Cyclamen aldehyde nano-emulsion, preparation method thereof and application of cyclamen aldehyde nano-emulsion in preparation of antibacterial agent

Abstract

The invention discloses cyclamen aldehyde nano-emulsion, a preparation method thereof and application of the cyclamen aldehyde nano-emulsion in preparation of an antibacterial agent, and the preparation method comprises the following steps: (1) dissolving a surfactant in water, and stirring to form a surfactant solution with the volume concentration of 1-9%; (2) dropwise adding cyclamen aldehyde into the surfactant solution obtained in the step (1), and stirring to form a coarse emulsion; and (3) enabling the crude emulsion obtained in the step (2) to form a nano-emulsion under the action of ultrasonication, so as to obtain the cyclamen aldehyde nano-emulsion. The emulsion can effectively inhibit growth of vegetative cells of bacillus cereus, formation of biological membranes and germination of spores, and can be applied to preparation of antibacterial agents. The nano-emulsion prepared by the invention is non-toxic and harmless, good in storage stability, high in antibacterial efficiency, simple and convenient. The bacillus cereus strain has a good application prospect in solving the problem of bacillus cereus pollution in the food processing and production process.

Description

Technical Field

[0001] The invention belongs to the field of antibacterial and anticorrosive technology and nanoemulsion encapsulation, and particularly relates to a preparation method and antibacterial application of silymarin nanoemulsion. Background Art

[0002] Bacillus cereus is a Gram-positive bacterium widely distributed in the air and soil. As a foodborne pathogen, it can contaminate nearly all foods, including rice and flour products, meat products, and soy products. Furthermore, Bacillus cereus produces highly resistant biofilms and spores. Biofilms can form on food-contact surfaces made of diverse materials, including rubber, glass, conveyor belts, and stainless steel. These biofilms are resistant to mechanical stress and extreme acid and alkaline environments. These contaminants can persist during food processing, potentially leading to food spoilage and threatening human health. Furthermore, due to their unique structure and physiological processes, spores are highly resistant to various sterilization treatments. Their resistance to high temperatures, high pressure, desiccation, and ultraviolet light also makes them difficult to completely remove during food production. Therefore, effectively preventing and controlling contamination by Bacillus cereus vegetative cells, biofilms, and spores has become a pressing issue for the food industry.

[0003] 3-(4-isopropylphenyl)-2-methylpropanal (CA) is a synthetic fragrance approved for inclusion in the National Food Safety Standard for Food Additives (GB-2760-2024). As an aromatic aldehyde, it is widely used in chemical industries such as flavors and fragrances. However, ca-aldehyde's hydrophobicity and high volatility result in low water solubility and poor stability, inevitably limiting its application in the food industry. Summary of the Invention

[0004] In order to solve the above-mentioned related problems, the primary purpose of the present invention is to provide a preparation method and antibacterial application of oxalicum aldehyde nanoemulsion.

[0005] Another object of the present invention is to provide a nanoemulsion obtained by the above-mentioned preparation method. The nanoemulsion can effectively improve the low water solubility and poor stability of silymarin, prevent the direct volatilization of silymarin, improve its utilization rate, and achieve the purpose of high-efficiency antibacterial.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for preparing a sedge aldehyde nanoemulsion comprises the following steps:

[0008] (1) dissolving a surfactant in water and stirring to form a surfactant solution with a volume concentration of 1% to 9%;

[0009] (2) adding pyralidin dropwise to the surfactant solution obtained in step (1), stirring to form a crude emulsion;

[0010] (3) The crude emulsion obtained in step (2) is subjected to ultrasonic crushing to form a nanoemulsion, thereby obtaining the oxaliplatin nanoemulsion.

[0011] Preferably, the surfactant in step (1) is one or more of Tween 80, Span 80 and Tween 20.

[0012] Preferably, the concentration of the surfactant solution in step (1) is 3-5%.

[0013] Preferably, the rate of dropwise addition of pyralidin in step (2) is 1±0.5 drops / s; and the stirring time is 5±2 min.

[0014] Preferably, the final concentration of sylvataldehyde in step (2) is 0.05-5%, preferably 2%.

[0015] Preferably, the frequency of ultrasonic fragmentation in step (3) is 400±200W, the time is 10±5min, the working time is 5±3s, and the rest time is 2±1s.

[0016] Preferably, the frequency of ultrasonic fragmentation in step (3) is 400±200W, the time is 10±5min, the working time is 5±3s, and the rest time is 2±1s.

[0017] In subsequent antibacterial tests using Bacillus cereus as the test subject, the results showed that the nanoemulsion prepared by this method had a good inhibitory effect on Bacillus cereus vegetative cells, biofilms and spores. The use of the oxalicum aldehyde nanoemulsion of the present invention in the preparation of antibacterial agents.

[0018] Preferably, the bacteria inhibited by the antimicrobial agent include Bacillus cereus.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention uses Tween 80 as a surfactant and prepares pyralidin into a nanoemulsion by ultrasonic emulsification. The prepared nanoemulsion has the advantages of small particle size, good dispersibility, good stability, simple process and easy large-scale production.

[0021] (2) The nanoemulsion prepared by the present invention successfully encapsulates pyralidone, overcoming the shortcomings of pyralidone such as poor water solubility and volatility. It has a good inhibitory effect on Bacillus cereus vegetative cells, biofilms and their spores, and is of great significance for food preservation applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The particle size, dispersibility index (PDI) (a) and zeta potential diagram (b) of the pyralid nanoemulsion prepared with different types of surfactants in Example 1.

[0023] Figure 2 The particle size, PDI (a) and Zeta potential diagram (b) of the oxalicum aldehyde nanoemulsion at different concentrations of Tween 80 in Example 2.

[0024] Figure 3 These are transmission electron micrographs of the oxalicum aldehyde nanoemulsion in Example 3 at different magnifications.

[0025] Figure 4 This is the effect of shear force (a) and viscosity (b) of the oxaliplatin nanoemulsion in Example 4 as a function of shear rate.

[0026] Figure 5 These are the particle size and PDI value graphs of the oxalicum aldehyde nanoemulsion in Example 5 stored in 4°C (a) and 25°C (b) environments for 28 days.

[0027] Figure 6 This is the effect of the rabbit ear aldehyde nanoemulsion in Example 6 on the growth curve of Bacillus cereus.

[0028] Figure 7 1 is a scanning electron micrograph of Bacillus cereus vegetative cells before and after treatment with the oxalicum aldehyde nanoemulsion in Example 7.

[0029] Figure 8 This is the effect of the rabbit ear aldehyde nanoemulsion in Example 8 on the leakage of protein (a) and nucleic acid (b) from Bacillus cereus vegetative cells.

[0030] Figure 9 These are the inhibitory (a) and clearing (b) effects of the rabbit ear aldehyde nanoemulsion on Bacillus cereus biofilm in Example 9.

[0031] Figure 10 This is the effect of the rabbit ear grass aldehyde nanoemulsion in Example 10 on the motility of Bacillus cereus.

[0032] Figure 11 These are the results of the determination of the minimum inhibitory concentration (MIC) (a) and germination curve (b) of the rabbit ear aldehyde nanoemulsion against Bacillus cereus spores in Example 11.

[0033] Figure 12 This is the result of measuring the germination rate of Bacillus cereus spores by the rabbit ear grass aldehyde nanoemulsion in Example 12.

[0034] Figure 13 These are the test results of the heat resistance (a) and salt resistance (b) of the rabbit ear aldehyde nanoemulsion on Bacillus cereus spores in Example 13.

[0035] Figure 14 This is the result of measuring the hydrophobicity of the rabbit ear aldehyde nanoemulsion on Bacillus cereus spores in Example 14. DETAILED DESCRIPTION

[0036] The present invention will be described in further detail below with reference to specific examples, but the embodiments of the present invention are not limited thereto. The reagents and methods used in the present invention are all common reagents and conventional methods in the art unless otherwise specified.

[0037] The Bacillus cereus has a deposit number of CMCC 63303.

[0038] Example 1: Effect of surfactant type on particle size, PDI and potential of oxalicum aldehyde nanoemulsion

[0039] Span 80, Tween 80, and Tween 20 were selected as surfactants for preparing nanoemulsions for screening. A 3% volume fraction of each surfactant was dissolved in deionized water. Sulfamethoxazole was added to the above solution at a rate of 1 drop / s to a final concentration of 2% (v / v). The mixture was magnetically stirred at room temperature for 5 minutes. The crude emulsion was emulsified in an ultrasonic mill at a frequency of 400W, with an operating frequency of 5 seconds and a rest period of 2 seconds. After 10 minutes, the mixture was immediately placed in an ice-water bath to cool, producing a milky white nanoemulsion. An appropriate amount of the prepared nanoemulsion was diluted 100-fold and placed in a sample cuvette of a Malvern particle size analyzer. The particle size, PDI, and potential were measured.

[0040] A stable emulsion system requires appropriate droplet size, PDI, and sufficiently high electrostatic repulsion. Figure 1 The results in (a) and (b) show that different surfactant types affect these three parameters of the pyralid nanoemulsion. Compared with Span 80 and Tween 20, the nanoemulsion prepared with Tween 80 has a smaller particle size and higher potential, indicating that the nanoemulsion system prepared with Tween 80 is more stable. Therefore, Tween 80 is the optimal surfactant.

[0041] Example 2: Effect of Tween 80 concentration on particle size, PDI and potential of oxalicum aldehyde nanoemulsion

[0042] 100, 300, 500, 700, and 900 μL of Tween 80 were added to 9.8 mL of deionized water to form 1%, 3%, 5%, 7%, and 9% Tween 80 solutions, respectively. The mixture was magnetically stirred at room temperature for 5 minutes. Subsequently, emulsions were prepared according to the experimental method in Example 1, and the particle size, PDI, and potential were measured.

[0043] Figure 2 The results showed that the particle size of the prepared oxalicum aldehyde nanoemulsion first increased and then decreased with increasing Tween 80 concentration. When the Tween 80 concentration was 3%, the particle size of the nanoemulsion was 160.867nm, the PDI was 0.27, and the Zeta potential was -30.67mV. The prepared oxalicum aldehyde nanoemulsion had a small particle size, a uniform dispersion, and good stability. Therefore, 3% is the optimal Tween 80 concentration.

[0044] Example 3: Transmission electron microscopy observation of the microstructure of oxalicum aldehyde nanoemulsion

[0045] The nanoemulsion was diluted 100-fold with deionized water, mixed thoroughly on a vortex, and then filtered through a 0.22 μm water filter. 5 μL of the solution was dropped onto a 200-mesh copper grid covered with a carbon film and negatively stained with 5 μL of a 2% phosphotungstic acid solution. The sample was allowed to dry at room temperature in a fume hood, and the microstructure of the nanoemulsion droplets was observed under a transmission electron microscope.

[0046] Figure 3 The results showed that the prepared oxalicum nanoemulsion particles were spherical with a relatively uniform size distribution. Transmission electron microscopy images showed that the nanoemulsion particle size was around 100 nm, which was inconsistent with the particle size measured by a laser particle size analyzer. This may be due to the influence of drying effects.

[0047] Example 4: Effect of shear force and viscosity of oxaliplatin nanoemulsion on shear rate.

[0048] The rheological properties of the rabbit ear grass aldehyde nanoemulsion were measured using a rheometer. The experiment used a 40mm diameter conical plate with a plate spacing of 150um. About 1mL of the emulsion was placed on the plate. The measurement program was set to steady-state shear mode, with the parameters set to temperature 25℃, shear rate (0.1~100s -1 ) Changes in apparent viscosity and shear force of the emulsion under .

[0049] When the fluid is a Newtonian fluid, its viscosity does not change with the change of shear rate. At this time, the fluid satisfies the Newtonian model in rheology, that is, τ = μ*γ, where τ is the shear force, μ is the viscosity of the fluid, and γ is the shear rate. Figure 4 (a) shows that there is a linear relationship between the shear force and shear rate of the nanoemulsion. Figure 4(b) shows that after the nanoemulsion becomes stable, the shear viscosity does not change with the increase of shear rate. The results indicate that the prepared rabbit ear grass aldehyde nanoemulsion is a Newtonian fluid with good fluidity and uniformity.

[0050] Example 5: Storage stability analysis of oxalis aldehyde nanoemulsion

[0051] The oxalicum aldehyde nanoemulsion was stored at 4°C and 25°C under sealed and light-proof conditions, and samples were taken at 0, 7, 14, 21, and 28 days to measure the average particle size and PDI.

[0052] Figure 5 The results showed that the average particle size and PDI of the prepared pyralidin nanoemulsion fluctuated slightly around the initial values ​​at 4°C. However, at 25°C, both the average particle size and PDI showed significant changes (p < 0.05). This indicates that the pyralidin nanoemulsion has good storage stability and is more suitable for storage at 4°C.

[0053] Example 6: Study on the antibacterial activity of oxalicum aldehyde nanoemulsion against Bacillus cereus vegetative cells

[0054] 1. MIC determination

[0055] In the suspension of Bacillus cereus (10 6 CFU / mL) was added to a final concentration of 0.06 ～ A 3.68 mg / mL pyralidin nanoemulsion was prepared. An equal volume of sterile phosphate buffered saline (PBS) was added to the control group as a control. The tubes were incubated at 37°C, 180 rpm for 24 hours, and the turbidity of the test tubes was observed. The pyralidin concentration in the nanoemulsion that inhibited visible Bacillus cereus was determined as the MIC.

[0056] Finally, it was concluded that the MIC of rabbit ear aldehyde nanoemulsion against Bacillus cereus was 0.115 mg / mL.

[0057] 2. Growth Curve Determination

[0058] Bacillus cereus bacterial solution (10 6 CFU / mL) was mixed with equal volumes of rabbit ear aldehyde nanoemulsion with a final concentration of 1 / 2MIC and MIC, and the control group was added with an equal volume of LB liquid medium. After being placed in a constant temperature shaking incubator at 37°C and 180 rpm, the culture OD was measured every 2 hours. 600 , draw the growth curve of Bacillus cereus.

[0059] Figure 6 The results showed that the control group showed a good growth trend. The OD of the bacterial solution was 600That is, it reached about 0.75. The bacterial growth in the 1 / 2MIC group was slow, which shows that the low concentration group can effectively delay the growth of Bacillus cereus, while the bacteria in the MIC group were completely inhibited from 2 hours onwards. This shows that the inhibitory effect of the rabbit ear aldehyde nanoemulsion on the growth of Bacillus cereus is concentration-dependent and has a strong inhibitory effect on Bacillus cereus vegetative cells.

[0060] Example 7: Scanning electron micrographs of Bacillus cereus before and after treatment with oxalicum aldehyde nanoemulsion

[0061] In the bacterial suspension (10 6 An equal volume of pyralidin nanoemulsion was added to a 1 / 2 MIC (microorganism-specific immunoglobulin CFU / mL) culture medium (microorganism-specific immunoglobulin CFU / mL) to a final concentration of 1 / 2 the MIC and MIC. The same volume of LB liquid medium was added to the control group. After 6 hours of incubation in a constant temperature shaking incubator at 37°C and 180 rpm, samples were collected and washed by centrifugation. The cells were then fixed overnight at 4°C by adding 3% glutaraldehyde. The next day, the pellet was dehydrated with 10%, 30%, 50%, 70%, 90%, and 100% ethanol, followed by dropwise transfer onto a cell slide to dry. The slide was then vacuum-coated with gold and observed using a scanning electron microscope.

[0062] like Figure 7 As can be seen, the bacteria in the control group were rod-shaped, structurally intact, and had clear boundaries between the bacteria, indicating that the bacteria were in a normal growth state. However, after treatment with 1 / 2 MIC, the surface of the bacteria was deformed and concave, and the bacteria were adhered. The deformation of the MIC-treated group was even more severe, with severe collapse and holes on the surface of the bacteria, and some bacteria were even difficult to discern from their original structure. This shows that the rabbit ear aldehyde nanoemulsion has a significant destructive effect on the morphological structure of Bacillus cereus vegetative cells, and this effect is dose-dependent.

[0063] Example 8: Effect of oxalicum aldehyde nanoemulsion on protein and nucleic acid leakage of Bacillus cereus

[0064] The rabbit ear aldehyde nanoemulsion was added to the final concentration of 1 / 2MIC, MIC, and the group without rabbit ear aldehyde nanoemulsion was used as the control. 6 CFU / mL) were mixed in equal volumes. Each group was placed in a constant temperature shaking incubator at 37°C and 180 rpm. 1 mL of bacterial solution was centrifuged (6000 rpm, 5 min) and the supernatant was diluted 10 times. The OD values ​​at 0, 2, 4, 6, and 8 h were measured. 260 ,OD 280 .

[0065] like Figure 8In (a) and (b), the nucleic acid and protein content in the control group remained largely unchanged, while the experimental group showed a significant increase compared to the initial values, particularly in the first 2 hours, when nucleic acid and protein leakage was severe. It is also clear that after 6 hours, the curve slows down and even declines. This is likely because in the early stages of oxadiazol treatment, the bacteria are sensitive to the antibacterial agent, increasing cell membrane permeability and allowing for rapid leakage of proteins and nucleic acids. However, as the reaction time increases, Bacillus cereus's own defense system begins to take effect, resulting in a dynamic balance between destruction and defense repair.

[0066] Example 9: Inhibitory and scavenging effects of oxalicum aldehyde nanoemulsion on Bacillus cereus biofilm

[0067] 1. Inhibitory effect of oxalicum aldehyde nanoemulsion on Bacillus cereus biofilm

[0068] The overnight culture of Bacillus cereus was diluted to a density of 10 8 CFU / mL. Then the bacterial suspension was mixed with a final concentration of 0.01 ～ An equal volume of 0.46 mg / mL pyralidin was added to a 96-well plate and incubated for 48 hours to culture the biofilm. An equal volume of LB medium was added to the control group. The medium was aspirated, washed twice with PBS, and then stained with 0.1% crystal violet for 30 minutes. After washing twice with PBS, 200 μL of 33% glacial acetic acid was added. The plate was placed in a 37°C incubator for 30 minutes, and the OD was measured. 570 .

[0069] The inhibition rate of oxalicum aldehyde nanoemulsion on biofilm was calculated according to formula (1):

[0070] Inhibition rate = (1-OD treatment / OD control )×100%(1)

[0071] Among them: OD treatment and OD control OD of the treatment group and the control group, respectively 570 .

[0072] like Figure 9 (a) With the exception of the 1 / 8 MIC (0.014 mg / mL) group, all other groups significantly inhibited biofilm formation relative to the control group (p < 0.05). Within a certain range, the inhibitory effect on biofilm formation was concentration-dependent. After 24 hours of treatment, the inhibition rates of the 1 / 4 MIC to 2 MIC oxalicum nanoemulsions against Bacillus cereus biofilm formation were 32.62%, 58.86%, 71.29%, and 93.19%, respectively, compared to the control group.

[0073] 2. Removal of Bacillus cereus biofilm by rabbit ear aldehyde nanoemulsion

[0074] Dilute the logarithmic phase Bacillus cereus to a density of 10 8 CFU / mL, mixed with LB medium and added to a 96-well plate, incubated at 37°C for 48 hours to cultivate a mature biofilm. After discarding the medium and washing with PBS, different concentrations of rabbit ear aldehyde nanoemulsion were added to each well, and the wells without rabbit ear aldehyde nanoemulsion were used as blank controls. After being placed in a 37°C incubator for 12 hours, the OD of each well was measured using crystal violet staining. 570 The clearance rate of different concentrations of oxalis aldehyde nanoemulsion on mature biofilm was calculated according to formula (2):

[0075] Clearance rate = (1-OD treatment / OD control )×100%(2)

[0076] Among them: OD treatment and OD control OD of the treatment group and the control group, respectively 570 .

[0077] Figure 9 (b) is the result of biofilm clearance test. The 1 / 4MIC to 2MIC groups can significantly improve the clearance rate of biofilm (p < 0.05). Figure 9 The results of (a) confirm each other.

[0078] In summary, a certain concentration of oxalicum aldehyde nanoemulsion can effectively inhibit the formation of Bacillus cereus biofilm, and also has a good elimination effect on mature biofilm.

[0079] Example 10: Effect of oxalicum aldehyde nanoemulsion on the motility of Bacillus cereus

[0080] To LB medium containing 0.3% agar, 0 (control), 1 / 4 MIC, 1 / 2 MIC, MIC, and 2 MIC of pyralidin nanoemulsion were added. 5 μL of a Bacillus cereus suspension was added to the center of the cooled LB agar. The culture was then allowed to stand in a laminar flow hood for 30 minutes before being transferred to a 37°C incubator. After 24 hours, bacterial motility was observed and the diameter (cm) of the motility zone was measured.

[0081] The results are as follows Figure 10As shown. The Bacillus cereus in the control group had a strong motility, and its movement diameter could reach 4.4 cm. When the concentration of the rabbit ear aldehyde nanoemulsion increased from 1 / 4MIC to 1 / 2MIC, the movement diameter decreased to 3.4 cm and 2.3 cm, respectively. When its concentration was MIC and 2MIC, no colonies were produced on the culture medium surface. The above results show that the rabbit ear aldehyde nanoemulsion inhibited the movement of Bacillus cereus. Therefore, it is reasonable to speculate that the rabbit ear aldehyde nanoemulsion is also capable of reducing the transfer and attachment of Bacillus cereus on food processing equipment, thereby improving the safety of food production.

[0082] Example 11: Inhibitory effect of oxalicum aldehyde nanoemulsion on Bacillus cereus spores

[0083] 1. Production of spores

[0084] Take 100.0μL of logarithmic phase Bacillus cereus bacterial suspension and spread it on tryptone soy agar medium (TSA) supplemented with 40mg / L manganese sulfate and 100mg / L calcium chloride. Place it in a 37℃ incubator and culture for 7 days to induce spore formation. Then scrape the spores with a sterilized slide and suspend them in PBS. Shake the washed bacterial suspension for 5 minutes to break up the bacterial clumps. Filter with sterile gauze to remove the agar clumps. Centrifuge at 6000rpm for 10 minutes, discard the supernatant, wash several times with sterile water, resuspend the spores in PBS, treat in a 70℃~80℃ water bath for 20 minutes to kill the nutrient cells, cool immediately, and then wash again several times. The prepared spore suspension should be stored at 4℃ for no more than 1 month.

[0085] 2. Determination of MIC of oxalicum aldehyde nanoemulsion against Bacillus cereus spores

[0086] The spore suspension (10 6 CFU / mL) was mixed with equal volumes of rabbit ear aldehyde nanoemulsion and the OD values ​​at 0 h and 24 h were measured. 600 The lowest concentration of pyralidin corresponding to the group with a numerical difference of less than 5% was identified as the MIC for Bacillus cereus spores.

[0087] like Figure 11 As shown in (a), when the concentration of pyralidin reaches 0.23 mg / mL, after inoculation with Bacillus cereus spores and culture for 24 h, the OD 600 The results showed that the MIC of the rabbit ear aldehyde nanoemulsion against Bacillus cereus spores was 0.23 mg / mL, which was not much different from the MIC test results for vegetative cells, indicating that the rabbit ear aldehyde nanoemulsion also has a good inhibitory effect on Bacillus cereus spores.

[0088] 3. Determination of spore germination curve

[0089] The rabbit ear aldehyde nanoemulsion was diluted to a final concentration of MIC and 2MIC and then mixed with the spore suspension (10 6 CFU / mL) were mixed in equal volumes, and the control group was added with the same volume of LB medium. The OD was measured every 2 h. 600 , draw the germination curve of Bacillus cereus spores.

[0090] The spore core is in a state of severe dehydration under normal circumstances. When the spore germinates, the core absorbs water and swells, resulting in the OD value of the spore suspension. 600 It shows a short-term downward trend. Figure 11 In (b), the absorbance of the CK group decreased in the initial stage of culture, indicating that the spores were gradually germinating and growing under the culture medium conditions. However, the MIC group (0.23 mg / mL) and the 2MIC group did not experience any significant fluctuations during the entire culture process, and always maintained the initial OD 600 This indicates that 0.23 mg / mL and 0.46 mg / mL of oxalicum aldehyde nanoemulsion can inhibit the germination and growth of spores.

[0091] Example 12: Determination of the germination rate of Bacillus cereus spores by using rabbit ear aldehyde nanoemulsion

[0092] After washing and resuspending the Bacillus cereus spores, they were diluted to a concentration of 10 6 CFU / mL. Then, the mixture was mixed with an equal volume of rabbit ear aldehyde nanoemulsion to achieve a final concentration of 0, MIC, and 2MIC. The mixture was then incubated at 37°C for 6 hours, and the spore counts in each group were calculated by dilution and smearing. The spore germination rate was calculated according to formula (3):

[0093] Spore germination rate = number of spores in the treatment group / number of spores in the control group × 100% (3)

[0094] Bacillus cereus can always exist in the form of spores in harsh environments. Once nutrients or other factors such as dodecylamine and high pressure appear in the environment, the spores will germinate, which seriously threatens the safety of food industry production and the health of consumers. Therefore, it is a key point to explore whether the rabbit ear aldehyde nanoemulsion has the effect of inhibiting spore germination. Figure 12 As shown, compared with the control group, MIC and 2MIC of pyralidin exhibited a significant inhibitory effect on spore germination (p < 0.05), reducing the spore germination rate to 11.78% and 4.06%, respectively. This indicates that pyralidin has a good function of inhibiting spore germination.

[0095] Example 13: Determination of Heat and Salt Resistance of Bacillus cereus Spores by Rabbit Ear Aldehyde Nanoemulsion

[0096] After washing and resuspending the Bacillus cereus spores with sterile distilled water, they were diluted to a concentration of 10 6 CFU / mL. Then, it was mixed with an equal volume of rabbit ear aldehyde nanoemulsion to make the final concentration of the antibacterial agent 0, 1MIC, and 2MIC. It was then cultured at 37°C for 6 hours, centrifuged and washed again, and resuspended in distilled water. To determine the heat resistance of the spores, they were placed in a 90°C constant temperature water bath and heated for 10 minutes, then quickly cooled. After dilution, the spores were spread on TSA plates and the number of spores in each group was counted. To determine the salt resistance of the spores, they were diluted and spread on TSA plates containing NaCl (4%) and counted.

[0097] The heat resistance and salt resistance of spores are the main reasons why they are difficult to be completely removed during food production and processing. Figure 13 (a) shows the effect of oxalicum aldehyde nanoemulsion treatment on the heat resistance of spores. After pretreatment with MIC and 2MIC oxalicum aldehyde nanoemulsion, the total colony count decreased by 1.65 and 1.83 logarithms, respectively, compared with the control group. This indicates that oxalicum aldehyde nanoemulsion can increase the heat sensitivity of spores, significantly reducing their ability to withstand high temperatures. Figure 13 (b) shows the salt tolerance of spores. Compared to heat resistance, salt tolerance did not change significantly. However, when the concentration of pyralidin nanoemulsion reached 2 MIC, the number of spores decreased by 0.57 logs compared to the control group. This indicates that pyralidin nanoemulsion treatment reduces the resistance of spores, which has a significant advantage for the subsequent complete inactivation of spores.

[0098] Example 14: Determination of the hydrophobicity of oxalicum aldehyde nanoemulsion on Bacillus cereus spores

[0099] The spores of Bacillus cereus were washed and resuspended with sterile PBS solution and diluted to 10 6 CFU / mL. Then, it was mixed with the rabbit ear aldehyde nanoemulsion in equal volumes to make the final concentration of the antibacterial agent 0, 1MIC, and 2MIC, and the spore OD in the system was measured. 600 The value of is set as A0. Then it is placed in a 37℃ environment for 6 hours. 3.00mL of spore solution is mixed with 0.60mL of n-hexadecane, mixed thoroughly and allowed to stand for 15 minutes. The water phase is taken to measure OD 600 is A1.

[0100] The ratio of hydrophobic spores (RHS) was calculated according to equation (4):

[0101] RHS(%)=(A0-A1) / A0×100% (4)

[0102] A0: OD of the initial spore suspension 600(Total spore count, including hydrophobic and hydrophilic spores).

[0103] A1: OD of the aqueous phase after mixing with n-hexadecane 600 (Amount of remaining hydrophilic spores).

[0104] Surface hydrophobicity is one of the important factors that determine the adhesion of Bacillus cereus spores. Figure 14 Untreated Bacillus cereus spores exhibited strong hydrophobicity (78.67%). However, after treatment with the oxalicum aldehyde nanoemulsion, the hydrophobicity of the MIC and 2MIC groups decreased to 56% and 47.27%, respectively. This indicates that the interfacial adhesion of the treated spores to hydrophobic surfaces is reduced, which provides a potential possibility for reducing Bacillus cereus spore contamination in food processing.

[0105] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a rabbit ear grass aldehyde nanoemulsion, characterized in that: The steps include: (1) dissolving a surfactant in water and stirring to form a surfactant solution with a volume concentration of 1% to 9%; (2) adding pyralidin dropwise to the surfactant solution obtained in step (1), stirring to form a crude emulsion; (3) The crude emulsion obtained in step (2) is subjected to ultrasonic crushing to form a nanoemulsion, thereby obtaining the oxaliplatin nanoemulsion.

2. The preparation method according to claim 1, characterized in that The surfactant in step (1) is one or more of Tween 80, Span 80 and Tween 20.

3. The preparation method according to claim 2, characterized in that The concentration of the surfactant solution in step (1) is 3-5%.

4. The preparation method according to claim 3, characterized in that The rate of dropwise addition of the pyralidin in step (2) is 1±0.5 drops / s; and the stirring time is 5±2 min.

5. The preparation method according to claim 1, 2, 3 or 4, characterized in that: The final concentration of the pyralidin in step (2) is 0.05-5%, preferably 2%.

6. The preparation method according to claim 1, 2, 3 or 4, characterized in that: The frequency of ultrasonic fragmentation in step (3) is 400±200W, the time is 10±5min, the working time is 5±3s, and the rest time is 2±1s.

7. The preparation method according to claim 5, characterized in that The frequency of ultrasonic fragmentation in step (3) is 400±200W, the time is 10±5min, the working time is 5±3s, and the rest time is 2±1s.

8. The cyperus aldehyde nanoemulsion prepared by the method according to any one of claims 1 to 7.

9. Use of the pyralid nanoemulsion according to claim 8 in the preparation of an antibacterial agent.

10. The use according to claim 9, characterized in that The bacteria inhibited by the antimicrobial agent include Bacillus cereus.

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