Application of strychnos nux-vomica essential oil and carum carvi essential oil in preparation of staphylococcus aureus antibacterial agent

Abstract

This invention belongs to the field of antibacterial product technology, specifically relating to the application of strychnine essential oil and caraway essential oil in the preparation of an antibacterial agent against Staphylococcus aureus. In the antibacterial agent, the concentration ratio of strychnine essential oil to caraway essential oil is 38-63:9-28. The combination of caraway essential oil and strychnine essential oil in this ratio exhibits superior antibacterial effects against Staphylococcus aureus.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial product technology, specifically relating to the application of strychnine essential oil and caraway essential oil in the preparation of Staphylococcus aureus antibacterial agents. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Staphylococcus aureus ( Staphylococcus aureus , S. aureus *Clostridium difficile* is a facultative anaerobic Gram-positive bacterium widely distributed in the natural environment and on the skin and cavities of humans and animals. It is a common foodborne pathogen. Its cells are spherical, approximately 0.8 μm in diameter, arranged in grape-like clusters. It lacks flagella and spores, and most lack capsules. The cell wall is mainly composed of peptidoglycan and teichoic acid. This bacterium is highly adaptable, growing over a wide range of temperatures and pH levels, and is resistant to drying, salt, and low temperatures. Its pathogenicity is extremely high, primarily due to the secretion of various virulence factors, such as heat- and acid / alkali-resistant enterotoxins (causing food poisoning) and hemolysins (damaging cells). It can also form biofilms to enhance drug resistance and environmental resistance. This bacterium easily contaminates meat, fruits, and vegetables during food processing, transportation, and storage, especially rapidly multiplying in nutrient-rich substrates such as cooked chicken. The enterotoxins it produces are difficult to inactivate with conventional heating, making it one of the major foodborne pathogens globally and posing a significant threat to food safety.

[0004] Natural antibacterial products are widely favored due to their advantages such as being green and safe, having low toxicity and side effects, broad antibacterial spectrum, being less likely to induce drug resistance, and not affecting the nutritional value of food. However, at present, the antibacterial effect of natural antibacterial products against Staphylococcus aureus is poor, making it difficult to meet the requirements of practical applications. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of strychnine essential oil and caraway essential oil in the preparation of Staphylococcus aureus antibacterial agents.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides the application of nux vomica essential oil and caraway essential oil in the preparation of a Staphylococcus aureus antibacterial agent, wherein the concentration ratio of nux vomica essential oil to caraway essential oil in the antibacterial agent is 38-63:9-28.

[0007] Secondly, the present invention provides a Staphylococcus aureus antibacterial essential oil composition, comprising nux vomica essential oil and caraway essential oil, wherein the concentration ratio of nux vomica essential oil to caraway essential oil is 38-63:9-28.

[0008] In some embodiments, the concentration ratio of strychnine essential oil to caraway essential oil in the antibacterial essential oil composition is 38-60:10-28.

[0009] Preferably, in the antibacterial essential oil composition, the concentration ratio of nux vomica essential oil to caraway essential oil is 38-55:15-28.

[0010] More preferably, in the antibacterial essential oil composition, the concentration ratio of nux vomica essential oil to caraway essential oil is 45-55:15-20.

[0011] More preferably, in the antibacterial essential oil composition, the concentration of strychnine essential oil is 45-55 μL / L, and the concentration of caraway essential oil is 15-20 μL / L.

[0012] In a further preferred embodiment, the concentration of strychnine essential oil in the antibacterial essential oil composition is 50-55 μL / L, and the concentration of caraway essential oil is 17-20 μL / L.

[0013] Specifically, in the antibacterial essential oil composition, the concentration of strychnine essential oil is 51 μL / L, and the concentration of caraway essential oil is 18 μL / L.

[0014] Thirdly, the present invention provides a Staphylococcus aureus antibacterial essential oil composition comprising 2,3-butanedione and strychnine essential oil, wherein the concentration ratio of 2,3-butanedione and strychnine essential oil is 25-50:10-40, or 6-10:60-65.

[0015] In some embodiments, the concentration ratio of 2,3-butanedione to strychnine essential oil in the antibacterial essential oil composition is 40-50:10-15, or 8-10:62-64.

[0016] Fourthly, the present invention provides a Staphylococcus aureus antibacterial essential oil composition comprising 2,3-butanedione and caraway essential oil, wherein the concentration ratio of 2,3-butanedione and caraway essential oil is 25-50:8-30, or 6-10:40-50.

[0017] In some embodiments, the concentration ratio of 2,3-butanedione to caraway oil in the antibacterial essential oil composition is 40-50:8-10, or 6-10:45-50.

[0018] Secondly, the present invention provides the use of the antibacterial essential oil composition as a food antibacterial additive.

[0019] Thirdly, an antibacterial spray prepared from the said antibacterial essential oil composition.

[0020] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: A combination of caraway essential oil and strychnine essential oil in a specific ratio exhibits superior antibacterial effects against Staphylococcus aureus.

[0021] The compound system showed stable antibacterial effects in five different food matrices, including apples, potatoes, and chicken. The residual strains were significantly lower than those in the single antibacterial agent treatment group, demonstrating good matrix adaptability. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 This is a comparison chart of the in vitro inhibitory effects of various antibacterial agents on Staphylococcus aureus in embodiments of the present invention; Figure 2 This is a comparison diagram of the antibacterial effects of the compound treatment of nux vomica oil and caraway oil on Staphylococcus aureus in an embodiment of the present invention. Figure 3 This is a comparison of the inhibitory effects of a combination of nux vomica oil and caraway oil on Staphylococcus aureus in different food matrices, as described in this embodiment of the invention. Figure 4 This is a comparative graph showing the effect of the compound treatment of nux vomica essential oil and caraway essential oil on the growth curve of Staphylococcus aureus in an embodiment of the present invention. Figure 5 This is a comparative diagram showing the effect of the compound treatment of nux vomica essential oil and caraway essential oil on the surface morphology of Staphylococcus aureus in an embodiment of the present invention. Figure 6 This is a comparative diagram showing the effect of the compound treatment of nux vomica essential oil and caraway essential oil on the relative conductivity of Staphylococcus aureus in an embodiment of the present invention. Figure 7 This is a comparative diagram showing the effect of the compound treatment of nux vomica essential oil and caraway essential oil on the extravasation of Staphylococcus aureus AKP in an embodiment of the present invention. Detailed Implementation

[0024] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] The present invention will be further described below with reference to the embodiments.

[0026] Example strains Staphylococcus aureus ATCC 25923 was purchased from Beijing Beina Chuanglian Biotechnology Research Institute. The test strain was streaked onto beef extract peptone agar plates and stored at 4 ℃. Single colonies were picked from the beef extract peptone agar plates and passaged to prepare working cultures.

[0027] Main reagents Beef extract peptone agar medium and LB broth medium were purchased from Qingdao Haibo Biotechnology Co., Ltd. Food-grade dimethyl sulfoxide (DMSO) was used and purchased from Jinan Lige Reagent Co., Ltd. Caraway oil, 2,3-butanedione, strychnos nux-vomica oil, wild chrysanthemum oil, artemisia oil, perilla leaf oil, spearmint oil, thyme oil, methyl salicylate, ylang-ylang oil, ginger oil, and sweet almond oil were purchased from Lusheng Holdings (Jinan) Co., Ltd. The AKP detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0028] Instruments and equipment Electronic balance (YP20002), purchased from Yuyao Jinno Balance Instrument Co., Ltd.; analytical balance (CP114), purchased from Ohaus Instruments Shanghai Co., Ltd.; vertical pressure steam sterilizer (YXQ-50G), purchased from Shanghai Boxun Medical Biological Instrument Co., Ltd.; double-person single-sided clean bench (SW-CJ-2F), purchased from Jiangsu Fuhong Technology Co., Ltd.; ultraviolet spectrophotometer (V1100D), purchased from Shanghai Meipuda Instrument Co., Ltd.; vortex mixer (SCI-VS), purchased from SCILOGEX (USA); biological microscope (UB100i), purchased from Chongqing Aopu Optoelectronic Instrument Co., Ltd.; biochemical incubator (SPX-50B-Z), purchased from Shanghai Boxun Industrial Co., Ltd.; constant temperature incubator shaker (HNY-200B), purchased from Tianjin Ounuo Instrument Co., Ltd.

[0029] Test methods Turbidimetric assay for the inhibitory effect of antibacterial agents on Staphylococcus aureus Single colonies of Staphylococcus aureus cultured on the plate were transferred to LB broth and incubated at 37°C and 180 rpm for 12 h using a constant temperature shaker. The number of Staphylococcus aureus in the culture was determined by the plate spread method, and the bacterial count was adjusted to 10⁻⁶. 6 CFU / mL available for use.

[0030] In a sterile environment, 10 μL of the prepared bacterial culture was added to LB broth medium, followed by 5 μL of each antibacterial agent. The blank control group was replaced with LB broth medium supplemented with an equal volume of the small-molecule solubilizer dimethyl sulfoxide. After mixing, the mixture was incubated in a constant-temperature shaker at 37 ℃ (180 rpm) for approximately 12 h. The absorbance was measured at 600 nm using a UV spectrophotometer (V1100D, China).

[0031] Antibacterial agents with good antibacterial effects were selected, and each agent was designed with concentration gradients of 0.2 mL / L, 0.1 mL / L, 0.05 mL / L, and 0.025 mL / L. Staphylococcus aureus was cultured and inhibited using the method described above. The absorbance at 600 nm was measured and recorded using a UV spectrophotometer. Finally, the inhibition rate, virulence regression equation, and EC50 for each treatment were calculated.

[0032] .

[0033] Agar diffusion test Add 20 mL of beef extract peptone agar medium to each petri dish. After solidification, spread 100 μL of bacterial suspension evenly on the dish. Then, place sterile filter paper covered with different antimicrobial agents on the inoculated agar surface and incubate at 37 °C for 24 h. Finally, measure the diameter of the inhibition zone using the cross-hatching method to indicate the antimicrobial activity of the agent; dimethyl sulfoxide (DMSO) was used as a control. Three replicates were set for each group, and the average value was taken.

[0034] Staphylococcus aureus in vivo antibacterial experiment Apples, cantaloupes, tomatoes, and potatoes with no mechanical damage or rot were selected and soaked in a 2% (v) NaClO solution for 2 minutes. During this time, they were gently turned and rubbed by hand to ensure good sterilization. Then, they were thoroughly rinsed with running water to avoid sodium hypochlorite residue affecting subsequent experiments. After air-drying in a sterile room, the peels of the apples, cantaloupes, tomatoes, and potatoes were removed with a sterile knife, and the central tissue was cut into 1 × 1 × 1 cm pieces. 3 The chicken was cleaned and cut into cubes using a sterile knife. The prepared substrate was placed in a pre-sterilized plastic container (length × width × height = 180 × 130 × 70 cm), and 5 μL of bacterial solution (10...) was added to the surface of the substrate. 6(CFU / mL). The antibacterial agent was dripped onto sterile filter paper fixed to adhesive tape and fumigated. Finally, the plastic box was placed in a 37 ℃ observation room for 24 h. The total bacterial count was determined according to the method in GB4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count". Three replicates were set for each group, and the average value was taken.

[0035] Determination of the growth curve of Staphylococcus aureus Staphylococcus aureus cultured to the logarithmic growth phase (OD600 = 0.5) was diluted to 0.1 and then centrifuged at 8000 r / min for 10 min at 4 ℃. The supernatant was removed, the bacterial cells were collected, washed with sterile PBS, and resuspended in fresh LB broth. Different antibacterial agents were added, and the bacterial suspension was cultured in a shaker at 37 ℃ and 180 r / min. The OD600 values ​​at 0, 2, 4, 6, 8, 10, 12, 16, 20, and 24 h were measured using a microplate reader. Each group was set up in triplicate, and the average value was taken.

[0036] Morphological observation of Staphylococcus aureus Staphylococcus aureus was cultured to the logarithmic growth phase, then centrifuged at 8000 r / min for 10 min at 4 ℃. The supernatant was removed, and the bacterial cells were collected and washed twice with sterile PBS. The precipitate was resuspended in PBS solution, and finally an antibacterial agent was added. The mixture was then incubated at 37 ℃ and 180 r / min for 6 h with shaking. After incubation, the sample was centrifuged at 8000 r / min for 10 min, washed with PBS, and then fixed with 2.5% glutaraldehyde for 20 h. The sample was dehydrated stepwise using a series of ethanol solutions (30, 50, 70, 80, 90, 95, and 100%) and vacuum dried. After gold sputtering, the sample was observed using a scanning electron microscope.

[0037] Determination of the leakage content of nucleic acid and protein in Staphylococcus aureus First, Staphylococcus aureus was cultured to the logarithmic developmental phase. Then, it was centrifuged at 8000 r / min for 10 min at 4 ℃, the supernatant was removed, and the cells were collected. The cells were washed with PBS and resuspended, and the OD600 value of the bacterial culture was adjusted to 0.5. Different antibacterial agents were added. The bacterial cultures were then incubated at 37 ℃ with shaking at 180 r / min for 6 h. After centrifugation, the OD260 and OD280 of the supernatant were measured. Three replicates were set for each group, and the average value was taken.

[0038] Determination of bacterial extracellular relative conductivity A 1% Staphylococcus aureus bacterial suspension was added to 50 mL of LB broth and cultured at 37 ℃ and 180 r / min in a shaker until the logarithmic growth phase. After culture, the suspension was aliquoted into centrifuge tubes and centrifuged at 8000 r / min for 10 min at 4 ℃. The bacterial precipitate was washed with 5% glucose solution to prepare an isotonic solution. An antibacterial agent was added to the 5% glucose solution, and a control group was set up. The conductivity was measured using a conductivity meter and recorded as follows. L1 Simultaneously, the same concentration of antibacterial agent was added to the isotonic bacterial solution, and the conductivity was measured every 2 hours and recorded as follows. L2 The bacterial suspension in 5% glucose solution was boiled in a water bath for 5 minutes, cooled, and then its relative conductivity was measured and recorded as follows. L0 .

[0039] .

[0040] Determination of Staphylococcus aureus cell wall integrity Cell wall integrity is assessed by measuring the activity of alkaline phosphatase (AKP) released by Staphylococcus aureus into the culture medium. A Staphylococcus aureus bacterial suspension (10... 6 The supernatant (CFU / mL) was treated with an antibacterial agent for 6 h, centrifuged at 8000 rpm for 10 min at 4 ℃, and the activity of AKP enzyme in the supernatant was detected using an AKP kit.

[0041] Data processing and analysis One-way ANOVA and Bonferroni test were used to determine the significance of differences. P <0.05 indicates a significant difference. All experiments were repeated three times, and the error bars are expressed as mean ± standard deviation.

[0042] Results and Discussion Screening of antibacterial agents Staphylococcus aureus, as a typical Gram-positive pathogen, has its growth-inhibiting effect, which is one of the core indicators for evaluating the activity of antibacterial agents. From Figure 1It can be seen that different antibacterial agents exhibit significant substance-specific effects on the growth regulation of Staphylococcus aureus. Caraway oil and 2,3-butanedione showed the strongest inhibitory activity against the strain, with corresponding OD600 values ​​of only 0.0665 and 0.0685, respectively, a decrease of over 95% compared to the control group. Strychnos nux-vomica oil and wild chrysanthemum oil followed, both with inhibition rates exceeding 87%. The inhibitory activities of Artemisia annua oil, Perilla frutescens leaf oil, Spearmint oil, Thyme oil, and methyl salicylate decreased sequentially. Ylang-ylang oil, ginger oil, and sweet almond oil showed relatively weak inhibitory effects, but their OD600 values ​​were still lower than the control group, indicating that these substances still have a certain degree of inhibitory effect on the growth of Staphylococcus aureus.

[0043] The aforementioned differences may be related to the different target sites and active ingredients of different essential oils when exerting their antibacterial effects. For example, the main components of geranium essential oil, citronellol and isomenthone, can disrupt the membrane structure of microorganisms and affect the normal physiological metabolism of microbial cells. Perilla leaf essential oil is mainly composed of terpenes and aromatic compounds, which can damage the membrane structure and interfere with metabolism. Carvacrol and thymol in thyme essential oil can interact with the phospholipid bilayer on the bacterial cell membrane, leading to distortion of the physical structure and increasing membrane fluidity and permeability, thereby inhibiting bacterial growth. Caraway seed essential oil, 2,3-butanedione, and strychnine seed essential oil were selected as subjects for subsequent research.

[0044] EC 50 Calculation To more effectively control Staphylococcus aureus, antibacterial agents with good individual inhibitory effects are used in combination to achieve the goal of reducing dosage and increasing efficacy.

[0045] The concentrations of each single antibacterial agent listed in Table 1 were logarithmically converted, and the growth inhibition rate of Staphylococcus aureus was converted into a probability value and subjected to linear regression to obtain the results in Table 2. Table 2 shows the virulence regression equations for the inhibitory effects of three antibacterial agents—2,3-butanedione, caraway oil, and strychnine oil—on Staphylococcus aureus, and the EC50 calculated from the equations. 50 From R in Table 2 2 The virulence equations of the three antibacterial agents against Staphylococcus aureus showed strong correlations, reaching 0.9815, 0.9997, and 0.991, respectively. The EC50 values ​​of the three antibacterial agents... 50 Arranged by size: Strychnos nux-vomica oil (76 μL / L) > 2,3-butanedione (55 μL / L) = caraway oil (55 μL / L).

[0046] Table 3 shows that in the combination of strychnine essential oil and caraway essential oil, when the concentration ratio is 51:18, the co-toxicity factor F value reaches 50.8, indicating that the combination exhibits a synergistic effect in inhibiting the growth of Staphylococcus aureus. At this combined concentration, the theoretical inhibition rate of strychnine essential oil + caraway essential oil against Staphylococcus aureus is 49.6%, and the actual inhibition rate is 74.8%. This result indicates that when the concentration ratio of strychnine essential oil to caraway essential oil is 51:18, the combination of the two has the best synergistic effect in inhibiting the growth of Staphylococcus aureus.

[0047] Table 1. Growth inhibition rate of single antibacterial agents against Staphylococcus aureus

[0048] Table 2. Toxicity equations of three antibacterial agents against Staphylococcus aureus antibacterial agent Toxicological equations <![CDATA[EC 50 ]]> 2,3-Butanedione Y = 1.9675 X + 1.5682, R² = 0.9815 55 μL / L Caraway essential oil Y = 2.0142 X + 1.4939, R² = 0.9997 55 μL / L Strychnos nux-vomica essential oil Y = 2.4442 X + 0.3931, R² = 0.991 76 μL / L Table 3 Evaluation of the synergistic effect of the two antibacterial agents

[0049] Effects of compound essential oils on the growth of Staphylococcus aureus The size of the inhibition zone directly reflects the strength of the antibacterial activity of the antibacterial agent. The antibacterial effects of strychnine essential oil and caraway essential oil, both individually and in combination, on Staphylococcus aureus were shown in the following results. Figure 2 As shown in Figures A and B, the inhibition zone diameter in the blank group was only 0.7 cm, indicating that the growth of the strain was not inhibited in the absence of exogenous antibacterial substances. Under the action of single antibacterial agents, the inhibition zone diameters of 69 μL / L Strychnos nux-vomica oil and 69 μL / L Caraway oil were 1.13 cm and 1.24 cm, respectively, with no significant difference between the two, indicating that the inhibitory activity of the two single antibacterial agents against Staphylococcus aureus at this concentration was at the same level. However, after using a combination of 51 μL / L Strychnos nux-vomica oil and 18 μL / L Caraway oil, the inhibition zone diameter reached 1.92 cm, which was significantly higher than that of the two single antibacterial agents. P The concentration of the compound group was <0.05%, and the total antibacterial agent concentration was consistent with that of the single group, indicating that the combination of the two produced a significant synergistic antibacterial effect.

[0050] Inhibitory effect of compound essential oils on Staphylococcus aureus The antibacterial effects of strychnine essential oil and caraway essential oil, both individually and in combination, on Staphylococcus aureus in different food matrices, such as... Figure 3As shown in Figures A and B. In all matrices, the residual levels of Staphylococcus aureus in the control group were consistently high, indicating that the strain could stably proliferate in different matrices without the presence of an antibacterial agent. Under the action of a single antibacterial agent, the antibacterial effects of 69 μL / L Strychnos nux-vomica oil and 69 μL / L Caraway oil showed matrix specificity: in apples, the inhibitory effects of both were similar, with residual levels of 3.57 lg CFU / g and 3.53 lg CFU / g, respectively; in potatoes, chicken, cantaloupe, and tomatoes, the antibacterial activity of Caraway oil was slightly better than that of Strychnos nux-vomica oil, but the overall difference was small. The compound system of 51 μL / L Strychnos nux-vomica oil + 18 μL / L Caraway oil showed superior antibacterial effects in all matrices. For example, in chicken, the residual levels of the strain in the compound group were significantly lower than those in the control group and the single Strychnos nux-vomica oil group. P <0.05).

[0051] Effect of compound essential oils on the growth curve of Staphylococcus aureus Growth curves can clearly reflect the effect of antimicrobial agents on the microbial growth process. Figure 4 It was found that the Staphylococcus aureus in the control group exhibited typical logarithmic growth characteristics: it entered the logarithmic growth phase after 2 h of culture, and the OD600 value rapidly increased from 0.1 to 1.35 at 24 h, indicating that the strain could continue to proliferate when not inhibited. Under the action of single antibacterial agents, both 69 μL / L Strychnos nux-vomica oil and 69 μL / L Caraway oil could delay the growth of the strain, but the inhibitory effect was limited. At 24 h of culture, the OD600 value of the Strychnos nux-vomica oil group was 0.89, and that of the Caraway oil group was 0.7. Neither completely blocked the proliferation of the strain, but only reduced the growth rate.

[0052] The combination of 51 μL / L strychnos nux-vomica oil and 18 μL / L caraway oil showed a stronger and more sustained inhibitory effect on the growth of Staphylococcus aureus. Throughout the culture process, the OD600 value of the combination group remained at a low level, only 0.48 at 24 h, significantly lower than the control group and the single antibacterial agent group. In terms of growth stages, the combination group not only prolonged the lag phase of the strain (OD600 value of only 0.21 at 4 h of culture), but also significantly inhibited the proliferation rate in the logarithmic growth phase.

[0053] Effects of compound essential oils on the surface morphology of Staphylococcus aureus The morphological changes of Staphylococcus aureus were evaluated by SEM analysis. Figure 3Image A shows SEM images of Staphylococcus aureus. Staphylococcus aureus cells treated with a compound system of 51 μL / L Strychnos nux-vomica oil + 18 μL / L Caraway oil exhibited irregular wrinkles on their surface, accompanied by broken, adherent, and aggregated damaged cells or cell debris. Furthermore, these cells were not uniform in size or distribution. In contrast, the bacterial cells in the control group had a regular, spherical morphology, a smooth surface, and were uniform in size and distribution. These results indicate that the Strychnos nux-vomica oil-caraway oil compound disrupts cell membrane integrity, alters the morphology and structure of Staphylococcus aureus, and thus affects the normal growth and metabolism of the bacteria.

[0054] Effects of compound essential oils on the cell membrane integrity of Staphylococcus aureus The cell membrane is a natural protective barrier for bacteria, playing a crucial role in maintaining stable intracellular metabolism and regulating selective substance exchange. When the cell membrane is damaged, intracellular macromolecules leak out. As shown in Table 4, in the control group, the OD260 and OD280 values ​​of the extracellular fluid of Staphylococcus aureus were 0.088 and 0.186, respectively, which are at a low level, indicating that under normal physiological conditions, the cell membrane of the strain can maintain a low leakage state of intracellular nucleic acids and proteins. After treatment with a single antibacterial agent, the degree of cell membrane damage increased: the OD260 and OD280 values ​​of the 69 μL / L Strychnos nux-vomica oil group increased to 0.127 and 0.251, respectively; the corresponding values ​​of the 69 μL / L Caraway oil group were 0.131 and 0.299, respectively. This indicates that both single antibacterial agents can disrupt cell membrane integrity, but the degree of damage is limited.

[0055] After treatment with the compound system of 51 μL / L Strychnos nux-vomica oil + 18 μL / L Caraway oil, the extracellular fluid OD260 and OD280 values ​​increased significantly to 0.193 and 0.428, respectively, which were 2.19 times and 2.30 times that of the blank group, and much higher than those of the single antibacterial agent group.

[0056] Strychnos nux-vomica oil is a monoterpene alcohol that can insert into the phospholipid bilayer of the cell membrane through hydrophobic interactions, disrupting its fluidity and barrier function. The ketone components of caraway oil further aggravate oxidative damage to the membrane structure. The synergistic effect of the two significantly increases cell membrane permeability, ultimately leading to a large leakage of intracellular nucleic acids and proteins.

[0057] Table 4. Effects of the combined treatment of Strychnos nux-vomica oil and Caraway oil on the leakage of nucleic acid and protein from Staphylococcus aureus.

[0058] Effect of compound essential oils on the relative conductivity of Staphylococcus aureus The cell membrane's barrier function is fundamental for bacteria to maintain intracellular ion homeostasis, while changes in extracellular relative conductivity directly reflect the cell membrane's permeability to small molecule ions. Results are as follows... Figure 6 As shown, the extracellular relative conductivity of the control group remained at a low level throughout, only 13.2% after 10 h of culture, indicating that the cell membrane of the strain can effectively block the outflow of intracellular ions and maintain a low permeability state under normal physiological conditions.

[0059] After treatment with a single antibacterial agent, the cell membrane permeability showed a slow upward trend: the conductivity of the 69 μL / L Strychnos nux-vomica oil group was 21.1% after 10 h of culture, and that of the 69 μL / L Caraway oil group was 23.9%, indicating that both single antibacterial agents could mildly damage the cell membrane structure, but the rate and extent of ion leakage were limited.

[0060] Treatment with a compound system of 51 μL / L Strychnos nux-vomica oil + 18 μL / L Caraway oil resulted in a significant time-dependent increase in extracellular relative conductivity: after 2 h of culture, the conductivity reached 15.3%, significantly higher than that of the single antibacterial agent group at the same time point. P <0.05); after 10 h of cultivation, the conductivity increased to 45.5%, which was 3.45 times and 2.16 times that of the blank group and the strychnine essential oil group, respectively.

[0061] The results showed that the compound essential oils could disrupt bacterial cell membranes, increase their permeability, and lead to intracellular K+. + Na + The large outflow of small molecular ions eventually manifests as a rapid increase in relative conductivity.

[0062] Cell wall integrity AKP is an enzyme bound to the cell membrane, mainly distributed between the cell wall and the cell membrane. When the bacterial cell wall is damaged, AKP is released in large quantities. Therefore, AKP is an important indicator of bacterial cell wall integrity. Figure 7 As shown, the extracellular AKP activity in the blank group was only 0.67 U / L, which is at a low level, indicating that the cell wall of Staphylococcus aureus is intact under normal physiological conditions.

[0063] After treatment with a single antibacterial agent, the extracellular AKP activity increased to varying degrees: the AKP activity in the 69 μL / L Strychnos nux-vomica oil group increased to 0.93 U / L, and the AKP activity in the 69 μL / L Caraway oil group was 1.04 U / L, indicating that both single antibacterial agents could disrupt cell wall integrity, leading to the leakage of some AKP into the extracellular space.

[0064] The extracellular AKP activity of the compound system of 51 μL / L Strychnos nux-vomica oil + 18 μL / L Caraway oil significantly increased to 1.72 U / L, which was 2.57 times that of the control group and much higher than that of the two single antibacterial agent groups. Damage to the bacterial wall by antibacterial substances usually occurs in conjunction with cell membrane damage, but the antibacterial mechanisms of different antibacterial substances against different bacteria also differ.

[0065] in conclusion The antibacterial activity of 12 common antibacterial agents was systematically analyzed using turbidimetric assay. By comparing the OD600 values ​​of the antibacterial agents, single antibacterial agents with good antibacterial effects were identified, and the optimal compound ratio was further screened to obtain the best results. The results showed that caraway oil, 2,3-butanedione, and strychnine oil exhibited superior antibacterial effects against Staphylococcus aureus. The synergistic antibacterial effect of strychnine oil and caraway oil was screened using the co-toxicity factor method, and the optimal compound volume ratio was determined to be 51 μL / L : 18 μL / L.

[0066] In addition, the antibacterial effect of compound essential oils on different food matrices was investigated. The results showed that the compound system exhibited stable antibacterial effects in five different food matrices, including apples, potatoes, and chicken. The residual amount of bacterial strains was significantly lower than that in the single antibacterial agent treatment group, demonstrating good matrix adaptability.

[0067] The antibacterial mechanism of the compound essential oil was then investigated. The results showed that the compound essential oil effectively inhibited the logarithmic growth phase of Staphylococcus aureus during the 6 h treatment period and further reduced the upper limit of growth after 12 h of treatment. Membrane permeability increased, cell contents leaked out, and relative conductivity increased. The cell wall membrane was significantly damaged, and AKP enzyme activity increased. This was consistent with the results of SEM observation, which showed that bacterial cells shrank and ruptured.

[0068] In summary, this invention clarifies the antibacterial activity and mechanism of action of the compound system of strychnine essential oil and caraway essential oil, providing a theoretical basis and technical support for the application of natural antibacterial agents in the food field.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of nux vomica essential oil and caraway essential oil in the preparation of Staphylococcus aureus antibacterial agent. The concentration ratio of nux vomica essential oil to caraway essential oil in the antibacterial agent is 38-63:9-28.

2. A Staphylococcus aureus antibacterial essential oil composition, characterized in that: It includes nux vomica essential oil and caraway essential oil, with a concentration ratio of 38-63:9-28.

3. The Staphylococcus aureus antibacterial essential oil composition according to claim 2, characterized in that: In the antibacterial essential oil composition, the concentration ratio of nux vomica essential oil to caraway essential oil is 38-60:10-28.

4. The Staphylococcus aureus antibacterial essential oil composition according to claim 3, characterized in that: In the antibacterial essential oil composition, the concentration ratio of nux vomica essential oil to caraway essential oil is 38-55:15-28; Preferably, in the antibacterial essential oil composition, the concentration ratio of nux vomica essential oil to caraway essential oil is 45-55:15-20.

5. The Staphylococcus aureus antibacterial essential oil composition according to claim 4, characterized in that: In the antibacterial essential oil composition, the concentration of nux vomica essential oil is 45-55 μL / L, and the concentration of caraway essential oil is 15-20 μL / L. Preferably, in the antibacterial essential oil composition, the concentration of strychnine essential oil is 50-55 μL / L, and the concentration of caraway essential oil is 17-20 μL / L; Preferably, in the antibacterial essential oil composition, the concentration of strychnine essential oil is 51 μL / L and the concentration of caraway essential oil is 18 μL / L.

6. A Staphylococcus aureus antibacterial essential oil composition, characterized in that: It includes 2,3-butanedione and strychnine essential oil, with a concentration ratio of 25-50:10-40 or 6-10:60-65.

7. The Staphylococcus aureus antibacterial essential oil composition according to claim 6, characterized in that: In the antibacterial essential oil composition, the concentration ratio of 2,3-butanedione to strychnine essential oil is 40-50:10-15, or 8-10:62-64.

8. A Staphylococcus aureus antibacterial essential oil composition, characterized in that: It includes 2,3-butanedione and caraway essential oil, with a concentration ratio of 25-50:8-30 or 6-10:40-50. Preferably, in the antibacterial essential oil composition, the concentration ratio of 2,3-butanedione to caraway essential oil is 40-50:8-10, or 6-10:45-50.

9. The use of the antibacterial essential oil composition according to any one of claims 2-8 as an antibacterial additive in food.

10. An antibacterial spray, characterized in that: It is prepared from the antibacterial essential oil composition according to any one of claims 2-8.

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