A compound antibacterial agent, its preparation method and application

Abstract

This invention belongs to the field of food technology and discloses a compound antibacterial agent, its preparation method, and its application. The compound antibacterial agent is obtained by compounding garlic essential oil nanoemulsion and ε-polylysine solution. The garlic essential oil nanoemulsion is an oil-in-water type, obtained by encapsulating garlic essential oil in the oil-in-water type using ultrasonic emulsification technology. This invention uses an emulsifier to encapsulate garlic essential oil using ultrasonic emulsification technology, which not only effectively improves the stability of garlic essential oil but also improves its solubility and application feasibility in high-moisture food systems. Furthermore, based on the fence theory, by adding ε-polylysine solution to the garlic essential oil nanoemulsion, the activity of garlic essential oil can be effectively enhanced, thereby greatly reducing the amount of garlic essential oil added in practical applications and avoiding any impact on the original flavor of the food.

Description

Technical Field

[0001] This invention belongs to the field of food technology, and specifically relates to a compound antibacterial agent, its preparation method, and its application. Background Technology

[0002] Food is highly susceptible to microbial contamination and spoilage during processing, transportation, and storage, leading to economic losses and even foodborne illnesses. Currently, most traditional food preservatives are chemically synthesized, and long-term, excessive use can not only easily induce food poisoning incidents but also pose potential risks such as carcinogenicity and teratogenicity, seriously endangering human health.

[0003] Natural antibacterial agents, characterized by their green and safe properties, are gradually becoming a focus of attention in related fields. Plant essential oils, as stimulating metabolites carrying special aromatic odors, have significant antibacterial activity and are highly promising natural antibacterial agents. Among them, garlic essential oil, a pale yellow oily liquid extracted from garlic bulbs, is inexpensive and readily available. It can effectively inhibit the growth of various foodborne pathogens and spoilage bacteria. However, it is highly volatile, poorly water-soluble, and when used in excessive amounts, its strong garlic odor can affect the original flavor of food. Therefore, there is an urgent need to seek effective stabilization and antibacterial enhancement technologies. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a compound antibacterial agent, its preparation method and application, to solve the technical problems that exist when garlic essential oil is used as a food preservative, such as poor water solubility, high volatility, and large dosage when used alone, which can easily affect the original flavor of food.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a compound antibacterial agent, which is obtained by compounding garlic essential oil nanoemulsion and ε-polylysine solution; wherein, the garlic essential oil nanoemulsion is an oil-in-water garlic essential oil nanoemulsion, which is obtained by encapsulating garlic essential oil using ultrasonic emulsification technology.

[0007] Furthermore, the particle size of the garlic essential oil nanoemulsion is 123.59-187.25 nm.

[0008] Furthermore, the preparation process of the garlic essential oil nanoemulsion is as follows:

[0009] Garlic essential oil, emulsifier and water are mixed to obtain a premix;

[0010] The premixture is subjected to high-speed shearing and then ultrasonic emulsification to obtain a white emulsion, which is the garlic essential oil nanoemulsion.

[0011] Further, by weight, 5-10 parts of garlic essential oil, 1-2 parts of emulsifier, and 88-94 parts of water are homogenized and emulsified to obtain the premix.

[0012] Furthermore, the emulsifier is Tween 80.

[0013] Furthermore, when the premixed mixture is subjected to high-speed shearing, the shearing rate is 16000-18000 r / min and the time is 2-4 min.

[0014] Furthermore, during the ultrasonic emulsification process, the ultrasonic power is 500-600W and the time is 10-15 minutes.

[0015] Furthermore, the process of compounding garlic essential oil nanoemulsion with ε-polylysine aqueous solution is as follows:

[0016] The garlic essential oil nanoemulsion was diluted 195-781 times to obtain a diluted solution of antibacterial agent monomer A; wherein the mass fraction of garlic essential oil in the garlic essential oil nanoemulsion was 5%-10%;

[0017] The ε-polylysine solution was diluted 200-750 times to obtain a diluted solution of antibacterial agent monomer B; wherein the concentration of the ε-polylysine solution was 1.6-12 mg / mL.

[0018] The diluted solution of antibacterial agent monomer A and the diluted solution of antibacterial agent monomer B are mixed evenly to obtain the compound antibacterial agent.

[0019] The present invention also provides a compound antibacterial agent for the inhibition of Listeria monocytogenes.

[0020] This invention also provides an application of a compound antibacterial agent in food preservation, food packaging, pet food, or livestock feed.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention provides a compound antibacterial agent, its preparation method, and its application. It employs an emulsifier and ultrasonic emulsification technology to encapsulate garlic essential oil, effectively improving its stability and solubility in high-moisture food systems. Secondly, based on the palisade theory, by adding ε-polylysine and compounding it with garlic essential oil nanoemulsion, the activity of garlic essential oil can be effectively enhanced, thereby significantly reducing the amount of garlic essential oil needed in practical applications and avoiding any impact on the original flavor of the food. Specifically, ε-polylysine and... Garlic essential oil nanoemulsion exhibits excellent synergistic antibacterial effects, with a partial inhibitory concentration index (PII) of 0.375 between the two. When ε-polylysine and garlic essential oil nanoemulsion are combined, they can effectively disrupt the permeability and integrity of foodborne pathogens, such as Listeria monocytogenes cell membranes, causing a large leakage of important intracellular substances and interfering with normal bacterial metabolism. This effectively inhibits bacterial growth and reproduction, and the effect is superior to the sum of the individual effects of the two. Therefore, the combined antibacterial agent can be used as a natural antibacterial agent in food preservation, food packaging, pet food, livestock and poultry feed, and other fields.

[0023] Furthermore, in the process of preparing garlic essential oil nanoemulsion, 5-10 parts by weight of garlic essential oil, 1-2 parts by weight of emulsifier and 88-94 parts by weight of water are mixed and subjected to high-speed shearing and ultrasonic emulsification. The amount of emulsifier used is relatively small, which effectively reduces the potential health risks of chemically synthesized emulsifiers and is conducive to their application in food. Attached Figure Description

[0024] Figure 1 A graph showing the effect of garlic essential oil nanoemulsion on the growth of Listeria monocytogenes.

[0025] Figure 2 The graph shows the effect of the compound antibacterial agent described in Example 1 on the growth of Listeria monocytogenes.

[0026] Figure 3 This is a diagram showing the effect of the compound antibacterial agent described in Example 2 on the intracellular nucleic acid proteins of Listeria monocytogenes;

[0027] Figure 4 The diagram shows the effect of the compound antibacterial agent described in Example 2 on the integrity of Listeria monocytogenes biofilm. Detailed Implementation

[0028] To make the technical problems solved by the present invention, the technical solutions, and the beneficial effects clearer, the following specific embodiments provide a further detailed description of the present invention. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of the invention.

[0029] This invention provides a compound antibacterial agent, which is obtained by compounding garlic essential oil nanoemulsion and ε-polylysine solution; wherein, the garlic essential oil nanoemulsion is an oil-in-water type garlic essential oil nanoemulsion, which is obtained by encapsulating garlic essential oil using ultrasonic emulsification technology; preferably, the particle size of the garlic essential oil nanoemulsion is 123.59-187.25 nm, and the mass fraction of garlic essential oil in the garlic essential oil nanoemulsion is 5%-10%; the concentration of the ε-polylysine solution is 1.6-12 mg / mL.

[0030] The preparation method of the compound antibacterial agent of the present invention includes the following steps:

[0031] Step 1: By weight, homogenize and emulsify 5-10 parts of garlic essential oil, 1-2 parts of emulsifier and 88-94 parts of water to obtain a premix; wherein the emulsifier is Tween 80.

[0032] Step 2: The premixture is subjected to high-speed shearing treatment, followed by ultrasonic emulsification treatment to obtain a white emulsion, which is the garlic essential oil nanoemulsion; wherein, during the high-speed shearing treatment of the premixture, the shearing rate is 16000-18000 r / min and the time is 2-4 min; during the ultrasonic emulsification treatment, the ultrasonic power is 500-600 W and the time is 10-15 min.

[0033] Step 3: Dissolve ε-polylysine in sterile water to obtain an ε-polylysine solution; wherein the concentration of the ε-polylysine solution is 1.6-12 mg / mL.

[0034] Step 4: Dilute the garlic essential oil nanoemulsion by 195-781 times to obtain a diluted solution of antibacterial agent monomer A; dilute the ε-polylysine solution by 200-750 times to obtain a diluted solution of antibacterial agent monomer B; mix the diluted solution of antibacterial agent monomer A and the diluted solution of antibacterial agent monomer B evenly to obtain the compound antibacterial agent.

[0035] Preparation process and antibacterial principle:

[0036] The compound antibacterial agent and its preparation method described in this invention first address the application defects of garlic essential oil, such as its volatility and poor water solubility. High-speed shearing combined with high-field-strength ultrasonic emulsification technology is used to encapsulate garlic essential oil in a nanoemulsion, obtaining a garlic essential oil nanoemulsion with a particle size of 123.59-187.25 nm. After encapsulating the garlic essential oil with an emulsifier, not only is the stability of the garlic essential oil effectively improved, but its solubility and application feasibility in high-moisture food systems are also enhanced. Comparative analysis shows that the antibacterial activity of the garlic essential oil nanoemulsion is more than double that of pure garlic essential oil in inhibiting Listeria monocytogenes. Furthermore, compared to existing nanoemulsions, this invention uses Tween 80 as an emulsifier for encapsulating garlic essential oil, resulting in an extremely low content, effectively reducing the potential health risks of chemically synthesized emulsifiers and making it more suitable for application in food.

[0037] Secondly, based on the encapsulation treatment of garlic essential oil with emulsifiers to enhance its antibacterial activity, this invention, based on the palisade theory, further enhances the activity of garlic essential oil by adding ε-polylysine, thereby reducing the amount of garlic essential oil added in practical applications and lowering usage costs. Specifically, ε-polylysine and garlic essential oil nanoemulsion exhibit a good synergistic antibacterial effect, with a partial inhibitory concentration index (PII) of 0.375 between the two. Furthermore, the combination of ε-polylysine and garlic essential oil nanoemulsion effectively disrupts the permeability and integrity of foodborne pathogens, such as Listeria monocytogenes cell membranes, causing a large leakage of important intracellular substances and interfering with normal bacterial metabolism, thus effectively inhibiting bacterial growth and reproduction. The effect is superior to the sum of the individual effects of the two.

[0038] The compound antibacterial agent described in this invention has significant inhibitory activity against Listeria monocytogenes and can be applied to the preservation of various traditional foods, pre-made foods, pet foods, etc. through direct addition or active packaging. Compared with the use of garlic essential oil nanoemulsion or ε-polylysine alone, the antibacterial effect is significantly improved, greatly reducing the amount of garlic essential oil and ε-polylysine used, reducing the cost of use, and having little impact on the flavor of the food itself.

[0039] Example 1

[0040] This embodiment 1 provides a compound antibacterial agent, which is obtained by compounding garlic essential oil nanoemulsion with ε-polylysine; wherein, the garlic essential oil nanoemulsion is an oil-in-water garlic essential oil nanoemulsion, which is obtained by encapsulating garlic essential oil using ultrasonic emulsification technology; the particle size of the garlic essential oil nanoemulsion is 123.59 nm.

[0041] The preparation method of the compound antibacterial agent described in Example 1 includes the following steps:

[0042] Step 1: According to the weight proportions, homogenize and emulsify 5 parts garlic essential oil, 1 part Tween 80 and 94 water to obtain a premix.

[0043] Step 2: The premixture is subjected to high-speed shearing, followed by ultrasonic emulsification to obtain a white emulsion, which is the garlic essential oil nanoemulsion. The high-speed shearing treatment of the mixture is performed at a shear rate of 16000 r / min for 4 min; the ultrasonic emulsification process uses an ultrasonic power of 600 W for 12 min; the garlic essential oil nanoemulsion contains 5% garlic essential oil by mass.

[0044] Step 3: Dissolve ε-polylysine in sterile water to obtain an ε-polylysine solution; wherein the concentration of the ε-polylysine solution is 1.6 mg / mL.

[0045] Step 4: Dilute the garlic essential oil nanoemulsion from Step 2 by 195 times to obtain a diluted solution of antibacterial agent monomer A; dilute the ε-polylysine solution from Step 3 by 200 times to obtain a diluted solution of antibacterial agent monomer B; mix the diluted solution of antibacterial agent monomer A and the diluted solution of antibacterial agent monomer B evenly to obtain the compound antibacterial agent.

[0046] Example 2

[0047] This embodiment 2 provides a compound antibacterial agent, which is obtained by compounding garlic essential oil nanoemulsion with ε-polylysine; wherein, the garlic essential oil nanoemulsion is an oil-in-water garlic essential oil nanoemulsion, which is obtained by encapsulating garlic essential oil using ultrasonic emulsification technology; the particle size of the garlic essential oil nanoemulsion is 159.37 nm.

[0048] The preparation method of the compound antibacterial agent described in Example 2 includes the following steps:

[0049] Step 1: According to the weight proportions, homogenize and emulsify 10 parts of garlic essential oil, 2 parts of Tween 80 and 88 water to obtain a premix.

[0050] Step 2: The premixture is subjected to high-speed shearing, followed by ultrasonic emulsification to obtain a white emulsion, which is the garlic essential oil nanoemulsion. The high-speed shearing treatment of the mixture is performed at a shear rate of 18000 r / min for 2 min; the ultrasonic emulsification process uses an ultrasonic power of 500 W for 10 min; the garlic essential oil nanoemulsion contains 10% garlic essential oil by mass.

[0051] Step 3: Dissolve ε-polylysine in sterile water to obtain an ε-polylysine solution; wherein the concentration of the ε-polylysine solution is 12 mg / mL.

[0052] Step 4: Dilute the garlic essential oil nanoemulsion from Step 2 by 781 times to obtain a diluted solution of antibacterial agent monomer A; dilute the ε-polylysine solution from Step 3 by 750 times to obtain a diluted solution of antibacterial agent monomer B; mix the diluted solution of antibacterial agent monomer A and the diluted solution of antibacterial agent monomer B evenly to obtain the compound antibacterial agent.

[0053] Example 3

[0054] This embodiment 3 provides a compound antibacterial agent, which is obtained by compounding garlic essential oil nanoemulsion with ε-polylysine; wherein, the garlic essential oil nanoemulsion is an oil-in-water garlic essential oil nanoemulsion, which is obtained by encapsulating garlic essential oil using ultrasonic emulsification technology; the particle size of the garlic essential oil nanoemulsion is 187.25 nm.

[0055] The preparation method of the compound antibacterial agent described in Example 1 includes the following steps:

[0056] Step 1: According to the weight proportions, homogenize and emulsify 8 parts of garlic essential oil, 1 part of Tween 80 and 91 water to obtain a premix.

[0057] Step 2: The premixture is subjected to high-speed shearing, followed by ultrasonic emulsification to obtain a white emulsion, which is the garlic essential oil nanoemulsion. The high-speed shearing treatment of the mixture is performed at a shear rate of 17000 r / min for 3 min; the ultrasonic emulsification process uses an ultrasonic power of 550 W for 15 min; the garlic essential oil nanoemulsion contains 8% garlic essential oil by mass.

[0058] Step 3: Dissolve ε-polylysine in sterile water to obtain an ε-polylysine solution; wherein the concentration of the ε-polylysine solution is 5 mg / mL.

[0059] Step 4: Dilute the garlic essential oil nanoemulsion from Step 2 by 312 times to obtain a diluted solution of antibacterial agent monomer A; dilute the ε-polylysine solution from Step 3 by 312 times to obtain a diluted solution of antibacterial agent monomer B; mix the diluted solution of antibacterial agent monomer A and the diluted solution of antibacterial agent monomer B evenly to obtain the compound antibacterial agent.

[0060] The compound antibacterial agents obtained by combining garlic essential oil nanoemulsion and ε-polylysine solution in Examples 1-3 of this invention have a significant synergistic effect. They can effectively inhibit the growth and reproduction of bacteria by disrupting the permeability and integrity of the cell membrane of foodborne pathogens and interfering with the normal metabolism of the bacteria. They can be used as natural antibacterial agents in food preservation, food packaging, pet food, livestock and poultry feed and other fields.

[0061] Performance verification test:

[0062] The antibacterial performance of the compound antibacterial agent described in Example 2 is verified and explained through verification experiments 1-3. The synergistic antibacterial mechanism of garlic essential oil nanoemulsion and ε-polylysine in the compound antibacterial agent described in Example 2 is explained through verification experiments 4-5, as follows:

[0063] Verification Experiment 1

[0064] This verification experiment 1 specifically includes the following steps:

[0065] S1. Preparation of Nutrient Agar Plates

[0066] Dissolve the nutrient agar thoroughly in distilled water and sterilize it in an autoclave at 121°C for 20 minutes. Then, cool the sterilized nutrient agar to 50-55°C. After that, pour the cooled nutrient agar onto plates and let it cool for later use.

[0067] S2. Preparation of bacterial suspension

[0068] Pick a single colony of Listeria monocytogenes into a centrifuge tube containing 30 mL of TSB liquid medium and incubate at 37 °C for 24 h. After incubation, adjust the OD value to 0.5 using a UV spectrophotometer at a wavelength of 600 nm with TSB liquid culture, and set aside for use.

[0069] S3. Preparation of filter paper

[0070] Select a qualitative filter paper with uniform texture and strong water absorption. Use a punching machine to punch out circular paper pieces with a diameter of 6mm from the filter paper. Sterilize them under high pressure and place them in a clean bench for later use.

[0071] S4. Determination of inhibition zone

[0072] The inhibition zone was determined using the filter paper disc method, as follows:

[0073] Add 80 μL of each of the three prepared bacterial suspensions to a nutrient agar plate and spread evenly. After a preset time, use sterile tweezers to pick up a sterilized 6 mm circular filter paper and place it on the nutrient agar plate. Perform three parallel experiments simultaneously. Then, use a pipette to pick up 20 μL of garlic essential oil nanoemulsion, 20 μL of ε-polylysine solution, and 20 μL of the compound antibacterial agent described in Example 1 and place them on the filter paper. Incubate in a constant temperature incubator at 37°C for 24 hours. Afterward, observe and measure the diameter (mm) of the inhibition zone of each experimental sample with vernier calipers and calculate the average value.

[0074] Table 1. Measurement results (cm) of the inhibition zone diameter for Listeria monocytogenes.

[0075]

[0076] As can be seen from Table 1 above, when Listeria monocytogenes is inhibited using the compound antibacterial agent described in Example 1, the inhibitory effect of the compound antibacterial agent on Listeria monocytogenes is stronger than that of the two when used alone, due to the synergistic antibacterial effect of garlic essential oil nanoemulsion and ε-polylysine.

[0077] Verification Experiment 2

[0078] In this validation experiment 2, the bacterial strain used was Listeria monocytogenes; the reagents included TSB liquid culture medium, bromocresol green indicator, and sterile physiological saline; the experimental instruments and supplies included 96-well plates, autoclave, laminar flow hood, constant temperature incubator, and ultraviolet spectrophotometer.

[0079] The experimental steps for this verification experiment 2 are as follows:

[0080] S1. Preparation of bacterial suspension

[0081] Pick a single colony of Listeria monocytogenes into a centrifuge tube containing 30 mL of TSB liquid medium and incubate at 37 °C for 24 h. After incubation, adjust the OD value to 0.5 using a UV spectrophotometer at a wavelength of 600 nm with TSB liquid culture, and set aside for use.

[0082] S2, Sample addition

[0083] Garlic essential oil nanoemulsion and ε-polylysine solution were added to a 96-well plate in the following order: From left to right, columns 1-8 each contained 50 μL of garlic essential oil nanoemulsion with final concentrations of 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, and 0 μg / mL; from top to bottom, columns A and H each contained 50 μL of garlic essential oil nanoemulsion with a final concentration of... ε-polylysine solutions with concentrations of 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0 μg / mL were prepared. Next, 80 μL of TSB liquid medium and 20 μL of bacterial suspension were added to each well of the 96-well plate, with wells without antibacterial agents serving as controls. The 96-well plates were then incubated at 37°C for 24 hours, and the results were observed.

[0084] S3. Results Observation

[0085] Because garlic essential oil nanoemulsion is a milky white turbid liquid, its addition to the culture medium causes slight turbidity, interfering with the results of visual observation. Therefore, one drop of bromocresol green indicator is added to each well, and the growth of bacteria is judged by the color change, where yellow-green indicates the presence of bacteria and blue indicates the absence of bacteria.

[0086] Table 2 Results of Minimum Inhibitory Concentration Test

[0087]

[0088] As shown in Table 2 above and in conjunction with the verification experiments, the compound antibacterial agent resulting from the mixture of garlic essential oil nanoemulsion and ε-polylysine exhibits a strong antibacterial effect, with a synergistic index of 0.375. The synergistic index is calculated using the following formula:

[0089]

[0090] Wherein, MIC A (A+B) represents the MIC when drug A is used in combination, MIC B (A+B) represents the MIC when drug B is used in combination, MIC A represents the MIC when drug A is used alone, and MIC B represents the MIC when drug B is used alone. It should be noted that when FIC < 0.5, it shows a synergistic effect; when 0.5 ≤ FIC < 1, it shows an additive effect; when 1 ≤ FIC < 2, it shows an irrelevant effect; and when FIC ≥ 2, it shows an antagonistic effect.

[0091] Verification Experiment 3

[0092] In this verification experiment 3, the bacterial strain used was Listeria monocytogenes; the reagents included TSB liquid culture medium and sterile physiological saline; the experimental instruments and supplies included an autoclave, a clean bench, a constant temperature incubator, an ultraviolet spectrophotometer, and several test tubes.

[0093] The experimental steps for this verification experiment 3 are as follows:

[0094] S1. Preparation of bacterial suspension

[0095] Pick a single colony of Listeria monocytogenes into a centrifuge tube containing 30 mL of TSB liquid medium and incubate at 37 °C for 24 h. After incubation, adjust the OD value to 0.5 using a UV spectrophotometer at a wavelength of 600 nm with TSB liquid culture, and set aside for use.

[0096] S2, Sample addition

[0097] Growth curve of S2.1 garlic essential oil nanoemulsion against Listeria monocytogenes

[0098] Garlic essential oil nanoemulsion and bacterial suspension were added to sterile centrifuge tubes at a mass ratio of 1:1, resulting in final concentrations of 1 / 16 MIC, 1 / 8 MIC, 1 / 4 MIC, 1 / 2 MIC, 1 MIC, and 2 MIC in the centrifuge tubes. A negative control group without antibacterial agent was also prepared, and three replicates were performed. The tubes were then incubated at 37°C, and samples were taken every 1 hour to measure the OD value at 600 nm, and a growth curve was plotted. Figure 1 As shown, attached Figure 1 The figure shows the effect curve of garlic essential oil nanoemulsion on the growth of Listeria monocytogenes; from the appendix Figure 1 As can be seen, garlic essential oil nanoemulsion completely inhibited the growth of Listeria monocytogenes at 1 MIC, and the sub-inhibitory concentration (1 / 16 to 1 / 2 MIC) of garlic essential oil nanoemulsion showed a dose-dependent inhibitory effect on bacterial proliferation. Furthermore, the higher the concentration of garlic essential oil nanoemulsion, the longer the hysteresis loop and the longer the time required to reach maximum density. Therefore, garlic essential oil nanoemulsion has a significant inhibitory effect on the growth of Listeria monocytogenes.

[0099] S2.2 Growth curve of Listeria monocytogenes synergistic with garlic essential oil nanoemulsion and ε-polylysine.

[0100] The compound antibacterial agent and bacterial suspension described in Example 1 were added to sterile centrifuge tubes at a 1:1 ratio, resulting in a mixture of 1 / 8 MIC garlic essential oil nanoemulsion, 1 / 4 MIC ε-polylysine solution, 1 / 8 MIC garlic essential oil nanoemulsion + 1 / 4 MIC ε-polylysine solution, and 0. A negative control group without the antibacterial agent was also prepared, and three parallel groups were performed. The tubes were then incubated at 37°C. Samples of the garlic essential oil nanoemulsion were taken at intervals, and their OD values ​​at 600 nm were measured. A growth curve was plotted. Figure 2 As shown, attached Figure 2The figure shows the effect curve of the compound antibacterial agent described in Example 1 on the growth of Listeria monocytogenes; from the appendix Figure 2 As can be seen, when 1 / 8 MIC of garlic essential oil nanoemulsion and 1 / 4 MIC of ε-polylysine were used alone, the maximum optical density of the bacteria was lower than that of the control group, but they did not completely inhibit the growth of Listeria monocytogenes during the growth period. When the two were used together, the maximum optical density of the bacteria remained basically unchanged within the measured 24-hour range, indicating that the combined use of the two at this concentration could completely inhibit the growth of the bacteria, demonstrating a synergistic effect.

[0101] Verification Experiment 4

[0102] In this validation experiment 1, the bacterial species was Listeria monocytogenes; the reagents included TSB liquid culture medium and sterile physiological saline; the experimental instruments and supplies included centrifuge tubes, autoclave, laminar flow hood, constant temperature incubator, refrigerated centrifuge, and ultraviolet spectrophotometer.

[0103] The experimental steps for this verification experiment 4 are as follows:

[0104] S1. Preparation of bacterial suspension

[0105] Pick a single colony of Listeria monocytogenes into a centrifuge tube containing 30 mL of TSB liquid medium and incubate at 37 °C for 24 h. After incubation, centrifuge with physiological saline (8000 r / min for 10 min) three times to adjust the OD value to 0.5 and set aside for use.

[0106] S2. Effects of garlic essential oil nanoemulsion synergistically with ε-polylysine on Listeria monocytogenes nucleic acid proteins.

[0107] Take 50mL centrifuge tubes, add 15mL of bacterial culture to each tube, and then add the pre-prepared compound antibacterial agent to each tube, bringing the final concentration to 2MIC of the compound antibacterial agent, 1 / 4MIC of garlic essential oil nanoemulsion, 1 / 2MIC of ε-polylysine, and 0. Place them in a constant temperature incubator for incubation.

[0108] At 0, 1, 3, and 6 hours of culture, 5 mL of bacterial culture was taken from each of five centrifuge tubes, centrifuged at 8000 r / min for 10 min, and the supernatant was collected. The absorbance of the supernatant in the five centrifuge tubes at 260 nm and 280 nm at different time points was measured using a UV-Vis spectrophotometer. Based on the recorded absorbance values ​​of single and combined antibacterial agents at different time points and concentrations, three parallel experiments were performed for each group, and the change curves were plotted. Figure 3 As shown, attached Figure 3 The diagram showing the effect of the compound antibacterial agent described in Example 2 on the intracellular nucleic acid proteins of Listeria monocytogenes is provided; from the appendix... Figure 3 As can be seen from the appendix Figure 3As can be seen, compared with the control group, Listeria monocytogenes showed a large amount of intracellular nucleic acid and protein leakage after treatment with 1 / 4MIC garlic essential oil nanoemulsion and 1 / 2MIC ε-polylysine. When 2MIC (1 / 4MIC garlic essential oil nanoemulsion and 1 / 2MIC ε-polylysine) were used in combination, the leakage amount increased significantly with the extension of treatment time compared with the use of the two alone.

[0109] Verification Experiment 5

[0110] In this validation experiment 5, the bacterial species was Listeria monocytogenes; the reagents included TSB liquid culture medium and sterile physiological saline; the experimental instruments and supplies included centrifuge tubes, autoclave, laminar flow hood, incubator, refrigerated centrifuge, ultraviolet spectrophotometer, and scanning electron microscope.

[0111] The experimental steps for this verification experiment 5 are as follows:

[0112] S1. Preparation of bacterial suspension

[0113] Pick a single colony of Listeria monocytogenes into a centrifuge tube containing 30 mL of TSB liquid medium and incubate at 37 °C for 24 h. After incubation, centrifuge with physiological saline (8000 r / min for 10 min) three times to adjust the OD value to 0.5 and set aside for use.

[0114] S2. Scanning electron microscopy observation of the integrity of Listeria monocytogenes membrane.

[0115] Transfer 30 mL of bacterial culture to a 50 mL sterile centrifuge tube, and add pre-prepared compound antibacterial agents to the mixture to achieve the following concentrations: 1 / 4 MIC garlic essential oil nanoemulsion, 1 / 2 MIC ε-polylysine, and 1 / 4 MIC garlic essential oil nanoemulsion + 1 / 2 MIC ε-polylysine (2 MIC). Incubate the mixed bacterial culture at 37°C on a shaker (135 r / min) for 4 h. Centrifuge the culture at 8000 r / min for 10 min to remove the supernatant. Wash the cells twice with physiological saline. Pre-cool the cells with glutaraldehyde at 4°C. After centrifugation, add 5-10 mL of 2.5% glutaraldehyde solution for suspension and fixation for 12 h (4°C). Wash the fixed cells three times with sterile physiological saline, then dehydrate with 30%, 50%, 70%, 90%, and 100% ethanol, each treatment lasting 10 min. Add 1 mL of isoamyl acetate to the centrifuged cell precipitate and fix at room temperature for 3 h. Take 30 μL of the well-mixed bacterial suspension into a 1 cm... 2 Place the petri dish on sterilized aluminum foil and dry it in an incubator (or at room temperature). After drying, sputter-coated the sample with gold and finally observe it using a scanning electron microscope; Figure 4 As shown, attached Figure 4The diagram showing the effect of the compound antibacterial agent described in Example 2 on the biofilm integrity of Listeria monocytogenes is provided; from the attached diagram... Figure 4 As can be seen, the control group of Listeria monocytogenes exhibits a typical rod-shaped morphology, with smooth cell surfaces, intact structure, and clear cell boundaries. (See Appendix for details.) Figure 4 A and Appendix Figure 4 a; After treatment with 1 / 4 MIC garlic essential oil nanoemulsion, Listeria monocytogenes cells showed slight shrinkage and deformation, and slight adhesion between cells. See appendix for details. Figure 4 B and appendix Figure 4 b; After treatment with 1 / 2 MIC ε-polylysine, cell deformation intensified, with some cells dissolving, breaking, and tightly adhering together. See Appendix for details. Figure 4 C and appendix Figure 4 c; When treated with a combination of 2MIC (1 / 4MIC garlic essential oil nanoemulsion and 1 / 2MIC ε-polylysine), the cell structure completely disintegrated, exhibiting a aggregated state. See Appendix for details. Figure 4 D and appendix Figure 4 d.

[0116] As can be seen from the above verification experiments 1-5, garlic essential oil nanoemulsions at MIC and 2MIC concentrations have significant inhibitory effects on Listeria monocytogenes; garlic essential oil nanoemulsions at 1 / 8MIC and ε-polylysine solution at 1 / 4MIC have significant inhibitory effects on Listeria monocytogenes under synergistic effects.

[0117] The compound antibacterial agent, its preparation method, and its application described in this invention are prepared by compounding garlic essential oil nanoemulsion with ε-polylysine. This compound antibacterial agent can effectively inhibit bacterial growth and reproduction by disrupting the permeability and integrity of the cell membranes of foodborne pathogens and interfering with normal bacterial metabolism. In addition to enhancing the antibacterial effect, the compound antibacterial agent also reduces the pungent odor of garlic essential oil and decreases the amount of each antibacterial agent required. The compound antibacterial agent described in this invention can significantly improve the antibacterial effect against Listeria monocytogenes. By preparing garlic essential oil into a nanoemulsion, the antibacterial activity of garlic essential oil can be improved while reducing its volatility and pungent odor, thus achieving a sustained antibacterial and bactericidal effect.

[0118] The above embodiments are merely one of the implementation methods for achieving the technical solution of the present invention. The scope of protection claimed by the present invention is not limited to this embodiment, but also includes any variations, substitutions and other implementation methods that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention.

Claims

1. A compound antibacterial agent, characterized in that, The compound antibacterial agent is obtained by compounding garlic essential oil nanoemulsion and ε-polylysine solution; wherein, the garlic essential oil nanoemulsion is an oil-in-water garlic essential oil nanoemulsion, which is obtained by encapsulating garlic essential oil using ultrasonic emulsification technology; The preparation process of the garlic essential oil nanoemulsion is as follows: Garlic essential oil, emulsifier and water are mixed to obtain a premix; The premixed material is subjected to high-speed shearing and then ultrasonic emulsification to obtain a white emulsion, which is the garlic essential oil nanoemulsion. The premix is ​​obtained by homogenizing and emulsifying 5-10 parts of garlic essential oil, 1-2 parts of emulsifier and 88-94% water by weight. The process of compounding garlic essential oil nanoemulsion with ε-polylysine aqueous solution is as follows: The garlic essential oil nanoemulsion was diluted 195-781 times to obtain a diluted solution of antibacterial agent monomer A; wherein the mass fraction of garlic essential oil in the garlic essential oil nanoemulsion was 5%-10%; The ε-polylysine solution was diluted 200-750 times to obtain a diluted solution of antibacterial agent monomer B; wherein the concentration of the ε-polylysine solution was 1.6-12 mg / mL. The diluted solution of antibacterial agent monomer A and the diluted solution of antibacterial agent monomer B are mixed evenly to obtain the compound antibacterial agent.

2. The compound antibacterial agent according to claim 1, characterized in that, The garlic essential oil nanoemulsion has a particle size of 123.59-187.25 nm.

3. The compound antibacterial agent according to claim 1, characterized in that, The emulsifier is Tween 80.

4. The compound antibacterial agent according to claim 1, characterized in that, When the premix is ​​subjected to high-speed shearing, the shearing rate is 16000-18000 r / min and the time is 2-4 min.

5. The compound antibacterial agent according to claim 1, characterized in that, During the ultrasonic emulsification process, the ultrasonic power is 500-600W and the time is 10-15 minutes.

6. A compound antibacterial agent as described in any one of claims 1-5 for the inhibition of Listeria monocytogenes.

7. The antibacterial application of a compound antibacterial agent as described in any one of claims 1-5 in food preservation, food packaging, pet food, or livestock feed.

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