not limited to tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0027] Preferably, the non-gastrointestinal dosage form includes but is not limited to an injection dosage form, a respiratory tract dosage form, a skin dosage form, a mucosal dosage form and a cavity dosage form.

[0028] Specifically, the antibacterial drug further comprises one or more pharmaceutically acceptable excipients.

[0029] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, adhesives, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, or antioxidants.

[0030] In a sixth aspect, the present invention provides an antibacterial daily product, which includes the above-mentioned antibacterial composition.

[0031] Preferably, the daily necessities include toiletries, household products, hygiene products, kitchen products or baby products.

[0032] The beneficial effects of the present invention are:

[0033] This invention provides an antimicrobial composition derived from natural plants. Composed of carvacrol, diallyl sulfide, and aluminum oxide nanoparticles, the composition significantly enhances its synergistic effect against Campylobacter jejuni in its viable nonculturable state (VBNC), effectively reducing the risk of drug resistance. Using a mathematical model-based dosage optimization method, the invention calculates the optimal ratio of antimicrobial agents, maximizing antimicrobial efficacy and ensuring stability in various processing environments. This invention addresses the existing problem of inability to inhibit VBNC bacteria, thereby ensuring food safety. Furthermore, the combination of natural plant ingredients and non-toxic nanoparticles is both safe and environmentally friendly, suitable for a variety of applications, including poultry processing, and has broad potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Figure 3 shows the antibacterial effects of carvacrol, diallyl sulfide, and alumina nanoparticles (Al2O3NPs) against VBNC Campylobacter jejuni in chicken juice under microaerobic conditions (37°C, 85% N2, 10% CO2, 5% O2); A in the figure is carvacrol; B is diallyl sulfide; and C is alumina nanoparticles.

[0035] Figure 2 The antibacterial effects of carvacrol (0.1 and 0.2 mg / mL) and diallyl sulfide (0.1 and 0.2 mg / mL) against VBNC Campylobacter jejuni in chicken juice at different time points under microaerobic conditions (37°C, 85% N2, 10% CO2, 5% O2); A in the figure is 0 h; B is 4 h; C is 8 h; D is 12 h; and E is 24 h.

[0036] Figure 3 The antibacterial effects of carvacrol (0.1 and 0.2 mg / mL) and alumina nanoparticles (Al2O3NPs, 0.05 and 0.1 mg / mL) on VBNC Campylobacter jejuni in chicken juice at different time points under microaerobic conditions (37°C, 85% N2, 10% CO2, 5% O2); A in the figure is 0 h; B is 4 h; C is 8 h; D is 12 h; and E is 24 h.

[0037] Figure 4 The antibacterial effects of diallyl sulfide (0.1 and 0.2 mg / mL) and alumina nanoparticles (Al2O3NPs, 0.05 and 0.1 mg / mL) on VBNC Campylobacter jejuni in chicken juice at different time points under microaerobic conditions (37°C, 85% N2, 10% CO2, 5% O2); A in the figure is 0 h; B is 4 h; C is 8 h; D is 12 h; and E is 24 h.

[0038] Figure 5 The antibacterial effects of carvacrol (0.1 mg / mL), diallyl sulfide (0.1 mg / mL) and alumina nanoparticles (Al2O3NPs, 0.05 and 0.1 mg / mL) on VBNC Campylobacter jejuni in chicken juice at different time points under microaerobic conditions (37°C, 85% N2, 10% CO2, 5% O2); A in the figure is 0 h; B is 4 h; C is 8 h; D is 12 h; and E is 24 h.

[0039] Figure 6 Figure 3 shows the concentration-effect curves of carvacrol, diallyl sulfide, alumina nanoparticles, and their mixture against VBNC Campylobacter jejuni in chicken juice under microaerobic conditions at 37°C for 24 h. The inhibitory effect is expressed as the percentage of inactivated bacteria. In the figure, A is carvacrol; B is diallyl sulfide; C is alumina nanoparticles; and D is their mixture.

[0040] Figure 7Figure 3 is the median effect graph of carvacrol, diallyl sulfide, and aluminum oxide nanoparticles (Al2O3NPs) in single, binary, and ternary combinations on VBNC Campylobacter jejuni in chicken juice at 37°C under microaerobic conditions for 24 h; AC in the figure represents the results of single or binary combination treatment; D represents the results of single or ternary combination treatment.

[0041] Figure 8 The Fa-CI diagram based on the Chou-Talalay method shows the synergistic effect evaluation of carvacrol, diallyl sulfide and alumina nanoparticles (Al2O3NPs) on VBNC Campylobacter jejuni in chicken juice after treatment with microaerobic conditions at 37°C for 24 h; A in the figure is carvacrol + diallyl sulfide; B is carvacrol + alumina nanoparticles; C is diallyl sulfide + alumina nanoparticles; D is carvacrol + diallyl sulfide + alumina nanoparticles. DETAILED DESCRIPTION

[0042] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0043] Example 1 Campylobacter jejuni ( Campylobacter jejuni ) and induction of VBNC state

[0044] This example used four Campylobacter jejuni strains: F38011 (Taxonomy ID: 1408176), 81-116 (Taxonomy ID: 407148), NCTC11168, and ATCC 33560. All of these strains are laboratory-derived collections. Approximately 50 μL of glycerol stock of each strain (F38011, 81-116, NCTC11168, and ATCC 33560) was plated onto Methionin (MH) plates containing 5% (v / v) defibrinated sheep blood and cultured at 37°C under microaerophilic conditions (85% N₂, 10% CO₂, 5% O₂) with regular passages. C. jejuni colonies were inoculated into MH broth and cultured at 37°C, 175 rpm, under microaerophilic conditions, until late logarithmic phase. Equal volumes of the cultures of the four strains were then mixed to obtain a mixture with an initial concentration of approximately 9 log CFU / mL. The culture medium was removed by centrifugation at 15,000 × g for 5 minutes, washed twice with PBS, and the cell pellet was resuspended in 7% (w / v) NaCl (concentration approximately 8 log CFU / mL) and incubated microaerophilically at 37°C, 175 rpm, for 48 hours. Viable bacterial counts were determined by propidium azidobromide (PMA) qPCR. Furthermore, 100 μL of the bacterial culture was evenly spread on MH plates containing 5% (v / v) defibrinated sheep blood using the plate count method. The plates were then inverted and incubated microaerophilically at 37°C for 18–24 hours. No bacterial colonies grew, indicating that Campylobacter jejuni had entered a specialized dormant state.

[0045] Example 2 Preparation of simulated chicken broth (simulating poultry processing environment)

[0046] Chicken juice was prepared using a freeze-thaw method: whole chickens (eviscerated) were frozen at -20°C, thawed in the refrigerator, and the exudate was collected. Large particles were removed from the exudate by centrifugation at 12,000 × g for 10 minutes. Furthermore, the supernatant was filtered through a 0.22 μm polyethersulfone syringe filter to remove microorganisms and stored at -20°C until use.

[0047] Simulated chicken juice was prepared by adding chicken juice (5% v / v) to MH broth. Campylobacter jejuni cultured for 48 hours in 7% (w / v) NaCl solution was then centrifuged at 15,000 × g for 10 minutes and resuspended in the simulated chicken juice for use in antimicrobial treatment.

[0048] Example 3 PMA treatment and qPCR analysis

[0049] PMA-qPCR was used to determine the number of viable VBNC C. jejuni cells remaining in samples after VBNC induction and antimicrobial treatment. PMA was dissolved in sterile ddH2O to a 1.5 mM stock solution and stored at -20°C. Samples (1 mL) at the end of VBNC induction and after antimicrobial treatment were centrifuged at 15,000 × g for 5 minutes at room temperature, and the pellet was resuspended in 1 mL of PBS. 5 μL of PMA and 100 μL of PMA enhancer (5× concentrate) were mixed with 395 μL of the C. jejuni suspension and incubated at room temperature for 10 minutes in the dark. At the end of the incubation, the sample was placed on ice and incubated under a 600-W halogen light source (at a distance of 20 cm) for 10 minutes. After centrifugation, the sample was eluted once with sterile double-distilled water, boiled at 100°C for 10 minutes, and quickly cooled in an ice bath to release DNA.

[0050] The qPCR reaction system consisted of 2 μL template DNA, 10 μL SensiFAST SYBR Lo-ROX reagent, and primers targeting the single-copy gene rpoB (100 nM; forward primer SEQ ID NO. 1: 5'-GAGTAAGCTTGCTAAGATTAAAG-3'; reverse primer SEQ ID NO. 2: 5'-AAGAAGTTTTAGAGTTTCTCC-3'). qPCR reaction conditions included incubation at 50°C for 2 minutes, followed by 10 minutes at 95°C, and 40 cycles of amplification (95°C for 15 seconds and 60°C for 1 minute).

[0051] Example 4 Preparation of Antimicrobial Agents and Antimicrobial Agent Stock Solutions

[0052] Carvacrol and diallyl sulfide were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions at a concentration of 32 mg / mL.

[0053] Al2O3 NPs (size: 40-50 nm; surface area: 32-40 m² / g) were dissolved in ddH2O to prepare a stock solution with a concentration of 20 mg / mL.

[0054] Example 5 Using mathematical models to study the interaction of antimicrobial agents

[0055] VBNC Campylobacter jejuni in simulated chicken juice was treated with different concentrations of carvacrol, diallyl sulfide, and Al2O3 NPs (or their combination) (see Tables 1-3). The mixture was placed at 37°C, 175 rpm, and microaerophilic conditions. Samples were taken at different time points, and the bacterial concentration was measured by PMA-qPCR to determine the antibacterial effect.

[0056] Table 1 Concentration of fungicides acting alone

[0057]

[0058] Table 2 Effective concentrations of combined fungicides

[0059]

[0060] Table 3 Concentration of fungicides with intermediate effect

[0061]

[0062] The combined effects of carvacrol, diallyl sulfide, and Al2O3 NPs were evaluated based on the median effect theorem of mass action. The equation is fa / fu = (D / Dm)^m, where fa represents the proportion of inactivated VBNC C. jejuni and fu represents the proportion of remaining VBNC C. jejuni. D represents the administered antimicrobial dose, Dm represents the median effect dose, and m represents the sigmoidal shape of the dose-effect curve. When m = 1, the dose-effect curve is hyperbolic; when m > 1, it is sigmoidal; and when m < 1, it is a flat sigmoidal curve.

[0063] The logarithmic form of the median effect theorem is log[fa / (1-fa)] = m log(D) - m log(Dm). By plotting y = log[fa / (1-fa)] against x = log(D), the slope "m" and the x-intercept log(Dm) can be determined. Concentration-effect curves and median effect plots were automatically generated using CompuSyn software. Furthermore, the Dm and m values ​​obtained with CompuSyn software were used to calculate the combination index (CI). A CI value < 1 indicates synergy, meaning that the two antimicrobial agents exhibit a stronger-than-expected additive effect when combined; a CI value = 1 indicates an additive effect; and a CI value > 1 indicates antagonism, meaning that the combination exhibits a weaker-than-expected additive effect.

[0064] Figure 1The results show the antimicrobial effects of carvacrol, diallyl sulfide, and alumina nanoparticles against VBNC Campylobacter jejuni in chicken juice under microaerobic conditions (37°C). Individual antimicrobials had minimal effects on VBNC C. jejuni. Even high concentrations of the antimicrobials reduced the number of VBNC C. jejuni by only 1-2 log CFU / mL, and this reduction was only observed in the following treatments: carvacrol (0.8 and 1.6 mg / mL), diallyl sulfide (1.6 mg / mL), and alumina nanoparticles (Al2O3 NPs, 0.3 and 0.4 mg / mL). Compared to alumina nanoparticles, higher concentrations of the plant-derived antimicrobials (carvacrol and diallyl sulfide) were required to achieve similar antimicrobial effects, indicating that alumina nanoparticles possess a stronger antimicrobial activity. In addition, when both carvacrol and diallyl sulfide were treated at a concentration of 1.6 mg / mL, the bacterial count in the carvacrol-treated group was approximately 1 log CFU / mL lower after 24 h, indicating that carvacrol had better antibacterial activity than diallyl sulfide.

[0065] Figure 2 Figure 3 shows the antibacterial effects of carvacrol (0.1 and 0.2 mg / mL) and diallyl sulfide (0.1 and 0.2 mg / mL) against VBNC Campylobacter jejuni in chicken juice at different time points under microaerobic conditions (37°C). Throughout the experiment, no significant differences in the number of VBNC Campylobacter jejuni were observed among the control, carvacrol-treated, diallyl sulfide-treated, and combined treatments. This suggests that the combination of carvacrol and diallyl sulfide has an additive effect against VBNC Campylobacter jejuni.

[0066] Figure 3 Figure 3 shows the antibacterial effects of carvacrol (0.1 and 0.2 mg / mL) and aluminum oxide nanoparticles (Al2O3 NPs, 0.05 and 0.1 mg / mL) against VBNC Campylobacter jejuni in chicken juice at different time points under microaerobic conditions (37°C). When low concentrations of the antimicrobial agents were used (0.1 mg / mL carvacrol and 0.05 mg / mL Al2O3 NPs), bacterial counts remained relatively stable, and no significant antibacterial effect was observed at the end of the experiment. When higher concentrations of the antimicrobial combination were used, bacterial counts in the treated groups were significantly lower than those in the control group (1 log CFU / mL). The strongest antibacterial effect was observed in the combination of 0.2 mg / mL carvacrol and 0.1 mg / mL Al2O3 NPs. However, bacterial counts in this combination were approximately 1 log CFU / mL lower than those in the treatments with either 0.2 mg / mL carvacrol or 0.1 mg / mL Al2O3 NPs alone, indicating that the effect was additive rather than synergistic.

[0067] Figure 4 Figure 2 shows the antibacterial effects of diallyl sulfide (0.1 and 0.2 mg / mL) combined with alumina nanoparticles (Al2O3 NPs, 0.05 and 0.1 mg / mL) against Campylobacter jejuni in VBNC form in chicken juice at different treatment times under microaerobic conditions (37°C). The combination of diallyl sulfide and Al2O3 NPs showed a similar trend, but required a longer treatment time (12 h) to achieve similar antibacterial efficacy as the carvacrol and Al2O3 NPs combination at 4 h.

[0068] Figure 5 The antibacterial effects of carvacrol (0.1 mg / mL), diallyl sulfide (0.1 mg / mL), and aluminum oxide nanoparticles (Al2O3 NPs, 0.05 and 0.1 mg / mL) against VBNC Campylobacter jejuni in chicken juice at different time points under microaerobic conditions (37°C) were shown. The bacterial count in the group treated with the combination of carvacrol, diallyl sulfide, and aluminum oxide nanoparticles was significantly reduced, indicating that these three substances have a strong inhibitory effect on VBNC Campylobacter jejuni.

[0069] Figure 6 The concentration-effect curves of carvacrol, diallyl sulfide, alumina nanoparticles, and their mixture against VBNC Campylobacter jejuni in chicken juice after treatment at 37°C under microaerophilic conditions for 24 hours. The inhibitory effect is expressed as the percentage of inactivated bacteria. The concentration-effect curves for all three antimicrobial agents exhibited similar S-shaped trends. The slope of the curve for alumina nanoparticles (Al2O3 NPs) was significantly steeper than that for the other antimicrobial agents, indicating their higher antimicrobial efficacy. Among the antimicrobial combinations, the combination containing alumina nanoparticles exhibited similar concentration-effect curve morphologies, while the combination of carvacrol and diallyl sulfide exhibited distinct curve characteristics. These results reveal differences in the effects of different antimicrobial combinations and their interactions, further highlighting the unique properties of the combination of alumina nanoparticles with carvacrol and diallyl sulfide.

[0070] Figure 7Figure 1 shows the median efficacy of carvacrol, diallyl sulfide, and aluminum oxide nanoparticles (Al2O3NPs) against VBNC Campylobacter jejuni in chicken juice after 24 hours of treatment at 37°C under microaerophilic conditions. The median effective dose (Dm, equivalent to IC50) of carvacrol, diallyl sulfide, and aluminum oxide nanoparticles (Al2O3NPs) alone was 0.24 mg / mL, 0.40 mg / mL, and 0.11 mg / mL, respectively. The Dm value of aluminum oxide nanoparticles was significantly lower than that of the other antimicrobial agents, indicating their high antimicrobial efficacy. Among the antimicrobial combinations, the Dm values ​​for carvacrol + diallyl sulfide, carvacrol + alumina nanoparticles, diallyl sulfide + alumina nanoparticles, and carvacrol + diallyl sulfide + alumina nanoparticles were 0.30, 0.15, 0.15, and 0.11 mg / mL, respectively. This indicates that alumina nanoparticles contributed significantly to the combination, and the three-antifungal combination had the strongest antimicrobial effect. Analysis of the slope (m-value) of the concentration-effect curves assessed the effect of varying antimicrobial concentration on antimicrobial efficacy. The m-values ​​for carvacrol, diallyl sulfide, and alumina nanoparticles ranged from 1.63 to 2.83, with the three-antifungal combination exhibiting the highest slope, indicating that bacterial survival decreased significantly with a slight increase in concentration.

[0071] In addition, the regression coefficients (r) of all curves exceeded 0.974, indicating that the model had a high degree of fit with the experimental data, verifying the accuracy and reliability of the method.

[0072] Figure 8 This Chou-Talalay-based Fa-CI plot shows the synergistic effect of carvacrol, diallyl sulfide, and aluminum oxide nanoparticles (Al2O3 NPs) against Campylobacter jejuni in VBNC form in chicken juice treated under microaerophilic conditions at 37°C for 24 hours. Combinations of carvacrol and diallyl sulfide exhibited effects ranging from additive to slightly antagonistic within the fa value range of 0.05 to 0.97. In contrast, all binary and ternary combinations containing aluminum oxide nanoparticles (Al2O3 NPs) exhibited significant synergistic effects (CI < 1) at fa values ​​exceeding 0.65. Furthermore, the shift from antagonism or additive to synergistic effects for all combinations was dose-dependent.

[0073] Among them, the ternary combination of carvacrol, diallyl sulfide, and alumina nanoparticles exhibited significant synergistic antibacterial activity, demonstrating that this combination can effectively inactivate Campylobacter jejuni in the VBNC state under simulated poultry processing conditions. Further analysis revealed that the concentration-effect curves of all synergistic combinations paralleled the curve trend of alumina nanoparticles, indicating that alumina nanoparticles made an important contribution to the observed synergistic effect.

[0074] Example 6 Effect of Antimicrobial Composition on Poultry Processing

[0075] In a poultry processing environment, the antimicrobial composition of the present invention is used to treat Campylobacter jejuni.

[0076] The antimicrobial composition is used as follows: (1) Carvacrol, diallyl sulfide, and aluminum oxide nanoparticles are mixed in an optimal ratio to form a solution, which is then evenly applied to the surface of the treated poultry. (2) The treated poultry carcasses are incubated in a microaerobic environment, and samples are taken regularly to test the survival rate and recovery of Campylobacter jejuni.

[0077] Effects: (1) Compared with traditional antimicrobial agents, the antimicrobial composition of the present invention can significantly reduce the survival rate of Campylobacter jejuni in the VBNC state. (2) By optimizing the ratio of antimicrobial agents through mathematical models, the antimicrobial effect is maximized and high antimicrobial performance is maintained in different processing environments. (3) The resurgence and regrowth of Campylobacter jejuni during poultry processing is effectively controlled, reducing food safety risks.

[0078] Example 7 Monitoring of antimicrobial effects during storage and transportation of poultry products

[0079] During the storage and transportation of poultry products, the inhibitory effect of the antibacterial composition (carvacrol, diallyl sulfide and aluminum oxide nanoparticles) of the present invention on VBNC bacteria is monitored in real time.

[0080] Monitoring process: (1) Poultry products were stored in a refrigerated environment and samples were taken regularly to monitor bacterial counts and antimicrobial efficacy. (2) Fluorescence imaging was used to monitor the efficacy of the antimicrobial composition and compare it with conventional antimicrobial agents.

[0081] Effects: (1) The antibacterial composition of the present invention exhibits excellent antibacterial effects under refrigerated conditions, effectively reducing the resurgence of Campylobacter jejuni in the VBNC state and maintaining the antibacterial effect for a long time. (2) Compared with traditional antibacterial agents, the composition of the present invention not only improves the inhibition efficiency of VBNC bacteria, but also reduces environmental pollution, and is environmentally friendly. (3) The composition can significantly reduce the contamination risk of poultry products during storage and transportation, ensuring the food safety of the products throughout the entire supply chain.

[0082] Example 8 Real-time detection of poultry product quality at the retail stage

[0083] During the retail stage of poultry products, the antimicrobial composition of the present invention is used for quality inspection and monitoring.

[0084] Testing process: (1) Apply the antimicrobial composition to the surface of poultry products and monitor the antimicrobial effect. (2) Ensure that poultry products remain safe and fresh at the retail stage by monitoring the effect of the antimicrobial agent in real time.

[0085] Effects: (1) This embodiment can accurately predict the quality changes of poultry products at the retail stage, monitor the antibacterial effect in real time, and ensure that poultry products remain safe before consumers purchase them. (2) By monitoring the antibacterial effect of poultry products in real time, the solution of the present invention not only improves the efficiency of quality inspection, but also ensures the quality and safety of poultry products, avoiding food safety hazards caused by manual inspection and delays. (3) This method can perform non-destructive inspection of poultry products without damaging their appearance or quality, thereby enhancing consumer trust and the market competitiveness of products.

[0086] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. An antibacterial composition for inhibiting bacteria in VBNC state, characterized in that: The antibacterial composition consists of carvacrol, diallyl sulfide and aluminum oxide nanoparticles; the diallyl sulfide is diallyl sulfide.

2. The antibacterial composition according to claim 1, characterized in that The antibacterial composition consists of 0.025-0.4 mg / mL carvacrol, 0.025-0.4 mg / mL diallyl sulfide and 0.025-0.4 mg / mL aluminum oxide nanoparticles; the diallyl sulfide is diallyl sulfide.

3. The antibacterial composition according to claim 2, characterized in that The antibacterial composition consists of 0.1 mg / mL carvacrol, 0.1 mg / mL diallyl sulfide and 0.1 mg / mL aluminum oxide nanoparticles; the diallyl sulfide is diallyl sulfide.

4. Use of the antibacterial composition according to any one of claims 1 to 3 in the preparation of antibacterial products, characterized in that: The antibacterial product is directed against Campylobacter jejuni in the VBNC state.

5. The use according to claim 4, characterized in that The antibacterial products include antibacterial drugs or antibacterial daily necessities.

6. An antibacterial drug, characterized in that: The antibacterial drug includes the antibacterial composition according to any one of claims 1 to 3.

7. The antibacterial drug according to claim 6, characterized in that The dosage form of the antibacterial drug includes a non-gastrointestinal dosage form or a gastrointestinal dosage form.

8. The antibacterial drug according to claim 6, characterized in that The antibacterial drug further comprises one or more pharmaceutically acceptable excipients.

9. An antibacterial daily product, characterized in that: The antibacterial daily necessities include the antibacterial composition according to any one of claims 1 to 3.

10. The antibacterial living product according to claim 9, characterized in that: The antibacterial daily necessities include household products or baby products.

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