Core-shell type plant essential oil disinfection microcapsule and preparation and application thereof

Abstract

This invention relates to a core-shell plant essential oil disinfectant microcapsule, its preparation method, and its application. Specifically, it relates to a core-shell plant essential oil disinfectant microcapsule, which, from the inside out, comprises: a core, a Pickering interface layer, an ionic crosslinking gel layer, and a covalently crosslinked polymer shell. The core is composed of a blend of Litsea cubeba essential oil and cinnamon essential oil. The Pickering interface layer coats the surface of the core and is stabilized by nanocellulose as a solid particle emulsifier. The ionic crosslinking gel layer coats the outside of the Pickering interface layer and is composed of sodium alginate crosslinked with calcium ions. The covalently crosslinked polymer shell coats the outer surface of the gel layer and is formed by covalently crosslinking chitosan and geniposide. This invention not only has strong academic value but also good industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of disinfection technology, specifically to a core-shell type plant essential oil disinfection microcapsule and its preparation and application. Background Technology

[0002] Disinfectants are an important technical measure to cut off the spread of pathogens, control livestock and poultry diseases, and ensure public health security. Traditional disinfectant types mainly include: chlorine-containing (sodium hypochlorite, chlorine dioxide), oxygen-containing (hydrogen peroxide, peracetic acid), aldehydes (glutaraldehyde, formaldehyde), and quaternary ammonium salts. These chemical agents usually have the advantages of rapid action and broad bactericidal spectrum, but a series of problems have also been exposed in the long-term application process: (1) Tolerance problem: some microorganisms show a tendency to tolerate under long-term low-dose exposure; (2) Safety problem: glutaraldehyde, formaldehyde, etc. have obvious irritant or potential carcinogenic risks; (3) Environmental problem: chlorine-containing agents are prone to forming toxic byproducts, and excessive discharge will cause pollution to water bodies and soil; (4) Compatibility problem: most chemical disinfectants have a corrosive or aging effect on metal equipment, plastic pipes and rubber seals; (5) Application limitation: the killing effect is significantly reduced under organic load conditions (such as manure, feed residue).

[0003] Plant essential oils, as naturally derived volatile active substances, have become a hot topic in disinfectant research in recent years due to their advantages such as broad-spectrum antibacterial activity, low likelihood of inducing drug resistance, high safety, and environmental friendliness. Litsea cubeba essential oil (LCEO) is a natural essential oil extracted by distillation from the fruit, leaves, or flowers of Litsea cubeba, a common medicinal plant in southern my country. Its main components include citral, limonene, and linalool. Existing studies have shown that LCEO has inhibitory or bactericidal effects on various pathogenic bacteria, including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and it is included in the list of permitted natural flavorings in my country's "Standards for the Use of Food Additives," and its safety has been verified.

[0004] Nevertheless, single essential oils face the following bottlenecks in practical applications: (1) Poor dispersion in aqueous phase: They are highly hydrophobic and easily flocculate, become turbid, and separate into layers in aqueous solutions; (2) Insufficient stability: They are easily affected by light, temperature, and oxidation, leading to component degradation and performance fluctuations; (3) Odor problems: High-concentration essential oils are often accompanied by strong odors, which limits their use in enclosed or densely populated places; (4) Decreased activity under organic load: In fecal or high-protein environments, active ingredients are easily adsorbed or deactivated, resulting in reduced sterilization efficiency; (5) High concentration required: To achieve effective sterilization, a higher concentration of essential oil is required, which increases the cost of use.

[0005] To overcome the aforementioned shortcomings, microencapsulation technology has been widely studied for the encapsulation and protection of essential oils. Existing technologies often employ methods such as complex coagulation, spray drying, or molecular inclusion (e.g., β-cyclodextrin). For example, gelatin and gum arabic or sodium carboxymethyl cellulose are used to form capsule walls through pH-induced complex coagulation. These methods improve the stability of essential oils and achieve sustained release to some extent, but the resulting microcapsule structures often have significant limitations: the mechanical strength of the wall materials (especially proteins) is generally insufficient, and the capsule walls are loose and porous, resulting in limited encapsulation efficiency and susceptibility to rupture under harsh conditions. More importantly, these traditional microcapsules based on the electrostatic interaction of hydrophilic colloids exhibit a sharp decline in stability in hard water environments with high ionic strength, with the capsule walls easily swelling and disintegrating, making it difficult to effectively maintain the activity and controlled-release performance of the internal essential oils. This fails to meet the industrial-grade disinfection requirements of farms, food processing plants, and other environments with high water hardness and organic interference. For example, patent CN120788004A discloses a method for encapsulating cinnamon-Litsea cubeba compound essential oil using a gelatin / sodium carboxymethyl cellulose system. Although this technology achieves preliminary encapsulation, the capsule wall still suffers from swelling and disintegration in hard water environments with high ionic strength, making it difficult to adapt to industrial applications in hard water environments.

[0006] Therefore, there is an urgent need in this field for a novel microencapsulation system that delivers plant essential oils with a more robust structure, a denser barrier, and significantly enhanced tolerance to environmental disturbances (especially hard water and organic matter), so as to truly transform the antibacterial potential of natural essential oils into stable, reliable disinfection products suitable for complex scenarios. Summary of the Invention

[0007] Based on the aforementioned technical shortcomings, this invention aims to provide a core-shell type plant essential oil disinfection microcapsule with a robust structure, strong environmental tolerance, and long-lasting bactericidal effect. This overcomes the technical difficulties in existing technologies, such as poor stability of microcapsules under hard water and organic load conditions, easy leakage of essential oils, and decreased bactericidal effect. At the same time, it achieves synergistic enhancement and controllable release of essential oils, meeting the high-efficiency disinfection needs in complex scenarios such as livestock and poultry farming, food processing, and medical environments.

[0008] The first inventive point of this invention is a core-shell type plant essential oil disinfectant microcapsule, which comprises, from the inside out: a core, a Pickering interface layer, an ionic crosslinking gel layer, and a covalently crosslinked polymer shell; the core is composed of Litsea cubeba essential oil and cinnamon essential oil; the Pickering interface layer is coated on the surface of the core and is stabilized by nanocellulose as a solid particle emulsifier; the ionic crosslinking gel layer is coated on the outside of the Pickering interface layer and is composed of sodium alginate and calcium ions crosslinked together; the covalently crosslinked polymer shell is coated on the outer surface of the gel layer and is formed by chitosan and geniposide crosslinked together by covalent bonds.

[0009] Furthermore, the microcapsule contains, by weight, 90-110 parts core, 2-5 parts Pickering interface layer, 10-30 parts ion-crosslinked gel layer, and 2-10 parts covalently crosslinked polymer shell.

[0010] Furthermore, the mass ratio of Litsea cubeba essential oil to cinnamon essential oil is 1:(0.8-1.2); the mass ratio of sodium alginate to calcium ions is 1:(0.1-1.2); and the mass ratio of geniposide to chitosan is (0.2-0.5):1.

[0011] Furthermore, the encapsulation rate of the essential oil in the microcapsules is not less than 70%; The second inventive point of this invention is a method for preparing core-shell type plant essential oil disinfectant microcapsules as described above, comprising the following steps: (1) The core oil phase containing Litsea cubeba essential oil and cinnamon essential oil is mixed and emulsified with the aqueous phase containing nanocellulose Pickering interface layer, so that the Pickering interface layer coats the core surface to obtain emulsion one; (2) Add sodium alginate solution to the emulsion obtained in step (1), stir evenly, and then introduce calcium ion solution to carry out ion cross-linking reaction to form an ion cross-linked gel layer that encapsulates the Pickering interface layer; and obtain gel particles. (3) Transfer the gel particles obtained in step (2) into the chitosan solution, adjust the pH to 5.0-6.0, add geniposide, and react at 35-40℃ for 1-3 hours to form a covalently cross-linked outer shell layer on the outside of the gel layer.

[0012] Furthermore, in step (1), The oil phase also contains the surfactant Tween-80 and medium-chain triglycerides, wherein the amount of Tween-80 added is 1%-10% of the oil phase mass, and the amount of medium-chain triglycerides added is 5%-20% of the oil phase mass. The aqueous phase also contains sodium citrate, a chelating agent, with a mass concentration of 0%-0.5% in the aqueous phase; the nanocellulose has a mass concentration of 0.2%-0.5% in the aqueous phase.

[0013] Further, in step (2), the mass concentration of sodium alginate in the reaction system is 0.5%-2.0%, and the concentration of calcium ion solution is 50-150 mM.

[0014] Further, in step (3), the mass concentration of chitosan in the reaction system is 0.1%-0.5%, and the mass concentration of geniposide in the reaction system is 0.05%-0.2%.

[0015] Furthermore, it also includes step (4): spray drying the microcapsule suspension obtained in step (3) to obtain microcapsule powder; the inlet temperature of the spray drying is 140-160℃ and the outlet temperature is 70-90℃.

[0016] The third invention is the application of the aforementioned core-shell type plant essential oil disinfectant microcapsules in the preparation of disinfectants for livestock and poultry breeding environments, food processing equipment or utensil surfaces, non-critical object surfaces in medical institutions, and hard surface disinfectants for homes or public places.

[0017] Compared with the prior art, the present invention has the following significant advantages: (1) Highly efficient synergistic antibacterial effect: Based on the synergistic effect of the core compound of "Litsea cubeba-cinnamon" essential oil and the cross-linked shell of "chitosan-geniposide", under suitable conditions (such as 37℃ for 20 min), it can achieve a log killing value of ≥5.00 against Staphylococcus aureus and Escherichia coli, which meets the high efficiency disinfection standard; (2) Structural innovation: The multi-layer encapsulation system without protein wall material is adopted, which overcomes the problem of instability of traditional microcapsules under high ionic strength and organic load; (3) High encapsulation rate and controlled release: The encapsulation rate of essential oil in microcapsules is ≥70%, the controlled release effect is significant, and the antibacterial activity is effectively prolonged; (4) Mild preparation process: Encapsulation at room temperature or low temperature can keep the active ingredients of essential oil from degrading, and the process is easy to scale up for production; (5) Good material compatibility: After 72 h of contact with stainless steel, PVC and silicone, there is no obvious corrosion or weight loss (≤ 0.1%); (6) Wide range of applications: It can still maintain high antibacterial activity in the presence of hard water and organic load (such as BSA, serum, etc.), and is suitable for a variety of complex environments. It can be used in livestock and poultry breeding, food processing, public health and home environments; (7) Environmental protection and safety: It uses natural biodegradable materials to ensure the green and environmentally friendly characteristics of the disinfectant.

[0018] The technical approach of this invention not only solves the problems of dispersion and stability of Litsea cubeba essential oil in application, but also obtains a highly efficient, safe, and environmentally friendly disinfectant product through scientific compound screening and synergistic evaluation, providing a practical new path for the engineering application of natural essential oils in the field of disinfectants.

[0019] Therefore, this invention not only has strong academic value, but also has good prospects for industrialization. Attached Figure Description

[0020] Figure 1 A schematic diagram of the checkerboard dilution method for the synergistic effect of Litsea cubeba essential oil combined with different synergists; Figure 2 Particle size distribution of the disinfection microcapsules of the present invention under dynamic light scattering detection; Figure 3Scanning electron microscope images of Staphylococcus aureus and Escherichia coli before and after the disinfection microcapsules of this invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0023] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0024] Example 1 This embodiment provides a core-shell type plant essential oil disinfection microcapsule. The microcapsule comprises, from the inside out: a core, a Pickering interface layer, an ionic crosslinking gel layer, and a covalently crosslinked polymer shell. The core is composed of Litsea cubeba essential oil and cinnamon essential oil. The Pickering interface layer covers the surface of the core and is stabilized by nanocellulose as a solid particle emulsifier. The ionic crosslinking gel layer covers the outside of the Pickering interface layer and is composed of sodium alginate and calcium ions crosslinked together. The covalently crosslinked polymer shell covers the outer surface of the gel layer and is formed by chitosan and geniposide crosslinked together by covalent bonds.

[0025] This microcapsule uses a blend of Litsea cubeba and cinnamon essential oils as its core, with an outer layer sequentially coated with a core-shell structure consisting of a cellulose nanofiber (CNC) stabilized Pickering interface layer, a sodium alginate-calcium ion crosslinked gel layer, and a chitosan-geniposide covalently crosslinked outer shell layer. This structure not only effectively enhances the stability of the essential oils but also achieves differentiated release through the synergistic effect of the essential oil components, thereby improving the antibacterial activity and durability of the microcapsule. Specifically, the main active ingredient in Litsea cubeba essential oil, citral (strongly polar), and the main active ingredient in cinnamon essential oil, cinnamaldehyde (strongly hydrophobic), have different affinities for the wall material. This characteristic lays the foundation for differentiated release of the two active ingredients, thus enhancing the synergistic antibacterial effect. Cellulose nanofiber (CNC), as a solid particle emulsifier, is irreversibly adsorbed at the oil-water interface, forming a dense physical barrier, thereby solving the problem of difficult dispersion of hydrophobic essential oils and providing a rigid framework for subsequent crosslinking.2+ The instantaneous cross-linking of sodium alginate triggers the formation of a three-dimensional network, endowing the microcapsules with mechanical strength and shape retention, thus achieving primary controlled release of essential oils. The outer shell is formed by covalent cross-linking of chitosan and geniposide. Under weakly acidic conditions (pH 5.0-6.0), geniposide undergoes ring-opening to expose active aldehyde groups, which react with the amino groups (-NH2) on the chitosan molecules in a Schiff base reaction, generating stable C=N double bonds (imine bonds), thereby constructing a dense three-dimensional covalently cross-linked network. This network effectively blocks water molecules, oxygen, and metal ions (such as Ca2+). 2+ Mg 2+ The penetration of chitosan into the microcapsules is crucial for maintaining their structural integrity and the stability of active ingredients in hard water and high-humidity environments. This invention utilizes the hydrophobic regions and hydrophilic channels in the chitosan-geniposide cross-linked network to achieve selective binding and sustained release of cinnamaldehyde, as well as stable diffusion of citral, thereby preserving the active ingredients and bactericidal effects of the essential oils in complex environments.

[0026] As a further preferred embodiment, the microcapsule contains, by weight parts: 90-110 parts core, 2-5 parts Pickering interface layer, 10-30 parts ion-crosslinked gel layer, and 2-10 parts covalently crosslinked polymer shell.

[0027] As a further preferred embodiment, the mass ratio of Litsea cubeba essential oil to cinnamon essential oil is 1:(0.8-1.2); the mass ratio of sodium alginate to calcium ions is 1:(0.1-1.2); and the mass ratio of geniposide to chitosan is (0.2-0.5):1.

[0028] Through extensive screening experiments, this invention has discovered that the combination of Litsea cubeba essential oil and cinnamon essential oil in this ratio not only has a synergistic antibacterial effect, but more importantly, its specific chemical composition (such as citral and cinnamaldehyde) produces good compatibility and synergistic protective effect with the multilayer wall material subsequently constructed in this invention, especially the outer shell layer crosslinked with geniposide.

[0029] The pickering interface layer, wrapped around the core layer, uses cellulose nanoparticles (CNC) as a solid particle emulsifier. It forms a dense physical barrier film at the oil-water interface through irreversible adsorption, and its addition amount can be 0.2%-0.5% of the water phase mass. This layer solves the problem of essential oil dispersion and provides a rigid framework for subsequent crosslinking.

[0030] An ion-crosslinked gel layer, composed of sodium alginate and calcium ions, encapsulates the emulsion layer. The concentration of sodium alginate is 0.5-2.0%, preferably 1.0%; the calcium ions are typically a calcium chloride (CaCl2) solution with a concentration of 50-150 mM, preferably 100 mM. Under gentle stirring, the calcium ion solution is introduced into the Pickering emulsion containing sodium alginate, instantly forming gel microspheres that encapsulate each emulsion droplet. This layer forms a gel with a three-dimensional network structure through an ion-crosslinking reaction, giving the microcapsules mechanical strength and shape retention, and providing a certain degree of controlled release of the internal essential oils.

[0031] The covalently cross-linked outer shell, serving as the outermost layer of the microcapsule, is a dense polymer shell formed by covalent cross-linking of chitosan and geniposide. The chitosan concentration can be 0.1-0.5%, preferably 0.2%; geniposide, as a natural cross-linking agent, can be 0.05-0.2%, preferably 0.1%. Under suitable conditions (e.g., pH 5.0-6.0, 35-40℃), the active groups of geniposide cross-link with the amino groups on the chitosan molecular chain, forming a dense network shell. This layer forms a dense and tough final barrier through chemical cross-linking. Its main functions are: ① preventing essential oil volatilization and oxidation; ② regulating essential oil release to achieve long-lasting effects; ③ resisting the penetration and damage of external environmental factors (such as calcium and magnesium ions in hard water, organic matter, and extreme pH).

[0032] As a further preferred embodiment, the microcapsules have an encapsulation rate of at least 70% for the essential oil.

[0033] The pickering interface layer reduces essential oil loss during emulsification through irreversible adsorption of particles. The calcium alginate gel layer rapidly forms a physical barrier on the outside of the emulsion droplets, preventing the essential oil from diffusing outward. The chitosan-geniposide covalent shell further seals the oil, reducing volatilization during drying and storage. The synergistic effect of these three layers ensures that the essential oil is efficiently locked inside the microcapsules, providing a material basis for subsequent sustained release and long-lasting sterilization.

[0034] As a further preferred embodiment, after the microcapsules are soaked in hard water of 250-350 mg / L CaCO3 for 5-9 days, the retention rate of essential oil encapsulation is not less than 95%.

[0035] As a further preferred embodiment, in the presence of an organic interfering agent containing 3-8% bovine serum albumin, the microcapsules achieve a logarithmic kill value of 4.5 or higher against Escherichia coli and Staphylococcus aureus after 25-35 minutes of action.

[0036] Example 2 This embodiment describes a method for preparing core-shell type plant essential oil disinfectant microcapsules according to Example 1, comprising the following steps: (1) The core oil phase containing Litsea cubeba essential oil and cinnamon essential oil is mixed and emulsified with the aqueous phase containing nanocellulose Pickering interface layer, so that the Pickering interface layer coats the core surface to obtain emulsion one; (2) Add sodium alginate solution to the emulsion obtained in step (1), stir evenly, and then introduce calcium ion solution to carry out ion cross-linking reaction to form an ion cross-linked gel layer that encapsulates the Pickering interface layer; and obtain gel particles. (3) Transfer the gel particles obtained in step (2) into the chitosan solution, adjust the pH to 5.0-6.0, add geniposide, and react at 35-40℃ for 1-3 hours to form a covalently cross-linked outer shell layer on the outside of the gel layer.

[0037] As a further preferred embodiment, in step (1), The oil phase also contains the surfactant Tween-80 and medium-chain triglycerides, wherein the amount of surfactant Tween-80 added is 1%-10% of the oil phase mass, preferably 3%-6%; The amount of medium-chain triglycerides added is 5%-20% of the oil phase mass; preferably 10%. The aqueous phase also contains sodium citrate, a chelating agent, with a mass concentration of 0.1%-0.2% in the aqueous phase; the nanocellulose has a mass concentration of 0.2%-0.5% in the aqueous phase.

[0038] As a further preferred embodiment, in step (2), the mass concentration of sodium alginate in the reaction system is 0.5%-2.0%, and the concentration of calcium ion solution is 50-150 mM.

[0039] As a further preferred embodiment, in step (3), the mass concentration of chitosan in the reaction system is 0.1%-0.5%, and the mass concentration of geniposide in the reaction system is 0.05%-0.2%.

[0040] As a further preferred embodiment, the method further includes step (4): spray drying the microcapsule suspension obtained in step (3) to obtain microcapsule powder; the inlet temperature of the spray drying is 140-160℃ and the outlet temperature is 70-90℃.

[0041] Example 3 This embodiment describes the application of the core-shell type plant essential oil disinfectant microcapsules described in Embodiment 1 in the preparation of disinfectants for livestock and poultry breeding environments, food processing equipment or utensil surfaces, non-critical object surfaces in medical institutions, and hard surface disinfectants for homes or public places.

[0042] Example 4 Experimental Materials and Methods: The tested bacterial strains were *Escherichia coli* (ATCC 25922) and *Staphylococcus aureus* (ATCC 6538), provided by the China Center for Type Culture Collection (CCTCC). The strains were frozen at -80℃ and, after thawing, cultured in LB broth (Solarbio, Beijing) at 37℃ with shaking.

[0043] The essential oils tested included: Litsea cubeba oil, Cinnamomum cassia oil, Thymus vulgaris oil, Origanum vulgare oil, Syzygium aromaticum oil, Melaleuca alternifolia oil, Peppermint oil, Lemon oil, and Thymol (containing thymol and other active ingredients). All were purchased from pharmaceutical-grade raw material suppliers, and GC-MS confirmed that the content of the main active ingredients was ≥95%.

[0044] The minimum inhibitory concentration (MIC) was determined using the microdilution method. Synergistic effects were assessed using the checkerboard assay: gradient dilutions of Litsea cubeba essential oil and candidate synergists were set up in the longitudinal and transverse directions of a 96-well plate, and after cross-combination, bacterial suspension was inoculated and incubated at 37°C for 24 h.

[0045] The formula for calculating the fractional inhibitory concentration index (FIC index) is as follows: (1) FIC A =MIC A (Combined use) / MIC A (Used alone); (2) FIC B =MIC B (Combined use) / MIC B (Used alone); (3) FIC = FIC A + FIC B The criteria for determination are shown in Table 1.

[0046] Table 1. Criteria for Judging the FIC Index

[0047] Experimental results The MIC values ​​of essential oils against Escherichia coli and Staphylococcus aureus were determined using the microdilution method, and the FIC index was calculated using the checkerboard dilution method. The results are shown in Table 2. Figure 1(In the diagram, A: Litsea cubeba essential oil; B: Cinnamon essential oil / Thyme essential oil / Oregano essential oil / Clove essential oil / Tea tree essential oil / Peppermint essential oil / Lemon essential oil / Thymol; A1=2 MIC, A2=1 MIC, A3=1 / 2 MIC, A4=1 / 4 MIC, B1=2 MIC, B2=1 MIC, B3=1 / 2 MIC, B4=1 / 4 MIC).

[0048] Table 2. MIC and FIC results of blends of different essential oils with Litsea cubeba essential oil.

[0049] Experimental conclusions This invention systematically evaluated the synergistic antibacterial effects of Litsea cubeba essential oil combined with eight common plant essential oils or active ingredients using the checkerboard dilution method. The results showed that the synergistic effect was most significant when cinnamon essential oil was combined with Litsea cubeba essential oil, with a FIC index as low as 0.375 against Escherichia coli and Staphylococcus aureus, far superior to thyme essential oil (FIC=0.5), oregano essential oil (FIC=0.44), peppermint essential oil (FIC=0.75), lemon essential oil (FIC=0.6), and thymol (FIC=0.5); while clove essential oil and tea tree essential oil showed no significant effect (FIC>1). Therefore, this invention selected the combination of cinnamon essential oil and Litsea cubeba essential oil, which exhibits the strongest synergistic effect, as the core active ingredient, ensuring the high-efficiency bactericidal performance of the microcapsules from the source, and laying a solid foundation for the subsequent construction of multilayer encapsulation structures and stable application in complex environments.

[0050] Example 5 Experimental Materials and Methods: The tested bacterial strains were *Escherichia coli* (ATCC 25922) and *Staphylococcus aureus* (ATCC 6538), provided by the China Center for Type Culture Collection (CCTCC). The strains were frozen at -80℃ and, after thawing, cultured in LB broth (Solarbio, Beijing) at 37℃ with shaking.

[0051] The essential oils tested included Litsea cubeba oil and Cinnamomumcassia oil.

[0052] Blending ratio settings: Set the mass ratio of Litsea cubeba essential oil to cinnamon essential oil as follows: 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5.

[0053] Using the same checkerboard dilution method as in Example 1, the MIC values ​​of each proportion of blended essential oils against the two test bacteria were determined, and the fractional inhibitory concentration index (FIC Index) was calculated.

[0054] Experimental results The MIC values ​​of the compound essential oils against Escherichia coli and Staphylococcus aureus were determined using the microdilution method, and the FIC index was calculated using the checkerboard dilution method. The results are shown in Table 3.

[0055] Table 3. FIC results for blended essential oils with different proportions

[0056] Experimental conclusions Litsea cubeba essential oil and cinnamon essential oil showed synergistic effects on both tested bacteria in a mass ratio range of 1:0.8 to 1:1.2 (FIC≤0.5), with the strongest synergistic effect observed at a ratio of 1:1.

[0057] Example 6 Experimental materials and methods: The test surfactants were Tween-20, Tween-80, and polyethylene glycol-40 hydrogenated castor oil (PEG-40), all of which were purchased from pharmaceutical grade reagent suppliers.

[0058] Preparation method: Based on the compound essential oil combination of Example 5 (Litsea cubeba essential oil and cinnamon essential oil in a 1:1 mass ratio), prepare each mixture to a final volume of 500 mL. Composition A: 10.0 mL of Litsea cubeba essential oil, 10.0 mL of cinnamon essential oil, 15.0 mL of Tween-20 (3%), and add water to make up to 500 mL. Stir and homogenize for 20 min. Composition B: 10.0 mL of Litsea cubeba essential oil, 10.0 mL of cinnamon essential oil, 30.0 mL of Tween-80 (6%), and add water to make up to 500 mL. Stir and homogenize for 20 min. Composition C: 10.0 mL of Litsea cubeba essential oil, 10.0 mL of cinnamon essential oil, 50.0 mL of polyethylene glycol-40 hydrogenated castor oil (PEG-40, 10%), and add water to make up to 500 mL. Stir and homogenize for 20 min.

[0059] Stability evaluation method: (1) Dispense the sample into transparent glass bottles and place them at 4 ℃ (low temperature), 37 ℃ (room temperature) and 54 ℃ (high temperature acceleration) for 24 h respectively; (2) Observe the appearance of the sample (clarity, turbidity, layering, etc.); (3) Use a dynamic light scattering instrument (DLS, Malvern Zetasizer Nano ZS90) to detect the particle size and PDI; (4) Score according to the stability scoring standard, as shown in Table 4.

[0060] Table 4 Stability Scoring Criteria

[0061] Experimental results The stability evaluation results are shown in Tables 5 and 6.

[0062] Table 5. Effects of different surfactants on system stability

[0063] Table 6. Effects of adding 6% Tween-80 at different temperatures on particle size and appearance stability.

[0064] Experimental conclusions The 6% Tween-80 used in this invention maintains a clear and transparent system under various temperature conditions (4℃, 37℃, 54℃), with a stable particle size of 180-195 nm, a PDI of less than 0.26, and a stability score of the highest of 5, demonstrating excellent emulsifying ability and thermodynamic stability. In contrast, Tween-20 shows slight turbidity at 37℃ and obvious stratification at 54℃, failing to meet the stability requirements of the formulation under different storage conditions; PEG-40, although clear at 4℃, shows slight turbidity and stratification at 37℃ and 54℃ respectively, with a stability score of only 3, significantly inferior to Tween-80. Therefore, this invention selects Tween-80 as a surfactant, which not only ensures the long-term stable dispersion of the compound essential oil system but also provides a uniform and stable emulsion template for the subsequent multilayer encapsulation process, making it one of the key excipients for ensuring the integrity of the microcapsule structure and encapsulation performance.

[0065] Example 7 Experimental materials and methods: The test chelating agents were sodium citrate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.) and disodium ethylenediaminetetraacetate (EDTA·2Na, analytical grade, Sigma-Aldrich).

[0066] The basic compound formulation was prepared using compound essential oil combination B from Example 6, with a final volume of 500 mL: 10.0 mL of Litsea cubeba essential oil, 10.0 mL of cinnamon essential oil, 30 mL of 6% Tween-80, 175 mL of 10% ethanol, 15 mL of 5% propylene glycol, and the remainder diluted to 500 mL with water. Based on this, 0%, 0.05%, 0.1%, and 0.2% sodium citrate or EDTA·2Na were added respectively (0 g, 0.25 g, 0.50 g, and 1.00 g), to prepare disinfectant samples with different chelating agent concentrations. All samples were homogenized by magnetic stirring for 20 min and then filtered through a 0.22 μm filter membrane for sterilization.

[0067] Test strains and culture conditions: *Escherichia coli* (ATCC 25922) and *Staphylococcus aureus* (ATCC 6538) were purchased from the China Center for Type Culture Collection. After resuscitation, the strains were cultured in LB broth at 37°C with shaking. The logarithmic growth phase culture (OD600≈0.6) was collected for later use.

[0068] Antibacterial effect test: Paper disc diffusion method was used: (1) Prepare bacterial suspension to 0.5 McFarland standard (approximately 1.5 × 10⁻⁶). 8 (2) Spread evenly on nutrient agar plates (90 mm in diameter, 4 mm in thickness); (3) Soak 6 mm diameter sterile filter paper discs in disinfectants with different chelating agent formulations for 30 s, remove excess solution, and place them on the surface of the plate; (4) Incubate in a 37℃ constant temperature incubator for 24 h; (5) Measure the diameter of the inhibition zone with vernier calipers, set 3 parallel replicates for each group, and take the average value, as shown in Table 7.

[0069] Table 7 Criteria for Determining the Diameter of the Inhibition Zone

[0070] Experimental results The inhibition zone diameters of each chelating agent against Escherichia coli and Staphylococcus aureus are shown in Table 8.

[0071] Table 8 Effect of chelating agents on the diameter of inhibition zone (mm, n=3, mean ± standard deviation)

[0072] Experimental conclusions The 0.2% sodium citrate used in this invention exhibited inhibition zone diameters of 18.5 mm and 19.2 mm against *Escherichia coli* and *Staphylococcus aureus*, respectively, with an inhibitory effect deemed "significant," significantly superior to other concentrations and the control chelating agent. From a concentration gradient perspective, the inhibitory effect of sodium citrate was concentration-dependent: at 0.05%, the inhibition zone was approximately 12-13 mm (moderate inhibition); at 0.10%, it increased to 15.8-16.4 mm (significant inhibition); and at 0.20%, it achieved the optimal effect (18.5-19.2 mm). In contrast, EDTA·2Na showed weaker inhibitory effects than sodium citrate at the same concentration: the inhibition zone of 0.20% EDTA·2Na was 15.3-16.1 mm, only equivalent to the level of 0.10% sodium citrate. Therefore, this invention selects 0.2% sodium citrate as a chelating agent, which not only softens hard water by chelating calcium and magnesium ions in the water and eliminates the interference of metal ions on the activity of essential oils, but also significantly enhances the antibacterial efficacy of compound essential oils, providing an important guarantee for the stable application of microcapsules in hard water environments.

[0073] Example 8 Experimental Materials and Methods: Basic Compound Formulation: The essential oil compound system obtained in Example 7 was used, with the following added: 50 mL of 10.0% medium-chain triglycerides (MCT), 1.50 g of 0.3% nanocellulose (CNC), 5.00 g of 1.0% sodium alginate, 7.35 g of 100 mM CaCl2, 1.00 g of 0.2% chitosan, 0.50 g of 0.1% geniposide, and 1.00 g of 0.2% sodium citrate. Water was added to bring the volume to 500 mL.

[0074] The preparation steps are as follows: (1) Oil phase preparation: Mix Litsea cubeba essential oil, cinnamon essential oil, MCT and Tween-80 evenly. (2) Aqueous phase preparation: Dissolve CNC and sodium citrate in part of the water. (3) Pickering emulsion formation: Mix the oil phase and the aqueous phase, and emulsify at 10,000 rpm for 3 minutes. (4) Gel inner layer formation: Add sodium alginate solution to the emulsion, stir evenly, add CaCl2 solution dropwise, and stir continuously for 15 minutes to form gel particles. (5) Cross-linked outer shell formation: Transfer the gel particles into chitosan solution, adjust the pH to 5.6, add geniposide, and cross-link at 37℃ for 2 hours. (6) Drying: Dry the microcapsule liquid through a spray dryer (inlet 150℃, outlet 80℃) to obtain microcapsule powder.

[0075] After the formulation was prepared, the microcapsule suspension was tested for appearance, particle size, PDI and stability, as shown in Table 9.

[0076] Table 9 Evaluation Indicators and Judgment Criteria

[0077] Experimental results The test results are shown in Table 10.

[0078] Table 10 Formulation characteristics of disinfectant microcapsules

[0079] Experimental conclusions like Figure 2 As shown, the microcapsules obtained in this embodiment have uniform particle size (particle size 185 nm, PDI 0.24), good stability under different temperature conditions, and an encapsulation efficiency of 82.5%. This indicates that the preparation process has good reproducibility.

[0080] Example 9: Verification of sterilization effect Experimental Materials and Methods: Test sterilization microcapsules: sterilization microcapsules prepared in Example 8; bacterial strains: *Escherichia coli* (ATCC 25922) and *Staphylococcus aureus* (ATCC 6538), purchased from the China Center for Type Culture Collection; culture media: LB broth, LB agar (Solarbio, Beijing); instruments: constant temperature water bath (37 ℃), sterile centrifuge tubes, pipettes, plate counter.

[0081] Experimental steps: ① After revival, the strain was cultured in NB broth until the logarithmic growth phase (OD600 ≈ 0.6), and then diluted to approximately 1×10⁻⁶. 6 CFU / mL; ② Take 9 mL of the test bacterial solution and mix it with 1 mL of sterilized microcapsules (microcapsules prepared in Example 8) (the final sterilized microcapsules are 10% of the total fraction), and incubate at 20℃, 37℃ and 40℃ for 10, 20 and 30 min respectively; ③ After the incubation is completed, immediately add a neutralizing agent (phosphate buffer containing 0.5% Tween-80 and 0.5% lecithin) to terminate the sterilization microcapsule incubation; ④ After a tenfold serial dilution, take 100 μL and spread it on NA plates, incubate at 37℃ for 24 h, and count the number of surviving colonies (CFU); ⑤ Calculation formula for log reduction (LR): Where: N0 = average colony count of the control group; N t = Average colony count of the treatment group, as shown in Table 11.

[0082] Table 11 Criteria for Determining Logarithmic Kill Value

[0083] Experimental results The bactericidal effects of Escherichia coli and Staphylococcus aureus under different conditions are shown in Table 12.

[0084] Table 12. Bactericidal effect of disinfectant microcapsules on bacteria (logarithmic kill value, n=3, mean ± standard deviation)

[0085] Experimental conclusions At commonly used ambient temperatures (20–40℃) and conventional contact times (10–30 min), the microcapsules exhibited logarithmic kill values ​​≥4.90 against both Escherichia coli and Staphylococcus aureus, reaching 5.20 after 20 min at 37℃, meeting the standards for highly efficient disinfection.

[0086] Experimental results show that even at lower temperatures (20℃) and shorter treatment times (10 min), the microcapsules of this invention can still achieve a bactericidal effect of nearly 5 log, demonstrating the synergistic antibacterial mechanism of the compound essential oils (Litsea cubeba: Cinnamomum cassia = 1:1) and the effective protection of essential oil activity by the multi-layered core-shell structure. Furthermore, the bactericidal effect of this invention is stable within the temperature range of 20–40℃, and the bactericidal efficacy further increases with increasing temperature and treatment time, indicating that the microcapsule shell (chitosan-geniposide covalently cross-linked layer) can regulate the essential oil release rate according to ambient temperature, achieving temperature-responsive controlled release. The synergistic effect of the core essential oil (FIC = 0.375), the essential oil dispersion protection of the Pickering interface layer (CNC stable), the mechanical support of the calcium alginate gel layer, and the barrier effect of the outermost covalently cross-linked network jointly ensure that the essential oil can continuously act on the target bacteria at an effective concentration during the bactericidal process, disrupting the cell membrane structure, thereby obtaining stable and efficient bactericidal performance.

[0087] Example 10 Effect of pH and Organic Load on Sterilization Efficacy Experimental Materials and Methods: Test disinfectant microcapsules: disinfectant microcapsules prepared in Example 8; bacterial strains: Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538); buffer solutions: phosphate-buffered saline (PBS) at pH 4.5, 6.5, 8.5, and 10.5, calibrated with a pH meter; organic load mimic: bovine serum albumin (BSA, ≥98%, Sigma-Aldrich).

[0088] Experimental steps: ① Prepare 0.5 McFarland bacterial suspension (approximately 1.5 × 10⁻⁶). 8 (CFU / mL); ② Dilute the bacterial suspension to approximately 1×10⁻⁶ CFU / mL. 6CFU / mL, and adjusted to different pH values ​​(4.5, 6.5, 8.5, 10.5); ③ An organic loading group was set up: 5% BSA was added at pH 6.5 to simulate an organic pollution environment; ④ Following the method in Example 8, 9 mL of bacterial solution was mixed with 1 mL of sterilized microcapsules and incubated at 37 ℃ for 20 min; ⑤ The reaction was terminated, diluted, plated, and the surviving colonies were counted; ⑥ The logarithmic kill value (LR) was calculated, as shown in Table 13.

[0089] Table 13 Criteria for Determining Logarithmic Kill Value

[0090] Experimental results The bactericidal effects under different pH and organic load conditions are shown in Table 14.

[0091] Table 14 Logarithmic kill values ​​of disinfectant microcapsules under different pH and organic load conditions (n=3, mean ± standard deviation)

[0092] Experimental conclusions As shown in Table 14, within the pH range of 4.5–8.5, the logarithmic kill value (LR) of the microcapsules against *Escherichia coli* and *Staphylococcus aureus* after 20 min was ≥5.0, meeting the high-efficiency disinfection standard. Even under strongly alkaline conditions at pH 10.5, the LR remained at 4.70–4.80, with only a slight decrease in bactericidal effect, still representing a significant bactericidal level. Regarding resistance to organic load, under conditions of pH 6.5 with the addition of 5% bovine serum albumin (BSA) to simulate a high-organic-pollution environment, the LRs of the microcapsules against the two tested bacteria were 4.60 and 4.55, respectively, still significantly higher than 4.5, meeting the technical indicators described in claim 5.

[0093] The dense outer shell (imine bond C=N) formed by the Schiff base reaction of chitosan and geniposide in this invention maintains structural stability over a wide pH range, effectively blocking H2O. + / OH - The penetration of organic macromolecules (such as BSA) prevents the capsule wall from swelling or disintegrating, ensuring that the core essential oil is not disturbed by the external environment. Even under highly alkaline or high organic load conditions, the Pickering interface layer (CNC stable) and the calcium alginate gel layer maintain structural integrity, working synergistically with the outer covalent network to achieve controlled release of the essential oil, avoiding a decrease in bactericidal activity caused by the instantaneous consumption of essential oil by organic matter. Litsea cubeba essential oil (citral) and cinnamon essential oil (cinnamaldehyde) exhibit a synergistic effect at a ratio of 1:0.8~1.2 (FIC≤0.5). Even under environmental stress conditions, they can still maintain highly efficient bactericidal activity through a dual mechanism of disrupting cell membranes and inhibiting enzyme activity.

[0094] Example 11 Experimental Materials and Methods: Test disinfectant microcapsules: disinfectant microcapsules prepared in Example 8; Strains: Staphylococcus aureus (ATCC 6538); Reagents: glutaraldehyde (2.5%, fixative), ethanol series (30%, 50%, 70%, 90%, 100%, gradient dehydration), tert-butanol (displacement solution), gold target used for gold sputtering treatment; Instruments: Scanning electron microscope (SEM, Hitachi S-4800, Japan).

[0095] Experimental steps: ① Prepare bacterial suspension (1×10⁻⁶) 8 Two portions were prepared: one portion was treated with sterilized microcapsules (final volume fraction 10%) for 20 min, and the other portion served as a blank control; ② After treatment, the bacterial cells were collected immediately by centrifugation (5000 rpm, 10 min) and washed twice with PBS; ③ The cells were fixed with 2.5% glutaraldehyde for 2 h and washed with PBS; ④ The cells were dehydrated sequentially with 30%, 50%, 70%, 90%, and 100% ethanol, 10 min for each stage; ⑤ The cells were replaced with tert-butanol and freeze-dried; ⑥ The samples were sputter-coated with gold on a gold target and observed by SEM. The judgment criteria are shown in Table 15.

[0096] Table 15 Evaluation Indicators and Judgment Criteria

[0097] Experimental results SEM observation results show that: Control group: Cells were spherical, with smooth and intact surfaces, and no obvious damage was observed (grade 0); Treatment group: Obvious pitting and cracking appeared on the cell surface (Grade 2), and some cells ruptured with leakage of contents (Grade 3). The results are summarized in Table 16.

[0098] Table 16 Morphological changes of Staphylococcus aureus before and after treatment with disinfection microcapsules

[0099] Experimental conclusions like Figure 3 As shown in the figure, SEM observation revealed that the surface of the bacteria in the treated group exhibited obvious depressions, cracks, and ruptures, confirming that the microcapsules exert their bactericidal effect by disrupting the cell membrane structure.

[0100] Example 12 Material Compatibility Test Experimental Materials and Methods: Tested disinfectant microcapsules: disinfectant microcapsules from Example 8; Material samples: ① Stainless steel sheet (304 type, 2 cm × 2 cm × 1 mm); ② Polyvinyl chloride (PVC sheet, 2 cm × 2 cm × 1 mm); ③ Medical silicone sheet (2 cm × 2 cm × 1 mm); Instruments and tools: Electronic balance (accuracy 0.1 mg, Sartorius), constant temperature incubator.

[0101] Experimental steps: ① Clean and dry each material sheet, weigh its initial weight (W0), and photograph its appearance; ② Immerse each material sheet in 100 mL of the test sterilization microcapsule solution, seal and place in a 37 ℃ constant temperature incubator; ③ Set the contact time to 72 h, remove, rinse with distilled water, dry, and weigh the final weight (W1); ④ Calculate the weight loss rate (%): ⑤ Simultaneously observe and record changes in appearance (discoloration, corrosion, cracks, etc.), and the judgment criteria are shown in Table 17.

[0102] Table 17 Material Compatibility Judgment Criteria

[0103] Experimental results The material compatibility results are shown in Table 18.

[0104] Table 18 Compatibility test of disinfectant microcapsules with different materials (n=3, mean ± standard deviation)

[0105] Experimental conclusions The results are shown in Table 18: After immersing stainless steel 304, polyvinyl chloride (PVC), and medical-grade silicone in the microcapsule suspension and contacting them at 37°C for 72 hours, the weight loss rates of the three materials were 0.10%, 0.10%, and 0.095%, respectively, all ≤0.1%, and there were no obvious changes in appearance (no discoloration, corrosion, or cracks). According to the judgment criteria in Table 17, all were evaluated as "good". The dense shell (imine bond C=N) formed by the Schiff base reaction of chitosan and geniposide in this invention is chemically stable and does not release corrosive small molecules, avoiding the oxidation or swelling effects of traditional chemical disinfectants (such as chlorine-containing agents and aldehydes) on metals and plastics. The continuous phase of the microcapsules in this invention is water, with the pH controlled in a weakly acidic range of 5.0-6.0, and it does not contain strong acids, strong bases, or strong oxidizing components, and has no swelling or aging-inducing effect on polymer materials (PVC, silicone). Even if the microcapsules are in long-term contact with the material surface, the calcium alginate gel layer and the Pickering interface layer (CNC stable) together form a physical buffer layer, which further blocks the direct contact between the core essential oil and the material matrix, and avoids the penetration and softening of the polymer material by organic solvents.

[0106] Example 13 Accelerated Stability Test Experimental Materials and Methods: Tested disinfectant microcapsules: Disinfectant microcapsules prepared in Example 8; Storage conditions: Samples were placed in a 54 ℃ constant temperature incubator for accelerated testing and stored for 7 days; 4 ℃ and 37 ℃ conditions were set as controls; Test indicators: ① Appearance changes (clarity / turbidity / layering); ② Particle size and polydispersity index (PDI) (dynamic light scattering instrument, Malvern Zetasizer Nano ZS90); ③ Bactericidal efficacy (quantitative bactericidal test of suspension against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC6538, calculating the logarithmic kill value LR); Test time points: 0 days, 3 days, and 7 days.

[0107] Table 19 Judgment Criteria

[0108] Experimental results The test results at each time point are shown in Table 20.

[0109] Table 20 Accelerated stability test results (54 ℃, n=3, mean ± standard deviation)

[0110] Experimental conclusions After being stored at 54 °C for 7 days, the microcapsules remained clear and transparent, with a particle size increase of only about 8%, and the PDI remained at around 0.26. The bactericidal efficacy against Escherichia coli and Staphylococcus aureus decreased by ≤0.2 log. Based on the results of the accelerated test, it is estimated that the microcapsules can be stably stored at room temperature for more than 12 months.

[0111] Example 14 Effect of different components on the overall performance of microcapsules Experimental Materials and Methods: Test disinfectant microcapsules: Experimental group: Disinfectant microcapsules prepared in Example 8; Comparative Example A (Chemical Crosslinking Agent Control): The natural crosslinking agent "geniposide" was replaced with an equimolar amount of the commonly used chemical crosslinking agent "glutaraldehyde", while other conditions remained unchanged; Comparative Example B (Single Active Ingredient Control I): The core essential oils "Litsea cubeba + Cinnamon" were replaced with an equal mass of "single Litsea cubeba essential oil", with other conditions remaining unchanged; Comparative Example C (Single Active Ingredient Control II): The core essential oils "Litsea cubeba + Cinnamon" were replaced with an equal mass of "single cinnamon essential oil", with other conditions remaining unchanged; Comparative Example D (natural polysaccharide cross-linking control) replaced "geniposide" with an equal mass of "xyglucan," a natural polysaccharide cross-linking agent commonly used to construct biopolymer networks, while keeping other conditions unchanged.

[0112] Experimental methods: (1) Initial encapsulation rate determination: The essential oil content was determined by ultraviolet spectrophotometry, and the encapsulation rate was calculated. Encapsulation rate (%) = (actual essential oil content in microcapsules / total amount of essential oil added) × 100%. (2) Hard water stability test: 100 mg of each microcapsule powder was weighed and placed in a 50 mL centrifuge tube. 30 mL of 300 mg / L CaCO3 hard water was added, and the microcapsules were soaked in a constant temperature shaker at 25℃ (100 rpm) for 7 days. After soaking, the microcapsules were collected by centrifugation, the remaining essential oil content was extracted and determined, and the encapsulation rate retention rate was calculated. Encapsulation rate retention rate (%) = (essential oil content in microcapsules after soaking / essential oil content in initial microcapsules) × 100%. (3) Determination of bactericidal activity under organic load: In phosphate buffer (pH 6.5) containing 5% bovine serum albumin (BSA), microcapsules were mixed with Staphylococcus aureus suspension and reacted for 30 minutes. The logarithmic kill value (LR) was calculated. (4) Odor evaluation: An appropriate amount of microcapsule powder was placed in a sealed glass bottle and left to stand at 25°C for 30 minutes. The bottle was then opened and the odor characteristics were recorded. (5) Determination of the stability of essential oil component ratio after high temperature storage: Microcapsules were stored in a constant temperature incubator at 54°C for 7 days. Microcapsules before and after storage were taken, and essential oils were extracted by breaking the capsules. The peak area ratio of Litsea cubeba essential oil (citric acid) and cinnamon essential oil (cinnamaldehyde) was analyzed by GC-MS to compare the relative content changes of the main active ingredients.

[0113] Experimental results The comparison results are shown in Table 21.

[0114] Table 21 Comparison of Key Performance

[0115] Experimental conclusions Comparative Example A (glutaraldehyde crosslinking) had a high initial encapsulation rate (75.2%), but after immersion in hard water for 7 days, the encapsulation rate retention rate plummeted to 70.5%, and the LR against Staphylococcus aureus at 5% BSA was only 3.9. It also left a pungent odor, indicating that although the chemical crosslinking agent could form a capsule wall, its crosslinking network lacked stability in hard water and posed a safety risk. Comparative Example B (single Litsea cubeba essential oil core) had an encapsulation rate retention rate of 68.5% after 7 days in hard water, and an LR of 4.1 at 5% BSA. Although better than the glutaraldehyde crosslinking group, it was still significantly lower than the present invention, confirming that the antibacterial spectrum and synergistic effect of the single essential oil core were limited. Comparative Example C (single cinnamon essential oil core) had the worst hard water stability (65.8%), with an LR of only 3.7 at 5% BSA, and a strong odor, indicating that single cinnamon essential oil was more volatile and lacked a synergistic complementary mechanism with Litsea cubeba essential oil. Comparative Example D (xyglucan crosslinking) had the lowest initial encapsulation rate (65.3%), and the encapsulation rate retention rate in hard water was only 52.5% after 7 days. The LR under 5% BSA was 3.1. All performance characteristics were significantly inferior to those of this invention, proving that the physical crosslinking network formed by xyglucan cannot effectively resist the penetration of hard water ions and organic interference.

[0116] In comparison, the microcapsules of this invention exhibit superior performance, demonstrating excellent hard water stability: after 7 days of soaking, the encapsulation rate remained as high as 96.2%, far exceeding that of the comparative groups (52.5%~70.5%); resistance to organic load bactericidal effects: the LR against Staphylococcus aureus under 5% BSA reached 4.8, significantly better than the comparative groups (3.1~4.1); controllable odor: the geniposide-crosslinked shell is mild and non-irritating, with no irritating residue of glutaraldehyde or the strong odor of single cinnamon essential oil; and high-temperature storage stability: the proportion of essential oil components remained highly stable, while the control groups all showed varying degrees of loss of active ingredients. These results fully verify the key role of the core technical solution of this invention: the synergistic effect of the compound core. The compounding of Litsea cubeba essential oil and cinnamon essential oil at a ratio of 1:0.8~1.2 not only broadens the antibacterial spectrum (FIC≤0.5), but also achieves differentiated release under complex environments through the difference in affinity of citral and cinnamaldehyde for the wall material, avoiding the rapid loss of single essential oils. The barrier function of the covalently cross-linked shell, the dense network (imine bond C=N) formed by chitosan and geniposide through the Schiff base reaction, and the Ca²⁺ barrier + Mg² + Hard water ions exhibit excellent barrier properties and low adsorption rate for organic macromolecules (BSA), ensuring the stable existence and continuous release of the core essential oil in harsh environments. The overall synergy of the multi-layered structure—the Pickering interface layer (CNC stabilization) provides a rigid framework, the calcium alginate gel layer imparts mechanical strength, and the outermost covalent network provides the ultimate barrier—requires no single layer to function.

[0117] Example 15: Comparative Experiment on Hard Water Stability Experimental Materials and Methods: Test disinfectant microcapsules: Control group A (gelatin / CMC-Na microcapsules): Prepared according to the formulation of Example 2 and the preparation method of Example 5 of Chinese Patent CN120788004A. The core is a compound of Litsea cubeba essential oil and cinnamon essential oil (mass ratio 1:1), and the wall material is gelatin and sodium carboxymethyl cellulose (CMC-Na). Control group B (CS / TPP ion microcapsules): Prepared using the conventional chitosan-sodium tripolyphosphate (CS / TPP) ion crosslinking method. The core process is the same as above: the compound essential oil is emulsified and then added dropwise to a chitosan acetate solution containing 0.5% sodium tripolyphosphate, stirred for crosslinking, washed, and dried. (3) Experimental group (core-shell microcapsules of the present invention): microcapsules prepared according to Example 8 of the present invention. Experimental method: (1) Sample processing: 100 mg of each of the three microcapsule powders were weighed and placed in a 50 mL centrifuge tube. 30 mL of the above-mentioned hard water was added and the mixture was gently shaken and mixed. (2) Soaking and sampling: The centrifuge tubes were placed in a constant temperature shaker at 25℃ (100 rpm) and samples were taken 1 day, 3 days and 7 days after soaking. (3) Test indicators: ① Encapsulation retention rate: After each sampling, the microcapsules were collected by centrifugation, the capsules were broken and the remaining essential oil content was determined. The retention rate relative to the initial encapsulation rate before soaking was calculated. Encapsulation retention rate (%) = (essential oil content in the microcapsules after soaking / essential oil content in the initial microcapsules) × 100%. ② Microscopic morphology (SEM): A small amount of soaked microcapsules were taken, freeze-dried and sputter-coated with gold, and the surface and cross-sectional morphology were observed. ③ Average particle size and polydispersity index (PDI): Take a small amount of microcapsule suspension and measure it with a laser particle size analyzer.

[0118] Experimental results The encapsulation efficiency retention rate, SEM images, and particle size changes of different microcapsules after immersion in hard water are shown in Tables 22 and 23.

[0119] Table 22 Encapsulation retention rate of different microcapsules after immersion in hard water (%, n=3)

[0120] Table 23 Changes in the physical properties of different microcapsules after soaking in hard water for 7 days

[0121] Experimental conclusions The microcapsules of this invention maintained an encapsulation efficiency of up to 96.2% after immersion in hard water for 7 days, significantly higher than that of the illumination adhesive / CMC-Na microcapsules (68.4%) and CS / TPP ion-crosslinked microcapsules (75.8%), and exhibited an intact microstructure with almost no change in particle size. This demonstrates that its multilayer core-shell structure effectively solves the stability problem of traditional microcapsules in hard water.

[0122] Example 16 Crosslinking Agent Comparison Experiment Experimental materials and methods: The sterilized microcapsules tested were exactly the same as those in Example 8 of this invention, except that the outermost cross-linking agent was different: (1) Experimental group (this invention): the cross-linking agent was geniposide; (2) Control group A (chemical cross-linking): the cross-linking agent was replaced with an equimolar amount of glutaraldehyde (GA); (3) Control group B (other natural cross-linking agents): the cross-linking agent was replaced with an equal mass of genipin (GP); (4) Control group C (ionic cross-linking control): no covalent cross-linking agent was used.

[0123] Crosslinking agent: Sodium tripolyphosphate (TPP) was used for ionic crosslinking only after chitosan coating. Detection indicators: (1) Initial encapsulation rate; (2) Hard water tolerance: The encapsulation rate retention rate after soaking in 300 mg / L hard water for 7 days was tested; (3) Bactericidal efficacy under organic load: The logarithmic killing value (LR) of microcapsules against Staphylococcus aureus was tested in phosphate buffer containing 5% BSA for 30 minutes; (4) Shell swelling degree: The ratio of the volume after water absorption and swelling to the dry volume was measured when the microcapsules were placed in deionized water; (5) Essential oil sustained release performance: The cumulative release rate of essential oil at different time points was measured by shaking at 37°C in a simulated use environment (pH 7.4 PBS).

[0124] Experimental results The effects of different crosslinking agents on the key properties of microcapsules are shown in Table 24. Table 24 Effects of different crosslinking agents on key properties of microcapsules

[0125] Experimental conclusions The "chitosan-geniposide covalently cross-linked shell" is key to the excellent water resistance and bactericidal properties achieved in this invention. Geniposide, as a natural cross-linking agent, not only endows the microcapsules with outstanding stability under hard water and organic loads (96.5% encapsulation retention rate), but also enables the continuous and controllable release of essential oils (62.3% release rate over 12 hours) while maintaining a mild odor. In practical applications, a difference of 0.5-1.0 log in bactericidal efficiency corresponds to a 3-10 times difference in disinfection effect. This confirms that geniposide is the key cross-linking agent enabling this invention to achieve its intended effects.

[0126] This invention successfully constructed a core-shell structured plant essential oil disinfectant microcapsule through systematic synergistic screening of compound essential oils, optimization of key excipients (surfactants and chelating agents), and an innovative Pickering emulsion template combined with sequential crosslinking process. The product formulation has undergone multi-dimensional experimental verification and possesses comprehensive advantages such as significant bactericidal effect, high physicochemical stability, strong environmental adaptability, and good material compatibility.

[0127] Compared with existing chemical disinfectants and traditional essential oil microcapsule products, the core-shell microcapsules provided by this invention exhibit the following outstanding characteristics: ① Green and safe: The core active ingredient and all wall materials are derived from natural biodegradable substances, with no toxic side effects or harmful residues, making them environmentally friendly. ② Highly efficient and broad-spectrum bactericidal performance: Through synergistic compounding and efficient delivery, it can achieve a logarithmic kill value of ≥5.0 against both Gram-positive and Gram-negative bacteria, meeting the standards for highly efficient disinfection. ③ Excellent stability and environmental tolerance: The unique core-shell structure allows it to maintain structural and efficacy stability under complex conditions such as temperature changes, accelerated aging, pH fluctuations (4.5-8.5), and the presence of hard water and organic loads. ④ Controllable preparation process: The preparation process has clear steps, mild conditions, and requires no special or complex equipment, making it suitable for large-scale production using conventional formulation production lines.

[0128] ⑤ Good material compatibility: It is non-corrosive to common facility materials such as stainless steel, PVC, and silicone, and has a wide range of applications.

[0129] Application Prospects: ① Livestock and Poultry Farming: The microcapsules of this invention can be used for disinfection of farm environments and drinking water systems. Specifically, in the spray disinfection of fattening pig houses and poultry houses with livestock / poultry present, it is recommended to use an aqueous suspension with a concentration of 0.5-1.0% (based on microcapsule dry powder) for spraying 2-3 times a week. This can effectively reduce the load of pathogens such as Staphylococcus aureus, Escherichia coli, and Salmonella in the environment, preventing the occurrence of bacterial diseases. In drinking water systems, continuous addition at a ratio of 0.2-0.5% can inhibit the growth of intestinal pathogens without corroding drinking water pipelines, ensuring the health of livestock and poultry. ② Food Processing and Cold Chain: Suitable for surface disinfection of equipment in slaughterhouses, dairy plants, and meat cold storage facilities. For example, during the intervals between slaughter line operations, wiping or spraying conveyor belts, knives, and workbenches with a 0.2-0.5% microcapsule suspension, followed by rinsing with water after 15-30 minutes, can significantly reduce the risk of cross-contamination by foodborne pathogens (such as Listeria monocytogenes and Salmonella). ③ Medical and Public Health: Suitable for daily disinfection of public areas such as hospitals, schools, and nursing homes. A 0.1-0.3% concentration is recommended for wiping object surfaces or spraying the environment, with an action time of 30 minutes. This effectively kills common infection-related bacteria and is gentle and non-irritating to staff and patients. ④ Home and Pet Care: Can be used as a safe household disinfectant, suitable for kitchens, bathrooms, floors, and pet-keeping environments. A 0.1-0.2% concentration is recommended. No rinsing is required after use. It is pet- and child-friendly and also has deodorizing properties.

[0130] The successful implementation of this invention not only provides a new technical solution for the application of plant essential oils in the field of disinfection, but also opens up a practical and feasible path for the large-scale and engineered application of natural active substances in the field of disinfection. This technology aligns with the policy orientation of green development and reducing antibiotic dependence, and while ensuring biosafety and public health, it also has significant economic benefits and social value.

[0131] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A core-shell type plant essential oil disinfectant microcapsule, characterized in that, The microcapsule comprises, from the inside out: a core, a Pickering interface layer, an ionic crosslinking gel layer, and a covalently crosslinked polymer shell; the core is composed of a blend of Litsea cubeba essential oil and cinnamon essential oil; the Pickering interface layer coats the surface of the core and is stabilized by nanocellulose as a solid particle emulsifier; the ionicly crosslinked gel layer coats the outside of the Pickering interface layer and is composed of sodium alginate and calcium ions crosslinked together; the covalently crosslinked polymer shell coats the outer surface of the gel layer and is formed by chitosan and geniposide crosslinked together by covalent bonds.

2. The disinfection microcapsule according to claim 1, characterized in that, The microcapsules contain, by weight, 90-110 parts core, 2-5 parts Pickering interface layer, 10-30 parts ion-crosslinked gel layer, and 2-10 parts covalently crosslinked polymer shell.

3. The disinfection microcapsule according to claim 2, characterized in that, The mass ratio of Litsea cubeba essential oil to cinnamon essential oil is 1:(0.8-1.2); the mass ratio of sodium alginate to calcium ions is 1:(0.1-1.2); and the mass ratio of geniposide to chitosan is (0.2-0.5):

1.

4. The core-shell type plant essential oil disinfectant microcapsule according to claim 1, characterized in that, The microcapsules have an encapsulation rate of at least 70% for the essential oil.

5. A method for preparing core-shell type plant essential oil disinfectant microcapsules according to any one of claims 1-4, characterized in that, Includes the following steps: (1) The core oil phase containing Litsea cubeba essential oil and cinnamon essential oil is mixed and emulsified with the aqueous phase containing nanocellulose Pickering interface layer, so that the Pickering interface layer coats the core surface to obtain emulsion one; (2) Add sodium alginate solution to the emulsion obtained in step (1), stir evenly, and then introduce calcium ion solution to carry out ion cross-linking reaction to form an ion cross-linked gel layer that encapsulates the Pickering interface layer. Gel particles were obtained; (3) Transfer the gel particles obtained in step (2) into the chitosan solution, adjust the pH to 5.0-6.0, add geniposide, and react at 35-40℃ for 1-3 hours to form a covalently cross-linked outer shell layer on the outside of the gel layer.

6. The method according to claim 5, characterized in that, In step (1), the oil phase further includes the surfactant Tween-80 and medium-chain triglycerides, wherein the amount of Tween-80 added is 1%-10% of the mass of the oil phase, and the amount of medium-chain triglycerides added is 5%-20% of the mass of the oil phase; The aqueous phase also contains sodium citrate, a chelating agent, with a mass concentration of 0%-0.5% in the aqueous phase; the nanocellulose has a mass concentration of 0.2%-0.5% in the aqueous phase.

7. The method according to claim 5, characterized in that, In step (2), the mass concentration of sodium alginate in the reaction system is 0.5%-2.0%, and the concentration of calcium ion solution is 50-150 mM.

8. The method according to claim 5, characterized in that, In step (3), the mass concentration of chitosan in the reaction system is 0.1%-0.5%, and the mass concentration of geniposide in the reaction system is 0.05%-0.2%.

9. The method according to claim 5, characterized in that, It also includes step (4): spray drying the microcapsule suspension obtained in step (3) to obtain microcapsule powder; the inlet temperature of the spray drying is 140-160℃ and the outlet temperature is 70-90℃.

10. The use of the core-shell type plant essential oil disinfectant microcapsules according to any one of claims 1-4 in the preparation of disinfectants for livestock and poultry breeding environments, disinfectants for the surfaces of food processing equipment or utensils, disinfectants for the surfaces of non-critical objects in medical institutions, and disinfectants for hard surfaces in homes or public places.

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