Antibacterial essential oil composition for escherichia coli and application thereof

Abstract

The invention belongs to the technical field of antibacterial essential oil, and discloses an antibacterial essential oil composition for escherichia coli and application of the antibacterial essential oil composition, 2, 3-butanedione and geranium essential oil are subjected to compounding treatment, so that the antibacterial essential oil composition has synergistic antibacterial activity on escherichia coli at low concentration, and growth of escherichia coli in various food matrixes can be effectively inhibited.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bacteriostatic essential oil, and particularly relates to a bacteriostatic essential oil composition for E. coli and application thereof. BACKGROUND

[0002] The statements herein are provided only to enhance understanding of the present application and are not necessarily intended to constitute the prior art.

[0003] E. coli is a gram-negative bacillus that can cause gastrointestinal infections, urinary tract infections, and other local tissue and organ infections. It can survive on the surface of agricultural products for a long time, and disinfection is considered a key step to reduce micro-organic pollutants in agricultural products to ensure safety. Chemical synthetic preservatives are widely used in the food industry to inhibit microbial growth, inhibit enzyme activity, and prevent oxidation. However, chemical preservatives are generally quite toxic and can even cause deformities and cancer.

[0004] Natural compounds such as plant essential oils have been widely used in food preservation due to their antibacterial activity. However, the use of single plant essential oils in antibacterial applications often requires a large amount, which has a strong odor and can cause sensory discomfort in people, reducing consumer acceptance. Moreover, the higher the concentration, the higher the cost. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application aims to provide a bacteriostatic essential oil composition for E. coli and application thereof.

[0006] To achieve the above-mentioned purpose, the present application is realized by the following technical solutions: In a first aspect, the present application provides a bacteriostatic essential oil composition for E. coli, comprising 2,3-butanedione and pelargonium essential oil, the concentration of 2,3-butanedione being 5-100 μL / L, the concentration of pelargonium essential oil being 5-100 μL / L, and the volume ratio of 2,3-butanedione to pelargonium essential oil being 1-20:35-80 or 35-50:5-25.

[0007] The solvent of the essential oil composition can be Tween 80, lecithin, PEG-40 hydrogenated castor oil, plant emulsifying wax, etc.

[0008] In some embodiments, the volume ratio of 2,3-butanedione to pelargonium essential oil is 35-45:10-22.

[0009] Preferably, the volume ratio of 2,3-butanedione to pelargonium essential oil is 37-42:15-20.

[0010] Further preferably, the volume ratio of 2,3-butanedione to pelargonium essential oil is 38-40:18-20.

[0011] More preferably, the concentration of 2,3-butanedione is 38-40 μL / L, and the concentration of pelargonium essential oil is 18-20 μL / L.

[0012] In some embodiments, the volume ratio of 2,3-butanedione and pelargonium essential oil is 5-15:35-50.

[0013] Preferably, the volume ratio of 2,3-butanedione and pelargonium essential oil is 7-12:40-50.

[0014] More preferably, the concentration of 2,3-butanedione is 7-12 μL / L, and the concentration of pelargonium essential oil is 40-50 μL / L.

[0015] More preferably, the concentration of 2,3-butanedione is 7-12 μL / L, and the concentration of pelargonium essential oil is 45-50 μL / L.

[0016] Specifically, the concentration of 2,3-butanedione is 10 μL / L, and the concentration of pelargonium essential oil is 48 μL / L.

[0017] In a second aspect, the present application provides the use of the bacteriostatic essential oil composition as a food bacteriostatic additive.

[0018] In a third aspect, a bacteriostatic spray is prepared from the bacteriostatic essential oil composition.

[0019] The beneficial effects achieved by one or more embodiments of the present application are as follows: Natural compounds represented by plant essential oils have significant antibacterial activity, but the use amount of a single plant essential oil in antibacterial applications is often large, has a strong odor, and is easy to cause people to feel uncomfortable. Through research on the bacteriostatic effect of 12 single bacteriostatic agents on Escherichia coli, and using the co-toxic factor method for compounding treatment to improve the bactericidal effect. The results show that when low-concentration 2,3-butanedione and low-concentration pelargonium essential oil are used in combination, they have excellent synergistic antibacterial properties against Escherichia coli. Compared with single 2,3-butanedione and pelargonium essential oil, the inhibition effect of the compounded essential oil on Escherichia coli is more obvious, which can achieve bacteriostasis by destroying the integrity of the cell wall and cell membrane, increasing the permeability of the cell membrane, leading to the leakage of intracellular proteins, nucleic acids, and other components, accompanied by the increase of extracellular alkaline phosphatase activity and the relative conductivity of bacterial suspension; at the same time, the scanning electron microscope results show that the compounding treatment can cause the bacterial morphology to appear serious shrinkage, concave distortion and rupture.

[0020] 2,3-butanedione is a naturally occurring and volatile alpha-diketone. Because of its aroma and flavor, it is often used as a food additive and is an important component of food flavorings. Geranium essential oil (GEO) is extracted from leaves, flowers and stems by distillation. Geranium essential oil is widely used in the formulation of perfumes, cosmetics and other fragrance products. Therefore, when the two active ingredients are synergistically added at a low dose, the sensory quality of the food is little affected, and the cost is low when added at a low concentration.

[0021] In addition to having a fragrance, geranium essential oil has certain biological activities, such as antibacterial activity, antioxidant capacity and anti-inflammatory activity. BRIEF DESCRIPTION OF DRAWINGS

[0022] The drawings constituting a part of the specification integrate to provide further understanding of the application, and the illustrative embodiments of the application and their description serve to explain the application without constituting an improper limitation of the application.

[0023] Figure 1 is a comparison chart of the in vitro inhibition effects of various bacteriostatic agents on E. coli; Figure 2 is a comparison chart of the bacteriostatic effects of 2,3-butanedione and geranium essential oil complex treatment on E. coli; Figure 3 is a comparison chart of the inhibition effects of 2,3-butanedione and geranium essential oil complex treatment on E. coli in different food matrices; Figure 4 is a comparison chart of the effects of 2,3-butanedione and geranium essential oil complex treatment on the growth curve of E. coli; Figure 5 is a comparison chart of the effects of 2,3-butanedione and geranium essential oil complex treatment on the surface morphology of E. coli; Figure 6 is a comparison chart of the effects of 2,3-butanedione and geranium essential oil complex treatment on the relative conductivity of E. coli; Figure 7 is a comparison chart of the effects of 2,3-butanedione and geranium essential oil complex treatment on the AKP exudation amount of E. coli. DETAILED DESCRIPTION

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

[0025] The present application will be further described below in conjunction with examples.

[0026] Materials and reagents Test strain: *Escherichia coli* (ATCC 25922), purchased from Beijing Beina Chuanglian Biotechnology Research Institute. The strain was stored in glycerol at -80 ℃.

[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 purchased from Jinan Lige Reagent Co., Ltd.; geranium essential oil, 2,3-butanedione, nerol, wild chrysanthemum essential oil, artemisia essential oil, perilla leaf essential oil, lemongrass essential oil, thyme essential oil, methyl salicylate, litsea cubeba essential oil, ginger essential oil, and spearmint essential 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 Name Model Place of origin or manufacturer analytical balance CP114 Ohaus Instruments (Shanghai) Co., Ltd. vertical pressure steam sterilizer YXQ-50G Shanghai Boxun Medical Biotech Instrument Co., Ltd. double single-side clean bench SW-CJ-2F Jiangsu Fuhong Technology Co., Ltd. biological microscope UB100i Chongqing Aupu Optoelectronic Instrument Co., Ltd. biochemical incubator SPX-250B-Z Shanghai Boxun Industry Co., Ltd. constant temperature incubation shaker HNY-200B Tianjin Ouno Instruments & Meters Co., Ltd. ultraviolet spectrophotometer V1100D Shanghai Mettler Toledo Instrument Co., Ltd. Test method Bacterial culture The lyophilized bacterial strains were activated according to the relevant ATCC operating guidelines. The lyophilized bacteria were inoculated into 100 mL of LB broth medium and then placed in a constant-temperature shaker at 37 ℃ and 180 rpm for 12 h. After activation, a small amount of the activated bacterial solution was streaked onto beef extract peptone agar medium using an inoculation needle and incubated at 37 ℃ for 12 h. Once single colonies appeared, the strains were stored at 4 ℃ for later use.

[0029] Determination of inhibition effect of bacteriostatic agent by turbidimetry First, take 5 μL of each of the 12 antibacterial agents and add them to 1% dimethyl sulfoxide. Then add 10 mL of LB broth medium, mix well, and then add 10 μL of bacterial suspension (10... 6 The culture was then placed in a constant temperature shaker at 37 °C (180 rpm) for 12 h. Antibacterial agents with good antibacterial effects were selected, and concentration gradients (0.2 mL / L, 0.10 mL / L, 0.05 mL / L, and 0.025 mL / L) were designed. The above steps were repeated to inhibit *E. coli*. Finally, the absorbance was measured at 600 nm using a UV spectrophotometer (V1100D, China) to calculate the inhibition rate. An equal volume of LB broth containing 1% dimethyl sulfoxide was added to the control group.

[0030] .

[0031] Selection and evaluation of bacteriostatic agent synergy EC50 Calculation The mass concentration of the antibacterial agent in LB broth medium was converted to logarithm. The corresponding probability values ​​were obtained from a biostatistical probability conversion table based on the bacterial growth inhibition rate. A linear regression was performed with the logarithm of the concentration as the x-axis and the probability values ​​from the table as the y-axis to obtain the antibacterial virulence equation. The effective median concentration (EC50) of each agent against *E. coli* was then calculated based on the virulence equation. 50 ).

[0032] Compound scheme design The optimal combination for inhibiting Escherichia coli growth was determined by combining 2,3-butanedione, geranium essential oil, and nerol in pairs. 50 Let a and b be the two components, respectively. Utilizing the additive effect in the co-toxicity factor method, five compound ratios were set at six equal points: 5a / 6+b / 6, 2a / 3+b / 3, a / 2+b / 2, a / 3+2b / 3, and a / 6+5b / 6. First, each antibacterial agent EC was prepared separately. 50 The solutions of different concentrations were then prepared and added to beef extract peptone liquid culture medium at volume ratios of 5:1, 4:2, 3:3, 2:4, and 1:5, respectively. The absorbance was measured at 600 nm using a UV spectrophotometer.

[0033] Evaluation of synergistic effect The co-toxicity factor method was used to evaluate whether the compound combination had a synergistic effect.

[0034] % In the above formula: F: co-toxic factor; M: actual inhibition rate of the mixed antibacterial agent (%); m: theoretical inhibition rate of the mixed antibacterial agent (%).

[0035] This method compares the actual inhibition rate obtained from the compound combination experiment with the calculated theoretical inhibition rate, and uses the co-toxicity factor F value to evaluate whether the combination of binary antibacterial agents has a synergistic effect in inhibiting the growth of Escherichia coli. When F>20, it indicates that the two mixed antibacterial agents have a synergistic effect; F≤-20, it indicates that the combination has an antagonistic effect; F between -20 and 20 indicates that the two mixed antibacterial agents have an additive effect.

[0036] Determination of inhibition zone diameter The antibacterial activity of each antibacterial agent against *Escherichia coli* was determined using the filter paper disc method. 0.1 mL of a 1×10⁻⁶ solution was taken. 6A bacterial suspension of CFU / mL was spread evenly on beef extract peptone agar medium using a sterile spreader to prepare bacterial plates. Sterile filter paper with different antibacterial agents was placed stably in the center of each petri dish. Dimethyl sulfoxide was used as a blank control (CK). Each plate was replicated in triplicate. After incubation at 37 °C for 24 h, the diameter of the inhibition zone was measured using the cross-sectional method.

[0037] In vivo bacteriostatic experiment Wash the apples, tomatoes, cantaloupe, and potatoes in sequence with tap water and sterile water. After air-drying them in a sterile room, remove the peels from the apples, tomatoes, cantaloupe, and potatoes with a sterile knife, and take the middle tissue portion, cutting it into 1×1×1cm pieces. 3 Cut the chicken into cubes using a sterile knife after washing. Place the prepared food substrate into a pre-sterilized plastic container (length × width × height = 180 × 130 × 70 cm), and aspirate 5 μL of bacterial solution (10) onto the substrate surface. 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 an observation room at 37 ℃ for 24 hours. 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".

[0038] Determination of E. coli growth curve Escherichia coli cultured to the logarithmic growth phase was diluted to 0.1% with 0.9% physiological saline, then centrifuged at 8000 r / min for 10 min at 4 °C. The supernatant was removed, and the bacterial cells were collected. The cells were washed with sterile PBS, and the E. coli suspension was inoculated into LB broth at an inoculation rate of 1% (V / V). Antimicrobial agents were added, and 1% DMSO was used as a blank control. The culture was incubated in a constant temperature shaker at 37 °C and 180 r / min. Sampling was performed at 0, 2, 4, 6, 8, 10, 12, 16, 20, and 24 h. The absorbance of the collected bacterial suspensions was measured at 600 nm, and growth curves were plotted.

[0039] Scanning electron microscope (SEM) analysis of E. coli To further investigate the morphological changes of *E. coli*, SEM analysis was performed. *E. coli* was cultured in LB broth medium with different antibacterial agents added to the bacterial suspension, and incubated at 37 °C in a shaker for 12 h. After incubation, the bacterial suspension was centrifuged at 4 °C and 8000 r for 10 min. The precipitate was washed twice with 0.1 M phosphate-buffered saline (PBS, pH 7.4). Bacterial cells were fixed in 2.5% glutaraldehyde at 4 °C for 12 h. Samples were dehydrated in sequential fractions of ethanol (30%, 50%, 80%, 90%, 100%) at 15-min intervals, followed by a final 30-min isovalerate wash. Finally, all samples were sputter-coated with gold for 2 min in an ion coater and then observed using a scanning electron microscope.

[0040] Cell membrane integrity Cell membrane integrity is assessed by measuring the extent to which cellular components (including nucleic acids and proteins) are released into the cell suspension. 5 mL of overnight cultured *E. coli* (10...) 6 The sample was transferred (CFU / mL) to a sterile centrifuge tube, centrifuged to obtain a precipitate, and washed three times with sterile PBS (phosphate-buffered saline). Different antibacterial agents were added. The samples were incubated at 37 °C and 180 r / min on a shaker for 6 h. After incubation, the supernatant was collected by centrifugation. The blank control group for each group was replaced with LB broth medium supplemented with an equal volume of the small molecule solubilizer DMSO. The absorbance values ​​at 260 nm (nucleic acid) and 280 nm (protein) wavelengths were measured using a UV spectrophotometer.

[0041] Relative conductivity determination Escherichia coli was cultured to the logarithmic growth phase and centrifuged at 8000 r / min for 10 min at 4 ℃. The bacterial precipitate was washed with 5% glucose solution and resuspended. The bacterial suspension was then treated in a boiling water bath for 5 min, cooled, and the relative conductivity was measured and recorded as L0. Different antibacterial agents were mixed with an equal volume of 5% glucose solution or the above bacterial suspension, and cultured in a shaker at 37 ℃ and 180 r / min. The measured conductivity values ​​were L2 and L1, respectively. Each experiment was conducted in triplicate.

[0042] .

[0043] Cell wall integrity determination Escherichia coli 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 cells were resuspended in fresh LB broth, and an antibacterial agent was added. The culture was then incubated at 37 ℃ and 180 r / min for 6 h with shaking. A 1 mL sample was taken, centrifuged at 12000 r / min for 5 min, and the supernatant was collected for assay using an AKP activity detection kit.

[0044] Data processing and analysis This paper uses SPSS (version 22.0) and Origin (version 9.5) to perform statistical analysis on the obtained data. One-way ANOVA and Bonferroni test were used to determine significant differences. P <0.05 indicates a significant difference. All experiments in this invention were repeated three times, and the error bar represents the mean ± standard deviation.

[0045] Results and analysis Inhibition effect of 12 bacteriostatic agents on E. coli growth The antibacterial activity of 12 antibacterial agents against Escherichia coli is as follows: Figure 1 As shown in the figure. After 12 hours of cultivation, all 12 antibacterial agents inhibited the growth of *Escherichia coli* compared to the control group. The antibacterial activities of the different agents showed significant differences. Geranium essential oil exhibited the best antibacterial effect, with an inhibition rate of approximately 98% against *E. coli*, followed by nerol essential oil and 2,3-butanedione, with inhibition rates of 90% and 83%, respectively. The inhibition rates of other antibacterial agents against *E. coli* were all below 50%. Therefore, geranium essential oil, nerol essential oil, and 2,3-butanedione were selected for further investigation of their antibacterial effects against *E. coli*.

[0046] Calculation of EC 50 Bacteriostatic agent The concentrations of each single antibacterial agent listed in Table 1 were logarithmically converted, and the *E. coli* growth inhibition rate was converted into a probability value and linearly regressed to obtain the results in Table 2. Table 2 shows that the concentration of each plant essential oil is highly correlated with its inhibitory effect on *E. coli*, with the inhibition rate significantly increasing with increasing concentration. The concentrations of nerol, geranium essential oil, and 2,3-butanedione showed high correlations with their antibacterial effects, with correlation coefficients of 0.9868, 0.977, and 0.9983, respectively. The EC50 values ​​for nerol, geranium essential oil, and 2,3-butanedione were 83, 57, and 58 μL / L, respectively. Based on the EC50 values, the antibacterial strength of the single antibacterial agents against *E. coli* was in the order of geranium essential oil > 2,3-butanedione > nerol.

[0047] Table 1. Growth inhibition rate of single antibacterial agents against Escherichia coli

[0048] Table 2. Toxicity equations of three antibacterial agents against Escherichia coli Toxicology equation 2,3-butanedione EC 50 ]]> Y = 2.0948X + 1.2988, R² = 0.9983 58 μL / L Geranium essential oil Y = 3.9548X - 1.9542, R² = 0.977 57 μL / L Nerol Y = 2.2976X + 0.5931, R² = 0.9868 83 μL / L Synergistic effect of compound bacteriostatic agent on E. coli Antibacterial activity of compound essential oil Based on the determination of the inhibitory effects of each antibacterial agent on Escherichia coli, the synergistic effect of each essential oil blend was determined using the co-toxicity factor method. Table 3 shows that when 2,3-butanedione and nerol were combined, the actual inhibition rates of both on Escherichia coli growth were significantly lower than the theoretical inhibition rates, exhibiting an antagonistic effect.

[0049] However, when geranium essential oil was combined with 2,3-butanedione and nerol, the actual antibacterial rate was significantly higher than the theoretical rate. For example, when the concentrations of 2,3-butanedione and geranium essential oil were 39 μL / L + 19 μL / L and 10 μL / L + 48 μL / L, respectively, the co-toxicity factor was greater than 20, indicating that the two mixed antibacterial agents had a synergistic effect. Similarly, when the concentrations of nerol and geranium essential oil were 69 μL / L + 10 μL / L, 28 μL / L + 38 μL / L, and 14 μL / L + 48 μL / L, respectively, the co-toxicity factor was also greater than 20, indicating that the two mixed antibacterial agents had a synergistic effect. The combination with the highest co-toxicity factor was selected for subsequent experiments.

[0050] Table 3 Evaluation of the synergistic effect of the two antibacterial agents

[0051] Figure 2 The size of the inhibition zone directly reflects the inhibitory effect of the antibacterial agent on Escherichia coli. In vivo inhibition effect of compound essential oil It can be seen that the diameter of the inhibition zone in the blank group was only 0.7 cm, indicating that the growth of Escherichia coli was not inhibited when no exogenous antibacterial substance was present. The diameter of the inhibition zone in the 58 μL / L 2,3-butanedione group was 1.13 cm, and the diameter of the inhibition zone in the 58 μL / L geranium essential oil group was 1.22 cm. Both showed certain antibacterial activity, but the effect was relatively limited.

[0052] The combination of 10 μL / L 2,3-butanedione and 48 μL / L geranium essential oil showed an inhibition zone diameter of 1.88 cm, significantly higher than that of the two single antibacterial agent groups and the control group. This result indicates that the combination of 2,3-butanedione and geranium essential oil exhibits a synergistic antibacterial effect.

[0053] Figure 3 The antibacterial effects of 2,3-butanedione, geranium essential oil, and their single and combined effects on Escherichia coli in different food matrices are as follows: Effect of compound essential oil on E. coli growth curve As shown in the figure, the E. coli colony count in the control group was at a relatively high level in all matrices, reaching 5.35 lg CFU / g in the chicken matrix, indicating that E. coli can proliferate in large quantities in different matrices under natural conditions. The inhibitory effect of the single antibacterial agent groups on E. coli showed matrix specificity. Specifically, the residual amount of chicken in the 58 μL / L 2,3-butanedione group was 5.02 lg CFU / g, while that in the 58 μL / L geranium essential oil group was 4.19 lg CFU / g, indicating a difference in the degree of inhibition of E. coli in chicken by the two groups.

[0054] In matrices such as apples and potatoes, although the colony counts of the single antibacterial agent group were lower than those of the control group, the reduction was relatively limited. In contrast, the compound antibacterial agent group showed more significant antibacterial activity in all matrices: for example, in the chicken matrix, the residue of the compound group was 4.19 lg CFU / g, significantly lower than the control group and the single 2,3-butanedione group; in the cantaloupe matrix, the compound group was 4.15 lg CFU / g, lower than the control group and the single antibacterial agent group. Furthermore, the antibacterial effect of the compound group was relatively stable in different matrices, while the activity of the single antibacterial agent group was more significantly affected by the matrix.

[0055] Figure 4 Growth curves can be used to further analyze the antibacterial activity of the antibacterial agent against *Escherichia coli*. Effect of compound essential oil on E. coli surface morphology As shown, the blank group exhibited no growth inhibition, with its OD600 continuously increasing with culture time, reaching 1.36 at 24 h, reflecting the normal growth process of logarithmic proliferation and stationary phase. In contrast, among the groups treated with single antibacterial agents, the growth rates of the 58 μL / L 2,3-butanedione group and the 58 μL / L geranium essential oil group were both slowed to varying degrees, with OD600 values ​​of 0.95 and 0.84 at 24 h, respectively. Although they could inhibit the proliferation of Escherichia coli, the effect was relatively limited.

[0056] In contrast, the combination of 10 μL / L 2,3-butanedione and 48 μL / L geranium essential oil showed a stronger inhibitory effect, with a 24-h OD600 of only 0.65, significantly lower than the blank group and the single antibacterial agent group. It can be concluded that the addition of essential oils mainly inhibited the division and proliferation of Escherichia coli during the logarithmic growth phase, resulting in a significant decrease in the number of viable bacteria, preventing them from reaching their normal growth peak. Furthermore, after treatment with the combined essential oils, the inhibitory effect on Escherichia coli was more pronounced due to the synergistic effect of 2,3-butanedione and geranium essential oil.

[0057] Figure 5 To more intuitively demonstrate the mechanism of action of the combined treatment group on *E. coli*, scanning electron microscopy (SEM) was used to observe the changes in the ultrastructure of *E. coli*. The SEM characterization results of *E. coli* are as follows: Effect of compound essential oil on E. coli cell membrane integrity As shown, the E. coli cells in the CK group exhibited typical bacillus morphology, with uniform size distribution, smooth and plump appearance, no cracks, intact structure, and were in a normal growth state.

[0058] After treatment with the compound, *E. coli* exhibited a rough, flattened morphology, with severe shrinkage, depressions, and distortions, leading to aggregation and adhesion. These observations further illustrate the bactericidal effect of the compound of 2,3-butanedione and geranium essential oil by disrupting the cell membrane and thus inhibiting cell growth.

[0059] Effect of compound essential oil on E. coli relative conductivity The cell membrane is a vital barrier protecting cells. When the cell membrane is damaged, small molecules are released first, followed by large molecules. Therefore, the extravasation of intracellular substances is a good indicator of cell membrane integrity.

[0060] As shown in Table 4, the OD260 and OD280 of E. coli in the blank group were 0.069 and 0.208, respectively, indicating that the leakage of intracellular substances was low under normal physiological conditions. After treatment with a single antibacterial agent, the leakage of intracellular substances of E. coli increased to varying degrees. Furthermore, the nucleic acid and soluble protein contents of the 2,3-butanedione-only treatment group and the geranium essential oil-only treatment group were significantly higher than those of the control group, indicating that both single antibacterial agents could disrupt cell membrane integrity.

[0061] After 6 hours of treatment with a combination of 2,3-butanedione and geranium essential oil, the contents of nucleic acid and soluble protein were 2.56 times and 2.65 times higher than those of the control group, respectively, with OD values ​​reaching 0.177 and 0.552, significantly higher than the blank group and the single antibacterial agent group. This result indicates that the combined antibacterial agent has a stronger damaging effect on the cell membrane. The antibacterial mechanism of 2,3-butanedione mainly involves transcriptionally downregulating phospholipid metabolism-related genes and directly binding to choline kinase PuCKI1, thereby disrupting cell membrane integrity, inducing lipid peroxidation, and causing intracellular leakage.

[0062] Table 4. Effects of the combination of 2,3-butanedione and geranium essential oil on nucleic acid and protein leakage in Escherichia coli.

[0063] Cell wall integrity When the cell membrane is damaged, it leads to disruption of various metabolic activities within the cell. Intracellular electrolytes, such as calcium... 2 + K + Na +Homeostasis is crucial for maintaining normal osmotic pressure and acid-base balance within cells. Disruption of the cell membrane leads to electrolyte leakage and an increase in conductivity. Therefore, changes in cell membrane permeability can be reflected by measuring changes in the conductivity of bacterial suspensions.

[0064] The results showed that the extracellular relative conductivity of E. coli in the blank control group remained at a low level, only 11.3% at 10 h, indicating that the cell membrane can effectively prevent ion outflow under normal physiological conditions. After treatment with a single antibacterial agent, the extracellular relative conductivity showed a slow upward trend: the conductivity of the 58 μL / L 2,3-butanedione group was 21.8% at 10 h, and that of the 58 μL / L geranium essential oil group was 25.7%, suggesting that both single antibacterial agents could mildly damage the cell membrane integrity, leading to the leakage of some intracellular ions, but the damage process was relatively mild.

[0065] After treatment with the compound antibacterial agent, the extracellular relative conductivity of Escherichia coli showed a rapid increase: the conductivity reached 10.4% after 2 hours of culture, significantly higher than that of the single antibacterial agent group at the same time point; after 10 hours, the conductivity rose to 47.7%, far higher than that of the blank group and the single antibacterial agent group. The results indicate that the compound antibacterial agent significantly disrupts the bacterial cell membrane and enhances its permeability.

[0066] Figure 7 The cell wall is a unique cellular structure that sustains bacterial cells; these structures are absent in the human body, making them an ideal target for antibacterial agents. Bacterial AKP is typically located between the cell wall and the cell membrane. Unless the bacterial cell wall is disrupted, the activity of this enzyme cannot be detected in the extracellular environment. Therefore, the degree of cell wall damage can be explained by the amount of intracellular AKP leaking into the bacterial suspension.

[0067] like ​ As shown, the extracellular AKP activity of *E. coli* in the control group was only 0.83 U / L. After treatment with a single antibacterial agent, the extracellular AKP activity increased to varying degrees: the AKP activity in the 58 μL / L 2,3-butanedione group increased to 1.03 U / L, and the AKP activity in the 58 μL / L geranium essential oil group increased to 1.12 U / L, indicating that both single antibacterial agents could disrupt cell wall integrity to some extent, leading to the leakage of some AKP into the extracellular space. After treatment with a combination of 10 μL / L 2,3-butanedione and 48 μL / L geranium essential oil, the extracellular AKP activity of *E. coli* significantly increased to 1.77 U / L, which was much higher than that in the control group and the single antibacterial agent group.

[0068] Generally, antibacterial substances exert their inhibitory effect on bacteria through multiple pathways, including disrupting cell membrane permeability and integrity, and inhibiting the synthesis of substances within the bacteria. This invention reveals that a combination of 2,3-butanedione and geranium essential oil can significantly affect the cell membrane structure of *Escherichia coli*.

[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. An antibacterial essential oil composition for Escherichia coli, characterized in that: It includes 2,3-butanedione and geranium essential oil, with the concentration of 2,3-butanedione being 5-100 μL / L and the concentration of geranium essential oil being 5-100 μL / L. The volume ratio of 2,3-butanedione to geranium essential oil is 1-20:35-80, or 35-50:5-25.

2. The antibacterial essential oil composition for Escherichia coli according to claim 1, characterized in that: The volume ratio of 2,3-butanedione to geranium essential oil is 35-45:10-22.

3. The antibacterial essential oil composition for Escherichia coli according to claim 2, characterized in that: The volume ratio of 2,3-butanedione to geranium essential oil is 37-42:15-20.

4. The antibacterial essential oil composition for Escherichia coli according to claim 3, characterized in that: The volume ratio of 2,3-butanedione to geranium essential oil is 38-40:18-20; Preferably, the concentration of 2,3-butanedione is 38-40 μL / L, and the concentration of geranium essential oil is 18-20 μL / L.

5. The antibacterial essential oil composition for Escherichia coli according to claim 1, characterized in that: The volume ratio of 2,3-butanedione to geranium essential oil is 5-15:35-50.

6. The antibacterial essential oil composition for Escherichia coli according to claim 5, characterized in that: The volume ratio of 2,3-butanedione to geranium essential oil is 7-12:40-50.

7. The antibacterial essential oil composition for Escherichia coli according to claim 6, characterized in that: The concentration of 2,3-butanedione was 7-12 μL / L, and the concentration of geranium essential oil was 40-50 μL / L.

8. The antibacterial essential oil composition for Escherichia coli according to claim 7, characterized in that: The concentration of 2,3-butanedione was 7-12 μL / L, and the concentration of geranium essential oil was 45-50 μL / L. Preferably, the concentration of 2,3-butanedione is 10 μL / L and the concentration of geranium essential oil is 48 μL / L.

9. The use of the antibacterial essential oil composition according to any one of claims 1-8 as an antibacterial food additive.

10. An antibacterial spray, characterized in that: It is prepared from the antibacterial essential oil composition according to any one of claims 1-8.

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