Preparation method and application of cinnamyl aldehyde-containing double-layer capsule wall microcapsule

Abstract

The invention discloses a preparation method and application of a cinnamyl aldehyde-containing double-layer capsule wall microcapsule. Uniformly mixing a cinnamyl aldehyde solution, the cinnamyl aldehyde-loaded porous starch and a beta-cyclodextrin solution, performing ultrasonic treatment for 30 minutes, and stirring for 30 minutes to 4 hours; then refrigerating and standing at 4 DEG C for 12h-2d, and then carrying out suction filtration and collecting microcapsules; and finally drying and grinding to obtain the porous starch and beta-cyclodextrin embedded cinnamyl aldehyde double-layer capsule wall microcapsule. The inner capsule wall of the double-layer capsule wall microcapsule is porous starch, and the outer capsule wall of the double-layer capsule wall microcapsule is beta-cyclodextrin. Core material cinnamyl aldehyde is loaded on the basis of the liquid characteristic of cinnamyl aldehyde and the high adsorption characteristic of internal pores of porous starch, then surface pores of the porous starch are encapsulated by utilizing the small molecule characteristic of beta-cyclodextrin, then cinnamyl aldehyde is subjected to double-layer embedding, and the microcapsules containing double capsule walls are prepared. The microcapsule has good embedding performance, bacteriostatic activity, recyclability and food preservative effect, and can be popularized and used as a bacteriostatic agent and a food preservative.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of preservatives in food, and particularly relates to a preparation method of a double-layer capsule wall microcapsule containing cinnamaldehyde and application thereof. BACKGROUND

[0002] Cinnamaldehyde is a low-molecular-weight hydrophobic aromatic aldehyde and is the main active ingredient of cinnamon essential oil. Studies have shown that cinnamaldehyde has good antibacterial activity against Fusarium solani, and its antibacterial activity is stronger than that of carvacol, perillic aldehyde, and potassium sorbate and carbendazim chemical inhibitors (Inhibitory effect of cinnamaldehyde on Fusarium solani and its application in postharvest preservation of sweet potato, Food Chemistry, 2023, 408: 135213). Studies have also found that cinnamaldehyde has good antibacterial activity against food spoilage bacteria Aspergillus flavus (Unveiling the influence of the Afatg3 gene: Autophagy’s role in sporulation, aflatoxin synthesis, pathogenicity, and drug stress response in Aspergillus flavus, Food Bioscience, 2025, 68: 106774). Since chemical antibacterial agents have recognized potential health and safety risks, cinnamaldehyde is a highly active natural antibacterial agent and has good application prospects for development as a green preservative.

[0003] Microcapsule technology is a technology for encapsulating solid, liquid or gas core materials in a small container, which can protect the core material from external air, light and other factors, and achieve controlled release of the core material. The commonly used wall material for encapsulating cinnamaldehyde is β-cyclodextrin, but the embedding rate of β-cyclodextrin for cinnamaldehyde is low. CN114522635A discloses an antibacterial microcapsule with controllable release of cinnamaldehyde and a preparation method thereof. Hollow V-shaped starch is dispersed in anhydrous ethanol containing cinnamaldehyde, and then deionized water is added to make the mass concentration of ethanol 40-60%; after sealing and stirring, cooling to room temperature, centrifugation, washing and drying, an antibacterial microcapsule with controllable release of cinnamaldehyde is obtained. Although this method achieves efficient embedding of cinnamaldehyde, the preparation process of hollow V-shaped starch is complex. Therefore, it is very important to provide a preparation method that is simple in process and can improve the stability of essential oils, embedding rate and antibacterial effect. SUMMARY

[0004] One of the objectives of this invention is to provide a method for preparing double-walled microcapsules containing cinnamaldehyde. The method is simple and can improve the stability, sustained-release properties, and antibacterial effects of plant essential oils such as cinnamaldehyde, thereby promoting the research and development of natural food preservatives.

[0005] The second objective of this invention is to provide an application for the cinnamaldehyde-containing double-walled microcapsules prepared by the above-mentioned method, thereby improving the preservative activity and recyclability of food preservatives prepared from plant essential oils such as cinnamaldehyde.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing a double-walled microcapsule containing cinnamaldehyde, comprising the following steps: Step 1: Preparation of cinnamaldehyde solution: Cinnamaldehyde is dissolved in anhydrous ethanol to obtain cinnamaldehyde solution; Step 2: Preparation of porous starch loaded with cinnamaldehyde: The porous starch was added to cinnamaldehyde and mixed thoroughly. After vacuum treatment, it was ultrasonically treated for 30 min, then refrigerated at 4℃ for 30 min to 2 h. After centrifugation, the supernatant was discarded to obtain porous starch loaded with cinnamaldehyde. Step 3: Preparation of β-cyclodextrin solution: β-cyclodextrin is added to water and heated at 60℃~80℃ until completely dissolved to obtain β-cyclodextrin solution; Step 4: Preparation of double-walled microcapsules: Cinnamaldehyde solution, porous starch loaded with cinnamaldehyde, and β-cyclodextrin solution were mixed and stirred for 30 min, followed by stirring for 30 min to 4 h. The microcapsules were then collected by filtration after being refrigerated at 4 °C for 12 h to 2 d. Finally, the microcapsules were dried and ground to obtain double-walled microcapsules with porous starch and β-cyclodextrin encapsulating cinnamaldehyde.

[0007] Preferably, in step one, the mass-to-volume ratio of cinnamaldehyde to anhydrous ethanol in the cinnamaldehyde solution is 2.5g~10g:20mL.

[0008] Preferably, in step two, the mass-to-volume ratio of the porous starch to cinnamaldehyde is 7.5g~12.5g:20mL.

[0009] Preferably, in step two, the centrifugation speed is 800~2000 rpm and the centrifugation time is 10 min.

[0010] Preferably, in step three, the mass concentration of the β-cyclodextrin solution is 0.075~0.125 g / mL.

[0011] Preferably, in step four, the mass ratio of cinnamaldehyde, porous starch, and β-cyclodextrin is 20~60:45~75:45~75.

[0012] More preferably, in step four, the mass ratio of cinnamaldehyde, porous starch, and β-cyclodextrin is 20:45:75, at which point the antibacterial effect is optimal.

[0013] Secondly, the present invention provides cinnamaldehyde-containing bilayer microcapsules prepared by the above method. The bilayer microcapsules have an inner wall composed of porous starch and an outer wall composed of β-cyclodextrin.

[0014] Thirdly, the present invention provides the application of the double-walled microcapsules prepared by the above method as an antibacterial agent for Fusarium solani.

[0015] Fourthly, the present invention provides the application of the double-walled microcapsules prepared by the above method as an antibacterial agent against Aspergillus flavus.

[0016] Fifthly, the present invention provides the application of the double-walled microcapsules prepared by the above method in food preservation.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The method for preparing double-walled microcapsules in this invention uses porous starch and β-cyclodextrin as wall materials, which can effectively encapsulate the core material cinnamaldehyde, and achieve a higher encapsulation rate of essential oils. Conventional β-cyclodextrin has an encapsulation rate of 2%~8% for essential oils (…). Figure 1 However, the essential oil encapsulation rate of the double-walled microcapsules of the present invention can reach 8%~24%, which is 200% higher (Table 2).

[0018] 2. The double-walled microcapsules prepared in this invention have a better inhibitory effect on food spoilage bacteria. When applied, these microcapsules allow the essential oils inside to naturally diffuse into the air, achieving non-contact gas-phase sterilization. The double-walled microcapsules of this invention have a better antibacterial effect against Aspergillus flavus and Fusarium solani than microcapsules prepared by a simple combination of β-cyclodextrin and porous starch. Figure 9 , Figure 10 ).

[0019] 3. The double-walled microcapsules prepared in this invention have a sustained-release antibacterial effect and can be recycled. Through volatilization antibacterial experiments, it was found that the double-walled microcapsules of this invention, compared to microcapsules prepared by a simple combination of β-cyclodextrin and porous starch, exhibited good antibacterial activity against Aspergillus flavus and Fusarium solani after one treatment and subsequent reuse twice. Figure 9 , Figure 10 ).

[0020] 4. The double-walled microcapsules prepared in this invention have better food preservation effects. When used, these microcapsules allow the essential oils inside to naturally diffuse into the food storage space, providing non-contact vapor-phase preservation, making them convenient to use. The double-walled microcapsules of this invention have a better inhibitory effect on peanut mold growth caused by Aspergillus flavus and the production of AFB1 than microcapsules prepared by a simple combination of β-cyclodextrin and porous starch. Figure 11 , Figure 12 ).

[0021] Therefore, compared with existing technologies, the double-walled microcapsules of this invention have excellent encapsulation performance, antibacterial activity, recyclability, and food preservation effects. This invention is beneficial to the research, development, and application of novel non-contact sustained-release food preservatives. Attached Figure Description

[0022] Figure 1 This study investigates the effect of different ratios of cinnamaldehyde (core material) and β-cyclodextrin (wall material) on the microcapsules and their oil loading capacity. *Represents... p <0.05, ** represents p <0.01, *** represents p <0.001; Figure 2 The effects of cinnamaldehyde, porous starch loaded with cinnamaldehyde in different proportions, and β-cyclodextrin on microcapsules; Figure 3 The effects of different ratios of core material cinnamaldehyde and cinnamaldehyde-loaded porous starch and β-cyclodextrin on microcapsules; Figure 4 The orthogonal experiment optimized the effect of the addition ratio of cinnamaldehyde to the wall material porous starch and β-cyclodextrin on the microcapsules. Figure 5 This is a microstructure diagram of a double-walled microcapsule containing cinnamaldehyde; Figure 6 This is a process flow diagram of the double-walled microcapsules containing cinnamaldehyde obtained through orthogonal experimental optimization; Figure 7 This study compares the antibacterial effects of cinnamaldehyde-containing double-walled microcapsules prepared by orthogonal experiments on Fusarium solani. Figure 8 This study compares the antibacterial effects of cinnamaldehyde-containing double-walled microcapsules prepared by orthogonal experiments against Aspergillus flavus. Figure 9 This study compares the antibacterial effects of double-walled microcapsules with those of simple combinations of double-walled and single-walled β-cyclodextrin microcapsules against Fusarium solani. (a)–(b): Diameter of the inhibition zone and its statistical graph after the first use of microcapsules; (c)–(d): Inhibition zone and its statistical graph after the recovery of microcapsules after one use; (e)–(f): Inhibition zone and its statistical graph after the recovery of microcapsules after two uses. Figure 10 This study compares the antibacterial effects of double-walled microcapsules with those of simple combinations of double-walled and single-walled β-cyclodextrin microcapsules against Aspergillus flavus. (a)–(b): Diameter of the inhibition zone and its statistical graph after the first use of microcapsules; (c)–(d): Inhibition zone and its statistical graph after the recovery of microcapsules after one use; (e)–(f): Inhibition zone and its statistical graph after the recovery of microcapsules after two uses. Figure 11 This study compares the preservative effects of double-walled microcapsules with those of simple combinations of double-walled and single-walled β-cyclodextrin microcapsules against peanut spoilage caused by Aspergillus flavus. Figure 12 This study compares the inhibitory effects of double-walled microcapsules and simple combinations of double-walled and single-walled β-cyclodextrin microcapsules on aflatoxin in peanuts. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0024] The chemical materials and reagents used in the following examples are all commercially available products.

[0025] Example 1: Preparation of cinnamaldehyde-containing microcapsules using β-cyclodextrin as the sole wall material Preparation of β-cyclodextrin solution: Add 10g of β-cyclodextrin to water and make up to 100mL. Heat in a water bath to 70℃ and stir until the β-cyclodextrin is completely dissolved.

[0026] 0 g, 1.25 g, 1.67 g, 2.5 g, and 5 g of cinnamaldehyde were dissolved in anhydrous ethanol to a final volume of 20 mL. The dissolved cinnamaldehyde was then added to a β-cyclodextrin solution to achieve core-to-material ratios of 0:120, 15:120, 20:120, 30:120, and 60:120, respectively. After mixing and sonicating for 30 min, the mixture was magnetically stirred at 400 rpm for 2.5 h and then allowed to stand at 4 °C for 12 h. The sample was poured into a Buchner funnel and collected by vacuum filtration. The sample was then dried in an oven at 37 °C for 6 h. Finally, the sample was ground in a mortar and pestle to obtain β-cyclodextrin-encapsulated cinnamaldehyde-containing microcapsules.

[0027] The cinnamaldehyde content loaded in the microcapsules was determined by differential gravimetric analysis. 0.1 g of microcapsule powder was weighed, added to 1 mL of anhydrous ethanol, sonicated for 1 h, and then soaked for 12 h to extract the essential oil embedded within the microcapsules. The sample was centrifuged at 12000 rpm for 5 min, the supernatant was discarded, and the precipitate was obtained. Then, 1 mL of anhydrous ethanol was added, sonicated for 1 h, and then soaked for 12 h. The precipitate was collected by centrifugation. It was dried at 80℃ to constant weight. The sample was weighed using an analytical balance; the difference in weight between the sample and the sample washed with ethanol was the essential oil content within 0.1 g of microcapsules. The percentage of the obtained essential oil content to the weight of the microcapsule sample not eluted with ethanol was the cinnamaldehyde loading of the β-cyclodextrin microcapsules. The loading was determined based on the color of the microcapsule powder (…). Figure 1 (a) and the essential oil loading of the microcapsules ( Figure 1 (b) The optimal mass ratio of core material cinnamaldehyde to wall material β-cyclodextrin is 30:120 (with the addition of 2.5g of cinnamaldehyde) to obtain the best microcapsules.

[0028] Example 2: Effect of the amount of porous starch loaded with cinnamaldehyde on the amount of microcapsules with double-layered walls Preparation of cinnamaldehyde solution: Dissolve 2.5g of cinnamaldehyde in 50mL of ethanol to obtain cinnamaldehyde solution for later use.

[0029] Preparation of porous starch loaded with cinnamaldehyde: Weigh 2.5g, 5g, 7.5g, 10g, and 12.5g of porous starch into 50mL centrifuge tubes, add 20mL of cinnamaldehyde, vortex to mix, place in a vacuum bag to remove internal air, sonicate for 30min, then place in a 4℃ refrigerator to stand for 30min, centrifuge at 800rpm for 10min, discard the supernatant (cinnamaldehyde not adsorbed by the porous starch), and obtain porous starch loaded with cinnamaldehyde for later use.

[0030] Preparation of β-cyclodextrin solution: Add 10g of β-cyclodextrin to water and make up to 100mL. Heat in a water bath to 70℃ to completely dissolve the β-cyclodextrin, and keep warm at 45℃ for later use.

[0031] Cinnamaldehyde solution and cinnamaldehyde-loaded porous starch were added to β-cyclodextrin solution to achieve cinnamaldehyde:porous starch:β-cyclodextrin ratios of 5:5:20, 5:10:20, 5:15:20, 5:20:20, and 5:30:20, respectively. The mixture was sonicated for 30 min and then magnetically stirred at 400 rpm for 2.5 h. The sample was then placed in a 4℃ refrigerator and allowed to stand for 12 h. Microcapsules were collected by vacuum filtration, dried in an oven at 37℃ for 6 h, and then ground to obtain β-cyclodextrin and porous starch-embedded cinnamaldehyde-containing bilayer capsules.

[0032] Figure 2The images show the actual products of double-walled microcapsules prepared with different proportions of porous starch. It can be seen that the double-walled microcapsules with proportions of 5:5:20 and 5:10:20 have better quality. As the proportion of porous starch gradually increases, the microcapsules clump together and stick to each other, indicating that an excessively high proportion of porous starch will affect the quality of the microcapsules.

[0033] Example 3: Effect of Cinnamaldehyde Addition on Bilayer Capsule Wall Prepared from Porous Starch and β-Cyclodextrin Preparation of cinnamaldehyde solution: Take 2.5g, 3.33g, 5g and 10g of cinnamaldehyde respectively, add them to anhydrous ethanol and make up to 20mL to dissolve them completely before use.

[0034] Preparation of β-cyclodextrin solution: Add 10g of β-cyclodextrin to water and make up to 100mL. Heat in a water bath to 70℃ to completely dissolve the β-cyclodextrin, and keep warm at 45℃ for later use.

[0035] Preparation of porous starch loaded with cinnamaldehyde: Weigh 10g of porous starch into a 50mL centrifuge tube, add 20mL of cinnamaldehyde, vortex mix, place in a vacuum bag to remove internal air, sonicate for 30min, then place in a 4℃ refrigerator to stand for 30min, centrifuge at 800rpm for 10min and discard the supernatant to obtain porous starch loaded with cinnamaldehyde for later use.

[0036] Ethanol solutions with different amounts of cinnamaldehyde and porous starch loaded with cinnamaldehyde were added to β-cyclodextrin solutions to achieve cinnamaldehyde:porous starch:β-cyclodextrin ratios of 15:60:60, 20:60:60, 30:60:60, and 60:60:60, respectively. The mixtures were sonicated for 30 min and then magnetically stirred at 400 rpm for 2.5 h. The samples were then placed in a 4℃ refrigerator and allowed to stand for 12 h. Microcapsules were collected by vacuum filtration, dried in an oven at 37℃ for 6 h, and ground to obtain β-cyclodextrin and porous starch as double-walled materials for encapsulating microcapsules with different amounts of cinnamaldehyde.

[0037] Figure 3 These are images of double-walled microcapsules prepared with different proportions of cinnamaldehyde. It can be seen that as the proportion of cinnamaldehyde in the double-walled encapsulation system increases, the powder surface color lightens, and the cinnamon aroma of the microcapsules decreases. This result indicates that when the ratio of core material to wall material in the double-walled encapsulation system is too high, cinnamaldehyde cannot be fully encapsulated, thus reducing the quality of the microcapsules. Therefore, the optimal ratios of cinnamaldehyde:porous starch:β-cyclodextrin in the double-walled encapsulation system are 15:60:60 and 20:60:60, respectively.

[0038] Example 4: Orthogonal design optimization of bilayer capsule wall microcapsule formulation Based on the above single-factor experimental results, the microcapsule formulation of the double-layer capsule wall embedding system was optimized by orthogonal experimental design using cinnamaldehyde, porous starch loaded with cinnamaldehyde, and β-cyclodextrin (see Table 1).

[0039] Table 1. Formulation optimization of double-walled microcapsules L93 3 Orthogonal array Preparation of cinnamaldehyde solution: Take 3.33g, 5g and 10g of cinnamaldehyde respectively, add them to anhydrous ethanol and make up to 20mL to dissolve and set aside.

[0040] Preparation of porous starch loaded with cinnamaldehyde: Weigh 7.5g, 10g, and 12.5g of porous starch into 50mL centrifuge tubes, add 20mL of cinnamaldehyde, vortex to mix, place in a vacuum bag to remove internal air, sonicate for 30min, then place in a 4℃ refrigerator to stand for 30min, centrifuge at 800rpm for 10min and discard the supernatant to obtain porous starch loaded with cinnamaldehyde for later use.

[0041] Preparation of β-cyclodextrin solution: Take 7.5g, 10g, and 12.5g of β-cyclodextrin respectively, add them to water, and make up to 100mL. Heat in a water bath to 70℃ to completely dissolve the β-cyclodextrin, and keep warm at 45℃ for later use.

[0042] Cinnamaldehyde solution and cinnamaldehyde-loaded porous starch and β-cyclodextrin solution were mixed separately, with cinnamaldehyde:porous starch:β-cyclodextrin ratios of 20:45:45, 20:60:75, 20:75:60, 30:45:75, 30:60:60, 30:75:45, 60:45:60, 60:60:45, and 60:45:75, respectively. After mixing and sonicating for 30 min, the mixture was magnetically stirred at 400 rpm for 2.5 h. The sample was then placed in a 4℃ refrigerator and allowed to stand for 12 h. Microcapsules were collected by vacuum filtration, dried in an oven at 37℃ for 6 h, and ground to obtain double-walled microcapsules of porous starch and β-cyclodextrin encapsulating cinnamaldehyde. Figure 4 ).

[0043] The cinnamaldehyde content loaded on the bilayer microcapsule was determined by differential gravimetric analysis. 0.1 g of microcapsule powder was weighed, added to 1 mL of anhydrous ethanol, sonicated for 1 h, and then soaked for 12 h. The mixture was centrifuged at 12000 rpm for 5 min, the supernatant was discarded, and the precipitate was collected. Then, 1 mL of anhydrous ethanol was added, sonicated for 1 h, and then soaked for 12 h. The precipitate was collected by centrifugation. It was dried at 80℃ to constant weight. The weight difference between the sample and the ethanol-washed sample was used to determine the essential oil content within 0.1 g of microcapsules. The percentage of essential oil content to the weight of the microcapsule sample not eluted with ethanol was the cinnamaldehyde loading of the bilayer microcapsule (Table 2).

[0044] Based on the range results, the influence on the quality of bilayer wall microcapsules was cinnamaldehyde > β-cyclodextrin > porous starch. The optimal formulation for the obtained bilayer wall microcapsules was cinnamaldehyde: porous starch: β-cyclodextrin = 3.33:7.5:12.5 (i.e., 20:45:75). The preparation process of cinnamaldehyde-containing bilayer wall microcapsules with this formulation is as follows: Figure 6 As shown. Figure 5 The scanning electron microscope shown reveals that porous starch has a good pore structure. The pores of the porous starch loaded with cinnamaldehyde are filled with liquid cinnamaldehyde. The porous starch loaded with cinnamaldehyde is further encapsulated with β-cyclodextrin, so that the pores of the microcapsules are covered, forming spherical double-walled microcapsules.

[0045] Table 2. Experimental results of orthogonal design optimization of bilayer capsule formulation. Example 5: Antibacterial effect of cinnamaldehyde-containing double-walled microcapsules prepared by orthogonal experiment against Fusarium solani and Aspergillus flavus. Fusarium solani ( Fusarium solani Aspergillus flavus is a major pathogen causing sweet potato root rot, potato dry rot, soybean root rot, and human fungal keratitis. Aspergillus flavus Aspergillus flavus is a major spoilage bacterium in agricultural products. Food contaminated with Aspergillus flavus also secretes aflatoxins, potent carcinogens, among which aflatoxin B1 (AFB1) is the most toxic and is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC).

[0046] Fusarium solani and Aspergillus flavus were cultured on potato dextrose agar (PDA) for 7 days for later use. Spores were washed with 0.85% physiological saline to prepare 5 × 10⁻⁶ spores. 6 A spore suspension of 100 μL / mL was prepared. 100 μL of the bacterial suspension was spread onto a petri dish containing 15 mL of PDA medium, and the dish was inverted. 0.02 g of microcapsule powder was placed in the center of the dish lid. After incubation at 28°C for 2 days, the antibacterial effect of each microcapsule was evaluated by observing the size of the inhibition zone.

[0047] Figure 7 (a) shows the inhibition zone of cinnamaldehyde-containing double-walled microcapsules against Fusarium solani. Figure 7 (b) shows the measurement results of the inhibition plaque diameter of Fusarium solani containing cinnamaldehyde-containing double-walled microcapsules.

[0048] Figure 8 (a) shows the inhibition zone of cinnamaldehyde-containing double-walled microcapsules against Aspergillus flavus. Figure 8 (b) shows the measurement results of the inhibition plaque diameter of the double-walled microcapsules containing cinnamaldehyde against Aspergillus flavus.

[0049] Depend onFigure 7 , Figure 8 As shown in Table 2, the size of the inhibition zone is closely related to the essential oil content of each microcapsule. The two groups with a mass ratio of cinnamaldehyde: porous starch: β-cyclodextrin of 30:45:75 and 30:60:60 have larger inhibition zones and higher essential oil content, and thus better antibacterial effects.

[0050] Example 6: Sustained-release antibacterial effect of double-walled microcapsules and simple combinations of double-walled and single-walled β-cyclodextrin microcapsules on Fusarium solani and Aspergillus flavus. Preparation of simple combined double-walled microcapsules: 5g of cinnamaldehyde was dissolved in anhydrous ethanol and the volume was adjusted to 100mL to obtain a cinnamaldehyde alcohol solution. 10g of β-cyclodextrin was added to RO water and the volume was adjusted to 100mL. The solution was heated to 70℃ to dissolve the β-cyclodextrin, preparing a 10% β-cyclodextrin aqueous solution. The cinnamaldehyde solution, 7.5g of porous starch, and the β-cyclodextrin solution were mixed separately, sonicated for 30min, and then magnetically stirred at 400rpm for 2.5h. The sample was then placed in a 4℃ refrigerator and allowed to stand for 12h. The microcapsules were collected by vacuum filtration, dried in an oven at 37℃ for 6h, and ground to obtain double-walled microcapsules of porous starch and β-cyclodextrin simply encapsulating cinnamaldehyde.

[0051] Fusarium solani and Aspergillus flavus were cultured on PDA medium for 7 days for later use. Spores were washed with 0.85% physiological saline to prepare 5 × 10⁻⁶ spores. 6 A spore suspension of 100 μL / mL was prepared. 100 μL of the bacterial suspension was spread onto a petri dish containing 15 mL of PDA medium, and the dish was inverted. 0.05 g of microcapsule powder was placed in the center of the dish lid. After incubation at 28°C for 2 days, the size of the inhibition zone was measured for each experimental group. The antibacterial effect of the microcapsules was evaluated based on the size of the inhibition zone.

[0052] Keeping the sample powder still, remove the petri dish cap containing the sample for microcapsule recovery. Using the petri dish cap, transfer the microcapsule sample to a new petri dish coated with *Fusarium solani* and *Aspergillus flavus* spore suspension, and continue culturing. After 2 days, measure the size of the inhibition zone for each experimental group. Keeping the sample powder still, remove the petri dish cap containing the sample for microcapsule recovery. Using the petri dish cap, transfer the microcapsule sample to a new petri dish coated with *Fusarium solani* and *Aspergillus flavus* spore suspension, and continue culturing for 2 days. Measure the size of the inhibition zone for each experimental group; the size of the inhibition zone is used to evaluate the sustained-release properties and antibacterial effect of the microcapsules.

[0053] The double-walled microcapsules prepared in this invention exhibit better inhibitory effects against food spoilage bacteria. When applied, these microcapsules allow the internal essential oils to naturally diffuse into the air, achieving non-contact gas-phase sterilization. The double-walled microcapsules of this invention show better antibacterial effects against Aspergillus flavus and Fusarium solani than microcapsules prepared by a simple combination of β-cyclodextrin and porous starch.Figure 9 , Figure 10 ).

[0054] Furthermore, the double-walled microcapsules exhibit sustained-release antibacterial effects and can be recycled for inhibiting the growth of food spoilage bacteria. Volatile antibacterial experiments revealed that, compared to microcapsules prepared from a simple combination of β-cyclodextrin and porous starch, the double-walled microcapsules of this invention showed better antibacterial activity against Aspergillus flavus and Fusarium solani when recycled and reused twice. Figure 9 , Figure 10 ).

[0055] Example 7: The effect of double-walled microcapsules and simple combinations of double-walled and single-walled β-cyclodextrin microcapsules on the prevention and control of peanut spoilage and toxin production caused by Aspergillus flavus. Break the peanuts open, remove the skins, and pour them into a 2% sodium hypochlorite solution. Sterilize by shaking at 28℃ and 180 rpm for 10 minutes. Then wash the peanuts twice with sterile water. Add 1×10⁻⁶... 5 Peanuts were soaked in a suspension of Aspergillus flavus spores / mL. The mixture was then incubated with shaking at 37℃ and 180 rpm for 30 min. The bacterial suspension was discarded, and the peanuts inoculated with Aspergillus flavus were placed into a 9 cm diameter petri dish using sterile forceps. 0.2 g of microcapsules with different treatment processes were placed on the lid of a 3.5 cm diameter petri dish and then placed inside the larger petri dish containing the peanuts. The petri dish was sealed with plastic wrap. After incubation at 28℃ for 4 days, the infection status of the peanuts with Aspergillus flavus was observed and photographed.

[0056] Take two peanut kernels from each group, place them in a 50mL centrifuge tube, add 6mL of sterile water, vortex vigorously for 3 minutes, then centrifuge at 8000rpm for 3 minutes. Collect the clear supernatant for later use. Transfer 800μL of the supernatant to a 2mL centrifuge tube and add 800μL of dichloromethane. Seal each centrifuge tube with sealing film, vortex for 1 minute, centrifuge at 9000rpm for 5 minutes, then aspirate 600μL of the lower dichloromethane layer and transfer it to a 1.5mL centrifuge tube. Place the tube in a fume hood and air dry overnight. Add 20μL of dichloromethane, rinse the centrifuge tube walls with a pipette, continuously pipetting until the dichloromethane evaporates to 10μL. Then, spot the evaporated dichloromethane onto a pre-marked thin-layer chromatography plate using a pipette. After adding 10 μL of AFB1 standard, the plate was placed in a fume hood and chromatography was performed in the prepared chromatography solution (5 mL acetone and 45 mL dichloromethane). When the chromatography solution reached two-thirds of the plate, the toxin production of each sample group was observed under the ultraviolet light of a gel imaging system and photographed for record.

[0057] The double-walled microcapsules prepared in this invention exhibit better food preservation effects. When used, these microcapsules allow the internal essential oils to naturally diffuse into the food storage space, providing non-contact vapor-phase preservation, and are convenient to use. The double-walled microcapsules of this invention have a better inhibitory effect on peanut mold caused by Aspergillus flavus than microcapsules prepared by a simple combination of β-cyclodextrin and porous starch. Figure 11 Under ultraviolet light, the brighter the sample, the higher the AFB1 content. Therefore, double-walled microcapsules have a better inhibitory effect on peanut toxin AFB1 than microcapsules prepared by a simple combination of β-cyclodextrin and porous starch. Figure 12 ).

[0058] This invention utilizes the liquid properties of cinnamaldehyde and the high adsorption characteristics of the internal pores of porous starch to load cinnamaldehyde as the core material. Then, the small molecule characteristics of β-cyclodextrin are used to encapsulate the surface pores of the porous starch, resulting in a double-layer encapsulation of cinnamaldehyde, thus preparing microcapsules with a double-layered wall. Non-contact antibacterial experiments revealed that, compared to single-wall encapsulation techniques using β-cyclodextrin and simple double-wall encapsulation techniques using a mixture of β-cyclodextrin and porous starch, this invention improves the antibacterial activity of cinnamaldehyde microcapsules against foodborne microorganisms such as *Aspergillus flavus* and *Fusarium solani*. Furthermore, recovery antibacterial experiments showed that the double-layered microcapsules exhibited good sustained-release antibacterial effects. Non-contact preservative experiments showed that the double-layered microcapsules had a good control effect on peanut spoilage caused by *Aspergillus flavus*. The double-layered microcapsules containing cinnamaldehyde essential oil of this invention have certain reference value for the research and application of novel non-contact sustained-release food preservatives.

[0059] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A process for the preparation of a cinnamaldehyde-containing double-layered wall microcapsule, characterized in that, Comprising the following steps: Step one, preparation of cinnamaldehyde solution: cinnamaldehyde is added to anhydrous ethanol to dissolve thoroughly, to obtain cinnamaldehyde solution; Step two, preparation of cinnamaldehyde-loaded porous starch: porous starch is added to cinnamaldehyde to mix thoroughly, vacuum treatment, then ultrasonic treatment for 30 min, 4℃ cold storage for 30 min~2h, centrifugation, and the supernatant is discarded to obtain cinnamaldehyde-loaded porous starch; Step three, preparation of β-cyclodextrin solution: β-cyclodextrin is added to water, heated to complete dissolution at 60℃~80℃, to obtain β-cyclodextrin solution; Step four, preparation of double-layer capsule wall microcapsule: cinnamaldehyde solution, cinnamaldehyde-loaded porous starch and β-cyclodextrin solution are mixed and mixed thoroughly, ultrasonic treatment for 30 min, then stirring for 30 min~4h; followed by 4℃ cold storage for 12h~2d, vacuum filtration to collect microcapsules; finally, drying and grinding to obtain double-layer capsule wall microcapsules of porous starch and β-cyclodextrin-embedded cinnamaldehyde.

2. A process for the preparation of a cinnamaldehyde containing double layer wall microcapsule according to claim 1, characterized in that, In step one, the mass-volume ratio of cinnamaldehyde to anhydrous ethanol in the cinnamaldehyde solution is 2.5g~10g:20mL.

3. The process for the preparation of a cinnamaldehyde containing double layer wall microcapsule according to claim 1, characterized in that, In step two, the mass-volume ratio of porous starch to cinnamaldehyde is 7.5g~12.5g:20mL.

4. The process for the preparation of a cinnamaldehyde containing double layer wall microcapsule according to claim 1, characterized in that, In step two, the centrifugal speed is 800~2000rpm, and the centrifugal time is 10min.

5. The process for the preparation of a cinnamaldehyde containing double layer wall microcapsule according to claim 1, characterized in that, In step three, the mass concentration of the β-cyclodextrin solution is 0.075~0.125g / mL.

6. The process for the preparation of a cinnamaldehyde containing double layer wall microcapsule according to claim 1, characterized in that, In step four, the mass ratio of cinnamaldehyde, porous starch and β-cyclodextrin is 20~60:45~75:45~75.

7. A process for the preparation of a cinnamaldehyde containing double layer wall microcapsule according to claim 6, characterized in that, In step four, the mass ratio of cinnamaldehyde, porous starch and β-cyclodextrin is 20:45:

75.

8. A cinnamaldehyde-containing double-layered wall microcapsule prepared by the method of any one of claims 1 to 7, characterized in that, The double-layer capsule wall microcapsule has an inner wall composed of porous starch and an outer wall composed of β-cyclodextrin.

9. The double-layer capsule wall microcapsule of claim 8 as a Fusarium solani inhibitor or a Aspergillus flavus inhibitor.

10. The double-layer capsule wall microcapsule of claim 8 for food preservation.

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