Antibacterial composition for inhibiting VBNC-state bacteria and application and product thereof

Abstract

The invention belongs to the technical field of food safety and poultry processing, and particularly relates to an antibacterial composition for inhibiting VBNC state bacteria and application and a product thereof. The invention provides the antibacterial composition, the antibacterial composition is composed of carvacrol, diallyl sulfide and aluminum oxide nanoparticles, through the synergistic effect, the inhibition effect on VBNC campylobacter jejuni is remarkably improved, the antibacterial activity is enhanced, and the drug resistance risk is effectively reduced. The optimal ratio of the antibacterial agent is calculated through a dosage optimization method based on a mathematical model, the antibacterial effect is maximized, and the stability of the antibacterial agent in different processing environments is ensured. The problem that VBNC bacteria cannot be inhibited in the prior art is solved, and food safety is guaranteed. In addition, the combination of natural plant components and non-toxic nanoparticles is safe and environment-friendly, is suitable for multiple fields such as poultry processing, and has a wide application prospect.

Description

Technical Field

[0001] The 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 is one of the main pathogens that cause foodborne gastroenteritis, and most of its infections are caused by contaminated poultry products. Typical symptoms of Campylobacter jejuni infection include diarrhea, abdominal pain, nausea, vomiting, and fever. In severe cases, it can lead to dehydration and shock, and is more threatening 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 spreads through the intestinal tract of poultry and environmental pollution, and can easily contaminate meat products such as chicken. Even if strict hygiene measures are taken during slaughter and processing, Campylobacter jejuni can still spread in the food chain through cross-contamination and other channels. Campylobacter jejuni has a strong ability to survive in the processing environment and has a certain tolerance to a variety of conventional disinfectants, which makes it highly challenging in food safety prevention and control.

[0004] The viable non-culturable state (VBNC state) refers to a dormant state that bacteria enter when they are subjected to environmental stress (such as nutrient deprivation, temperature fluctuations, pH changes, etc.). In this state, although the bacteria are still alive and maintain a certain degree of virulence, they cannot grow on conventional culture media. Studies have shown that Campylobacter jejuni is prone to enter the VBNC state during poultry processing, especially under adverse environmental conditions such as low-temperature storage or sterilization treatment. The bacteria can transform into the VBNC state and thus evade conventional detection methods.

[0005] Although bacteria in the VBNC state cannot be detected by traditional culture methods, they are still pathogenic and can be revived and restore their pathogenic activity under appropriate conditions. This characteristic makes VBNC bacteria a major hidden danger in food safety monitoring, especially in food processing, storage and transportation. The resurgence of VBNC bacteria may cause food contamination and cause foodborne diseases. Therefore, the control and monitoring of VBNC bacteria has become a major issue that needs to be urgently addressed in the current food safety field.

[0006] In the prior art, there is still a lack of effective antibacterial methods for bacteria in the VBNC state. Most of the existing antimicrobial agents are designed for bacteria in the viable state, and fail to fully consider the particularity of bacteria in the VBNC state. Therefore, traditional antimicrobial agents have limited effects in controlling VBNC bacteria, and cannot effectively inhibit their resuscitation and eliminate potential food safety risks.

[0007] In addition, most of the existing methods of using antimicrobial compositions lack systematic dosage optimization, especially when dealing with VBNC bacteria, and fail to accurately adjust the ratio and concentration of antimicrobial agents to maximize their antibacterial effect. Although there are some studies on the combined use of antimicrobial agents, many studies lack in-depth analysis of the synergistic effects between antimicrobial agents and fail to quantify the comprehensive inhibitory effects of different antimicrobial combinations on VBNC bacteria. This leads to the risk of unstable antimicrobial effects or bacterial resistance in actual applications of antimicrobial compositions.

[0008] In addition, the application of existing mathematical models in the optimization of antimicrobial agents is relatively limited, especially in the evaluation of the effects on VBNC bacteria. Most traditional models are based on the growth state of conventional bacteria and lack the precise quantification of the inhibitory effect under the VBNC state. Therefore, the optimization and formulation design of antimicrobial compositions have failed to make full use of mathematical models to achieve fine control, resulting in unsatisfactory results.

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

[0010] In view of the above-mentioned deficiencies, the present invention provides an antibacterial composition for inhibiting VBNC bacteria, and its application and product. The present invention provides an antibacterial composition, which is composed of carvacrol, diallyl sulfide and aluminum oxide nanoparticles, and significantly improves the inhibitory effect on VBNC Campylobacter jejuni through synergistic effect, which not only enhances the antibacterial activity, but also effectively reduces the risk of drug resistance. The present invention calculates the optimal ratio of the antibacterial agent 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 VBNC bacteria cannot be inhibited 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.

[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 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 comprises using the mean effect theorem to evaluate the combined effect of carvacrol, diallyl sulfide and aluminum oxide nanoparticles; the mean effect theorem equation is fa / fu=(D / Dm)^m.

[0016] Specifically, the fa represents the proportion of inactivated VBNC Campylobacter jejuni; fu represents the proportion of remaining VBNC Campylobacter jejuni; D represents the dose of the antimicrobial agent administered, 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)]=mlog(D)-m log(Dm) to draw a relationship diagram 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, and make a combined effect judgment; the m represents the slope; the log(Dm) represents the x-intercept; and the CI value represents the combination index value.

[0018] Preferably, the 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 antibacterial composition in the preparation of antibacterial products.

[0020] Specifically, the antibacterial products include antibacterial drugs, antibacterial food additives 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 bacterium is Campylobacter jejuni.

[0024] In a fourth aspect, the present invention provides an antibacterial food additive, wherein the antibacterial food additive comprises the above-mentioned antibacterial composition.

[0025] Preferably, the health food further comprises conventional food auxiliary materials, which include but are not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids or nutritional enhancers.

[0026] In a fifth aspect, the present invention provides an antibacterial drug, which includes the above-mentioned antibacterial composition.

[0027] Specifically, the dosage form of the antibacterial drug includes a parenteral dosage form or a parenteral dosage form.

[0028] 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.

[0029] Preferably, the non-gastrointestinal dosage forms include, but are not limited to, injection dosage forms, respiratory tract dosage forms, skin dosage forms, mucosal dosage forms and cavity dosage forms.

[0030] Specifically, the antibacterial drug further comprises one or more pharmaceutically acceptable excipients.

[0031] 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.

[0032] In a sixth aspect, the present invention provides an antibacterial daily necessity, wherein the antibacterial daily necessity comprises the antibacterial composition described above.

[0033] Preferably, the daily necessities include toiletries, household items, hygiene items, kitchen items or baby items.

[0034] The beneficial effects of the present invention are:

[0035] The present invention provides an antibacterial composition derived from natural plants, which is composed of carvacrol, diallyl sulfide and aluminum oxide nanoparticles. The inhibitory effect on Campylobacter jejuni in a viable non-culturable state (VBNC) is significantly improved through synergistic action, which not only enhances the antibacterial activity but also effectively reduces 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 VBNC bacteria cannot be inhibited 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The antibacterial effect of carvacrol, diallyl sulfide and alumina nanoparticles (Al2O3 NPs) 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; C is alumina nanoparticles.

[0037] Figure 2 The antibacterial effect of carvacrol (0.1 and 0.2 mg / mL) and diallyl sulfide (0.1 and 0.2 mg / mL) on VBNC Campylobacter jejuni in chicken juice under microaerobic conditions (37°C, 85% N2, 10% CO2, 5% O2) at different time points; A in the figure is 0h; B is 4h; C is 8h; D is 12h; E is 24h.

[0038] Figure 3 The antibacterial effect of carvacrol (0.1 and 0.2 mg / mL) and alumina nanoparticles (Al2O3 NPs, 0.05 and 0.1 mg / mL) on VBNC Campylobacter jejuni in chicken juice under microaerobic conditions (37°C, 85% N2, 10% CO2, 5% O2) at different time points; A in the figure is 0h; B is 4h; C is 8h; D is 12h; E is 24h.

[0039] Figure 4 The antibacterial effect 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 under microaerobic conditions (37°C, 85% N2, 10% CO2, 5% O2) at different time points; A in the figure is 0h; B is 4h; C is 8h; D is 12h; E is 24h.

[0040] Figure 5The antibacterial effect of carvacrol (0.1 mg / mL), diallyl sulfide (0.1 mg / mL) and alumina nanoparticles (Al2O3 NPs, 0.05 and 0.1 mg / mL) on VBNC Campylobacter jejuni in chicken juice under microaerobic conditions (37°C, 85% N2, 10% CO2, 5% O2) at different time points; A in the figure is 0h; B is 4h; C is 8h; D is 12h; E is 24h.

[0041] Figure 6 This is the concentration-effect curve of carvacrol, diallyl sulfide, aluminum oxide nanoparticles and their mixture after treatment with VBNC Campylobacter jejuni in chicken juice at 37°C microaerobic conditions for 24 hours. The inhibitory effect is the percentage of inactivated bacteria. A in the figure is carvacrol; B is diallyl sulfide; C is aluminum oxide nanoparticles; and D is a mixture of the three.

[0042] Figure 7 This is the median efficacy diagram of single treatment, binary combination and ternary combination of carvacrol, diallyl sulfide and aluminum oxide nanoparticles (Al2O3 NPs) on VBNC Campylobacter jejuni in chicken juice under microaerobic conditions at 37℃ for 24 hours; AC in the figure are the results of single treatment or binary combination treatment; D is the result of single treatment or ternary combination treatment.

[0043] Figure 8 The Fa-CI diagram based on the Chou-Talalay method shows the synergistic effect evaluation of carvacrol, diallyl sulfide and alumina nanoparticles (Al2O3 NPs) on VBNC-state Campylobacter jejuni in chicken juice after 24 h of treatment under microaerobic conditions at 37°C; 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

[0044] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0045] Example 1 Cultivation of Campylobacter jejuni and Induction of VBNC State

[0046] This example uses four strains of Campylobacter jejuni, F38011 (Taxonomy ID: 1408176), 81-116 (Taxonomy ID: 407148), NCTC11168 and ATCC 33560, for experiments. The above strains are all laboratory preserved strains. The glycerol stock solution (about 50 μL) of each of the four strains of Campylobacter jejuni, F38011, 81-116, NCTC11168 and ATCC 33560 was inoculated on a MH plate containing 5% (v / v) defibrotized sheep blood, and cultured at 37°C under microaerobic conditions (85% N2, 10% CO2, 5% O2) and passaged regularly. The Campylobacter jejuni colony was inoculated into MH liquid culture medium, cultured at 37°C, 175rpm, under microaerobic conditions to the late logarithmic period, and the culture solutions of the four strains were mixed in equal volumes to obtain a mixed solution with an initial concentration of about 9 log CFU / mL. The culture medium was removed by centrifugation at 15,000 × g for 5 min, PBS solution was added for elution twice, and the cell pellet was resuspended in 7% (w / v) NaCl solution (concentration was about 8 log CFU / mL), and cultured for 48 h at 37 ° C, 175 rpm, and microaerobic conditions. The viable bacterial content was detected by propidium azidobromide (PMA) qPCR. In addition, 100 μL of the bacterial solution was evenly spread on an MH plate containing 5% (v / v) defibrotized sheep blood using the plate count method, inverted in an incubator, and cultured microaerophilically at 37 ° C for 18-24 h. If there was no colony growth, it indicated that Campylobacter jejuni had entered a special dormant state.

[0047] Example 2 Preparation of simulated chicken sauce (simulated poultry processing environment)

[0048] Chicken juice was prepared by freeze-thaw method: whole chicken (eviscerated) was frozen to -20°C, thawed under refrigeration and the exudate was collected. Large particles in the exudate were removed by centrifugation at 12,000 × g for 10 min. In addition, microorganisms in the supernatant were removed by 0.22 μm polyethersulfone syringe filter and stored at -20°C for future use.

[0049] The simulated chicken juice was obtained by adding chicken juice (5% v / v) to the MH liquid medium. Then, Campylobacter jejuni cultured in 7% (w / v) NaCl solution for 48 hours was centrifuged at 15,000×g for 10 minutes and resuspended in the simulated chicken juice for use in antibacterial treatment.

[0050] Example 3 PMA treatment and qPCR analysis

[0051] PMA-qPCR was used to determine the number of viable VBNC C. jejuni cells remaining in the samples at the end of VBNC induction and antimicrobial treatment. PMA was dissolved in sterile ddH2O to prepare a stock solution at a concentration of 1.5 mM and stored at -20°C. The samples (1 mL) at the end of VBNC induction / antimicrobial treatment were centrifuged at 15,000 × g for 5 min at room temperature, and the pellet was resuspended in 1 mL of PBS solution. 5 μL of PMA and 100 μL of PMA enhancer (5× concentrate) were mixed with 395 μL of C. jejuni suspension and incubated at room temperature in the dark for 10 min. The samples at the end of incubation were placed on ice and incubated under a 600-W halogen light source (20 cm away) for 10 min. After centrifugation to remove the mixed solution, sterile double-distilled water was added for elution once, followed by boiling at 100°C for 10 min and rapid ice bath cooling to release DNA.

[0052] The qPCR reaction system included: 2 μL template DNA, 10 μL SensiFAST SYBR Lo-ROX reagent, primers for the single copy gene rpoB (concentration of 100 nM, forward primer SEQ ID NO.1: 5'-GAGTAAGCTTGCTAAGATTAAAG-3'; reverse primer SEQ ID NO.2: 5'-AAGAAGTTTTAGAGTTTCTCC-3'). qPCR reaction conditions: incubation at 50°C for 2 min, 95°C for 10 min, and 40 amplification cycles (95°C, 15 s; 60°C, 1 min).

[0053] Example 4 Preparation of Antimicrobial Agents and Antimicrobial Agent Stock Solutions

[0054] Carvacrol and diallyl sulfide were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions with a concentration of 32 mg / mL.

[0055] Al2O3 NPs (size: 40-50nm; surface area: 32-40m 2 / g) was dissolved in ddHO to prepare a stock solution with a concentration of 20 mg / mL.

[0056] Example 5 Using mathematical models to study the interaction of antimicrobial agents

[0057] 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), and the mixture was placed at 37°C, 175rpm, under microaerobic conditions. Samples were taken at different time points, and the bacterial concentration was measured by PMA-qPCR to determine the antibacterial effect.

[0058] Table 1 Concentration of fungicides acting alone

[0059] Fungicides Fungicide concentration (mg / mL) Carvacrol 0、0.05、0.1、0.2、0.4、0.6、0.8、1.6 Diallyl sulfide 0、0.05、0.1、0.2、0.4、0.6、0.8、1.6 <![CDATA[Al2O3 NPs]]> 0、0.05、0.1、0.2、0.3、0.4

[0060] Table 2 Combination fungicide concentration

[0061]

[0062]

[0063] Table 3 Concentration of fungicides based on the medium effect theorem

[0064]

[0065]

[0066] The combined effect of carvacrol, diallyl sulfide, and Al2O3 NPs was evaluated based on the median effect theorem of the law of mass action. The equation is fa / fu = (D / Dm)^m, where fa represents the proportion of inactivated VBNC Campylobacter jejuni and fu represents the proportion of remaining VBNC Campylobacter jejuni. D represents the dose of the antimicrobial agent applied, Dm represents the median effect dose, and m represents the S-shaped degree of the dose-effect curve. When m = 1, the dose-effect curve is a hyperbola; when m>1, it is an S-shaped curve; when m<1, it is a flat S-shaped curve.

[0067] The logarithmic form of the median effect theorem is log[fa / (1-fa)]=m log(D)-m log(Dm). By plotting the relationship between y=log[fa / (1-fa)] and x=log(D), the slope "m" and the x-intercept log(Dm) can be determined. The concentration-effect curve and the median effect plot were automatically generated by CompuSyn software. In addition, the combination index (CI) value was calculated using the Dm and m values ​​obtained by CompuSyn software. A CI value <1 indicates synergy, meaning that the two antimicrobial agents show a stronger additive effect than expected when combined; a CI value = 1 indicates an additive effect; and a CI value > 1 indicates antagonism, meaning that the combination shows a weaker additive effect than expected.

[0068] Figure 1The results show the antibacterial effects of carvacrol, diallyl sulfide, and alumina nanoparticles on VBNC Campylobacter jejuni in chicken juice under microaerobic conditions (37°C). When the fungicides acted alone, the effects on VBNC Campylobacter jejuni were small, and even high concentrations of the fungicides reduced the number of VBNC Campylobacter jejuni by only 1-2 logCFU / mL, and only in the following treatment groups: 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 with alumina nanoparticles, plant-derived antimicrobial agents (carvacrol and diallyl sulfide) required higher concentrations to achieve similar antibacterial effects, indicating that alumina nanoparticles have stronger antibacterial activity. In addition, when both carvacrol and diallyl sulfide were treated at a concentration of 1.6 mg / mL, the number of bacteria in the carvacrol-treated group was approximately 1 log CFU / mL lower after 24 h, indicating that the antibacterial activity of carvacrol was superior to that of diallyl sulfide.

[0069] Figure 2 The antibacterial effects of carvacrol (0.1 and 0.2 mg / mL) and diallyl sulfide (0.1 and 0.2 mg / mL) on VBNC Campylobacter jejuni in chicken juice under microaerobic conditions (37°C) at different time points. During the entire experiment, there was no significant difference in the number of VBNC Campylobacter jejuni in the control group, carvacrol treatment group, diallyl sulfide treatment group, and the combination of the two compounds. This indicates that the combination of carvacrol and diallyl sulfide has an additive effect in treating VBNC Campylobacter jejuni.

[0070] Figure 3 Figure 2 shows the antibacterial effect of carvacrol (0.1 and 0.2 mg / mL) and alumina nanoparticles (Al2O3 NPs, 0.05 and 0.1 mg / mL) on VBNC Campylobacter jejuni in chicken juice under microaerobic conditions (37°C) at different time points. When low concentrations of the antimicrobial agents were used (0.1 mg / mL of carvacrol and 0.05 mg / mL of alumina nanoparticles), the bacterial counts remained relatively stable, and no significant antibacterial effect was observed even at the end of the experiment. When a higher concentration of the antimicrobial combination was used, the bacterial counts in the treatment group were significantly lower than those in the control group (1 log CFU / mL), and the strongest antibacterial effect was seen in the combination treatment with a carvacrol concentration of 0.2 mg / mL and an alumina nanoparticle concentration of 0.1 mg / mL. However, the bacterial count of this combination was about 1 log CFU / mL lower than that of the treatment groups with 0.2 mg / mL carvacrol or 0.1 mg / mL alumina nanoparticles alone, indicating that this effect was additive rather than synergistic.

[0071] Figure 4The antibacterial effect 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 under microaerobic conditions (37°C) at different time points. The combination of diallyl sulfide and alumina nanoparticles also showed a similar trend, but required a longer treatment time (12 h) to achieve a similar antibacterial effect as the combination of carvacrol and alumina nanoparticles at 4 h.

[0072] Figure 5 The antibacterial effects of carvacrol (0.1 mg / mL), diallyl sulfide (0.1 mg / mL) and alumina nanoparticles (Al2O3 NPs, 0.05 and 0.1 mg / mL) on VBNC Campylobacter jejuni in chicken juice under microaerobic conditions (37°C) at different time points. The bacterial count in the group using a combination of carvacrol, diallyl sulfide and alumina nanoparticles decreased significantly, indicating that these three substances have a strong inhibitory effect on VBNC Campylobacter jejuni.

[0073] Figure 6 The concentration-effect curves of carvacrol, diallyl sulfide, alumina nanoparticles and their mixture against VBNC Campylobacter jejuni in chicken juice at 37°C microaerobic conditions for 24 h, and the inhibitory effect is the percentage of inactivated bacteria. The concentration-effect curves of the three antimicrobial agents all showed a similar S-shaped trend. Among them, the slope of the curve of alumina nanoparticles (Al2O3NPs) was significantly steeper than that of other antimicrobial agents, indicating that it has a higher antimicrobial efficacy. Among the antimicrobial combinations, the combination containing alumina nanoparticles showed a similar concentration-effect curve morphology, while the combination of carvacrol and diallyl sulfide showed completely different curve characteristics. These results reveal the differences in effects and interactions between different antimicrobial combinations, and further highlight the unique performance of alumina nanoparticles when combined with carvacrol and diallyl sulfide.

[0074] Figure 7The median efficacy of carvacrol, diallyl sulfide and alumina nanoparticles (Al2O3 NPs) on VBNC Campylobacter jejuni in chicken juice after 24 hours of treatment under microaerobic conditions at 37°C, including the antibacterial effects of single treatment, binary combination, and ternary combination. The median effective dose (Dm, equivalent to IC50) of carvacrol, diallyl sulfide and alumina nanoparticles (Al2O3NPs) when used alone was 0.24mg / mL, 0.40mg / mL and 0.11mg / mL, respectively. Among them, the Dm value of alumina nanoparticles was significantly lower than that of other antimicrobial agents, indicating that it has a higher antibacterial efficacy. In the antimicrobial agent combination, the Dm values ​​of 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 shows that alumina nanoparticles contribute more to the combination, and the antibacterial effect of the three combinations is the strongest. By analyzing the slope (m value) of the concentration-effect curve, the effect of changes in antimicrobial agent concentration on the antimicrobial effect can be evaluated. The m values ​​of carvacrol, diallyl sulfide, and alumina nanoparticles ranged from 1.63 to 2.83, among which the combination of the three antimicrobial agents showed the highest slope, indicating that the bacterial survival rate of the combination decreased significantly when the concentration increased slightly.

[0075] 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.

[0076] Figure 8 The Fa-CI plots based on the Chou-Talalay method show the synergistic effect evaluation of carvacrol, diallyl sulfide, and alumina nanoparticles (Al2O3 NPs) against VBNC Campylobacter jejuni in chicken juice after 24 h of treatment under microaerobic conditions at 37°C. The combination of carvacrol and diallyl sulfide showed a change from additive to slightly antagonistic effect in the fa value range of 0.05 to 0.97. In contrast, all binary and ternary combinations containing alumina nanoparticles (Al2O3 NPs) showed significant synergistic effects (CI < 1) when the fa value exceeded 0.65. In addition, the transition of the interaction from antagonism or additive effect to synergistic effect was dose-dependent for all combinations.

[0077] Among them, the ternary combination of carvacrol, diallyl sulfide and alumina nanoparticles showed significant synergistic antibacterial effects, indicating that the combination can effectively inactivate Campylobacter jejuni in the VBNC state under simulated poultry processing conditions. Further analysis found that the concentration-effect curves of all combinations showing synergistic effects were parallel to the curve trend of alumina nanoparticles, indicating that alumina nanoparticles made an important contribution to the observed synergistic effect.

[0078] Example 6 Effect of Antimicrobial Composition on Poultry Processing

[0079] In a poultry processing environment, the antimicrobial composition of the present invention is used to treat Campylobacter jejuni.

[0080] The use process of the antibacterial composition is as follows: (1) Carvacrol, diallyl sulfide and aluminum oxide nanoparticles are mixed in an optimal ratio to prepare a solution, and the solution is 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 detect the survival rate and recovery of Campylobacter jejuni.

[0081] 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) The ratio of antimicrobial agents is optimized by mathematical models to ensure the maximization of antimicrobial effects and maintain high antibacterial performance in different processing environments. (3) The recovery and regrowth of Campylobacter jejuni in poultry processing is effectively controlled, reducing food safety risks.

[0082] Example 7 Monitoring of antimicrobial effects during storage and transportation of poultry products

[0083] 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.

[0084] Monitoring process: (1) Store poultry products in a refrigerated environment and take samples regularly to monitor the bacterial count and antimicrobial efficacy. (2) Use fluorescence imaging technology to monitor the efficacy of the antimicrobial composition and compare it with traditional antimicrobial agents.

[0085] Effects: (1) The antibacterial composition of the present invention exhibits excellent antibacterial effect in a refrigerated environment, can effectively reduce the resuscitation of Campylobacter jejuni in the VBNC state, and maintain 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 has the characteristics of being green and 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 supply chain.

[0086] Example 8 Real-time detection of poultry product quality at the retail stage

[0087] In the retail stage of poultry products, the antimicrobial composition of the present invention is used for quality inspection and monitoring.

[0088] 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 during the retail stage by monitoring the effect of the antimicrobial agent in real time.

[0089] 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 the 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 non-destructively inspect poultry products without damaging their appearance or quality, thereby enhancing consumer trust and product market competitiveness.

[0090] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

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

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.

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.

4. The method for optimizing the dosage of the antibacterial composition according to claims 1 to 3, characterized in that: The dosage optimization method 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; The fa represents the proportion of inactivated VBNC Campylobacter jejuni; fu represents the proportion of remaining VBNC Campylobacter jejuni; D represents the dose of the antimicrobial agent administered, Dm represents the median effect dose, and m represents the S-shape of the dose-effect curve.

5. The dose optimization method according to claim 4, characterized in that: 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 diagram 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 and make a joint action judgment; The m represents the slope; the log(Dm) represents the x-intercept; and the CI value represents the combination index value.

6. The dose optimization method according to claim 5, characterized in that: The CI value <1 indicates a synergistic effect; a CI value = 1 indicates an additive effect; and a CI value > 1 indicates an antagonistic effect.

7. Use of the antibacterial composition according to any one of claims 1 to 3 in the preparation of antibacterial products.

8. The use according to claim 7, characterized in that: The antibacterial products include antibacterial drugs, antibacterial food additives or antibacterial daily necessities.

9. The use according to claim 7, characterized in that: The antibacterial product targets bacteria in the VBNC state.

10. The use according to claim 9, characterized in that: The bacteria include one or more of Gram-negative bacteria and Gram-positive bacteria.

11. The use according to claim 10, characterized in that: The bacteria is Campylobacter jejuni.

12. An antibacterial food additive, characterized in that: The antibacterial food additive comprises the antibacterial composition according to any one of claims 1 to 3.

13. The antibacterial food additive according to claim 12, characterized in that: The food includes human food and / or animal food.

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

15. The antibacterial drug according to claim 14, characterized in that: The dosage form of the antibacterial drug includes a non-gastrointestinal dosage form or a gastrointestinal dosage form.

16. The antibacterial drug according to claim 14, characterized in that: The antibacterial drug also includes one or more pharmaceutically acceptable excipients.

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

18. The daily necessities according to claim 17, characterized in that: The daily necessities include toiletries, household items, hygiene items, kitchen items or baby items.

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