Preparation method of an acid-responsive emulsion

Abstract

The present invention discloses a preparation method of an acid-responsive emulsion, which relates to the technical field of emulsion preparation. The preparation method of this acid-responsive emulsion comprises the following steps: S1. Prepare volatile oil; S2. Prepare a CNC suspension: Disperse microcrystalline cellulose in sulfuric acid and stir in a water bath; Subject the reaction product to dialysis treatment to displace the excess sulfuric acid to obtain a CNC suspension with a constant pH; S3. Prepare CNC-CaCO3 composite particles; S4. Prepare a composite emulsion: CNC affects the growth and shape of CaCO3 crystal grains through electrostatic action and complexation, thereby generating nanoscale composite particles. As a PE stabilizer, compared with CNC-PE and CaCO3-PE, CNC / Ca-PE has excellent stability. Due to the participation of CaCO3 in the system, CNC / Ca-PE exhibits pH-responsive behavior, manifested as an increase in the emulsion particle size, destruction of the shell layer and three-dimensional structure, and rapid release in an acidic medium.

Description

Technical Field

[0001] The present invention relates to the technical field of emulsion preparation, and particularly to a preparation method of an acid-responsive emulsion. Background Art

[0002] Pickering emulsion (PE) is an emulsion in which solid particles replace traditional surfactants as stabilizers. It has attracted much attention due to its environmental friendliness, easy preparation, good stability and other characteristics. At present, PE is widely used in fields such as food packaging and biomedicine to ensure, transport and control the release of active ingredients. Taking essential oil (EO) as an example, EO is a volatile aromatic oily liquid extracted from plants by water distillation. The PE prepared with cellulose, chitosan, protein as solid particles and Tilia amurensis, cinnamon, citrus, clove EO as the oil phase has excellent stability, which is beneficial to the protection and storage of EO active ingredients.

[0003] The main function of general PE is to stably deliver, protect and slowly release EO components by using solid particles, which is a very promising drug release pathway. Some biopolymers can form stable, pH-responsive emulsions to control the active release. Specific applications require the emulsion to respond to environmental stimuli to release active substances faster, such as food preservation and oral administration. The stimulus response of PE depends on the properties of solid particles and environmental factors, and the particle structure and physical and chemical properties endow it with a high-level response to environmental pH.

[0004] The responsive release system protects active substances through the core or three-dimensional structure. Cellulose and its nanoparticles are abundant biopolymers in nature and valuable stabilizers. Cellulose nanocrystals (CNC) are needle-shaped or rod-shaped functional nanomaterials with a large specific surface area, high aspect ratio, strong affinity and adsorption ability. However, CNC has high hydrophilicity, high charge and poor emulsifying performance, which hinders its adsorption at the oil-water interface and leads to unstable emulsions. At present, its emulsifying ability is mainly improved by hydrophobic modification or complexation with polysaccharides and proteins. The challenge lies in managing the release of active ingredients. CNC can be chemically modified or combined with other responsive substances to not only improve the emulsion stability but also make the emulsion responsive.

[0005] In recent years, inorganic materials such as calcium carbonate (CaCO3) have been used to enhance the stability and stimulus responsiveness of emulsions. CaCO3 is used as a PE emulsifier and has attracted much attention in the food and pharmaceutical industries due to its safety, low cost and degradability. Its acid responsiveness can achieve controllable drug targeted delivery, and it can also be converted into biocompatible Ca 2+ , but its drug loading capacity is limited. It has been found that CNC and Ca 2+ can be complexed by electrostatic attraction and ligand bonds, and a small amount of Ca 2+Aggregating on the CNC surface can shield the electrostatic repulsion and trigger complexation and bridging. Therefore, it is hypothesized that combining CNC with CaCO3 can form a network structure to maintain the super-stability of the emulsion and achieve responsive stimuli.

[0006] In this study, this application innovatively combines CNC with CaCO3 with responsive characteristics to successfully prepare CNC / CaCO3 (CNC / Ca) nanocomposites and uses them as stabilizers for PE. Through long-term stability evaluation and pH-responsive characterization of the emulsion, the results show that the emulsion stabilized by CNC / Ca not only significantly enhances its own stability but also endows the emulsion with excellent acid-responsive properties. In summary, this application has developed a new method to make the composite material have pH-responsive ability by promoting the formation of a nanocomposite material from CNC and CaCO3 and successfully applied it to maintain the long-term stability of the emulsion. Summary of the Invention

[0007] In view of this, in view of the deficiencies of the existing technology, the main purpose of the present invention is to provide a preparation method for an acid-responsive emulsion. CNC affects the growth and shape of CaCO3 crystal grains through electrostatic and complexation effects, thereby generating nanoscale composite particles. As a PE stabilizer, compared with CNC-PE and CaCO3-PE, CNC / Ca-PE has excellent stability. Due to the participation of CaCO3 in the system, CNC / Ca-PE exhibits pH-responsive behavior, manifested as an increase in the emulsion particle size, the destruction of the shell layer and three-dimensional structure, and rapid release in an acidic medium.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A preparation method for an acid-responsive emulsion, comprising the following steps:

[0010] S1. Prepare volatile oil;

[0011] S2. Prepare a CNC suspension: Disperse microcrystalline cellulose in sulfuric acid, stir in a water bath, and then terminate the reaction; Centrifuge the reaction product to separate the precipitate; Dialyze the reaction product to replace the excess sulfuric acid to obtain a CNC suspension with a constant pH.

[0012] S3. Prepare CNC-CaCO3 composite particles: Put Ca(OH)2 into pure water, stir at room temperature to obtain a Ca(OH)2 solution; Add the CNC suspension and stir at room temperature to mix the two evenly; Then introduce CO2 gas at a uniform flow rate and stir at room temperature to obtain CNC-CaCO3 composite particles.

[0013] S4. Preparation of composite emulsion: Use the volatile oil as the oil phase, take the CNC-CaCO3 composite particles as the water phase, drop the oil phase into the water phase containing the CNC-CaCO3 composite particles, shear and homogenize to obtain the composite emulsion.

[0014] As a preferred embodiment: In S2, the microcrystalline cellulose is dispersed in 20 times the amount of sulfuric acid with a mass fraction of 64%, stirred in a water bath at 45 °C and 400 rpm for 45 min, and then 10 times the amount of pure water is added to terminate the reaction; the reaction product is centrifuged at 4 °C and 5000 rpm for 10 min to separate the precipitate.

[0015] As a preferred embodiment: In S2, the reaction product is encapsulated in a regenerated cellulose dialysis bag for dialysis treatment to displace the excess sulfuric acid, and a CNC suspension with a constant pH is obtained after 5 days of dialysis.

[0016] As a preferred embodiment: In S3, Ca(OH)2 is put into pure water, and under normal temperature conditions, it is stirred at 500 rpm with a magnetic stirrer for 10 min to obtain a Ca(OH)2 solution with a mass fraction of 0.07%-0.14%.

[0017] As a preferred embodiment: In S3, the CNC suspension is added, and under normal temperature conditions, it is stirred at 500 rpm with a magnetic stirrer for 10 min to mix the two evenly; then CO2 gas is introduced at a uniform flow rate for 10 min, and under normal temperature conditions, it is stirred at 500 rpm with a magnetic stirrer for 10-12 h to obtain the CNC-CaCO3 composite particles.

[0018] As a preferred embodiment: In S4, the volatile oil includes tea oil and Mosla chinensis Maxim. volatile oil. The specific preparation process of the Mosla chinensis Maxim. volatile oil is as follows: Weigh the Mosla chinensis Maxim. medicinal materials, add 6-8 times the amount of water, distill and extract for 3-5 h, separate the water layer and the volatile oil layer, and add anhydrous sodium sulfate to the volatile oil layer for dehydration to obtain the Mosla chinensis Maxim. volatile oil.

[0019] As a preferred embodiment: In S4, shear at 22000 rpm for 5-10 min.

[0020] As a preferred embodiment: The homogenization conditions in S4 are as follows: Homogenize in a high-pressure homogenizer at pressures of 300 bar and 500 bar for 10-30 min each.

[0021] As a preferred embodiment: The CNC-CaCO3 composite particles in S3 include CNC / Ca2 composite particles and CNC2 / Ca composite particles.

[0022] As a preferred embodiment: The composite emulsion in S4 includes CNC / Ca2 composite emulsion and CNC2 / Ca composite emulsion.

[0023] The present invention has obvious advantages and beneficial effects compared with the prior art. Specifically, as can be seen from the above technical solutions, different mass ratios of CNC / Ca nanocomposite particles were successfully prepared in this study; CNC affects the growth and shape of CaCO3 crystal grains through electrostatic and complexation effects, thereby generating nanoscale composite particles, which are used as PE stabilizers. Compared with CNC-PE and CaCO3-PE, CNC / Ca-PE has excellent stability. In addition, all CNC / Ca-PEs remained stable for up to 4 months except for CNC1 / Ca2-PE and CNC2 / Ca3-PE. Due to the participation of CaCO3 in the system, CNC / Ca-PE exhibits pH-responsive behavior, manifested as an increase in the emulsion particle size, destruction of the shell layer and three-dimensional structure, and rapid release in acidic media. The CNC / Ca-PE nanocomposite particles loaded with EO exhibit good antibacterial activity, and their emulsifying properties and structure affect the antibacterial activity of the composite particles. CNC / Ca-PE has a high drug loading capacity and is expected to become a pH-responsive oral drug carrier.

[0024] To more clearly elaborate the structural features and efficacy of the present invention, the following will be described in detail with reference to the accompanying drawings and specific embodiments. Description of the Drawings

[0025] Figure 1 Infrared spectra of CNC / Ca nanocomposites, CNC, and CaCO3 in Examples 1 to 5 of the present invention;

[0026] Figure 2 XPS spectra of CNC / Ca nanocomposites, CNC, and CaCO3 in Examples 1 to 5 of the present invention;

[0027] Figure 3 Peak-fitted high-resolution C1s spectrum of CNC in Comparative Example 1 of the present invention;

[0028] Figure 4 Peak-fitted high-resolution C1s spectrum of CNC1 / Ca2 in Example 1 of the present invention;

[0029] Figure 5 Peak-fitted high-resolution C1s spectrum of CNC2 / Ca2 in Example 2 of the present invention;

[0030] Figure 6 Peak-fitted high-resolution C1s spectrum of CNC3 / Ca2 in Example 3 of the present invention;

[0031] Figure 7 Peak-fitted high-resolution C1s spectrum of CNC2 / Ca1 in Example 4 of the present invention;

[0032] Figure 8 It is the high-resolution C1s spectral peak fitting diagram of CNC2 / Ca3 in Example 5 of the present invention;

[0033] Figure 9 It is the XRD diffraction pattern of CNC / Ca nanocomposite, CNC and CaCO3 in Examples 1 to 5 of the present invention;

[0034] Figure 10 It is the AFM map of CNC in Comparative Example 1 of the present invention;

[0035] Figure 11 It is the AFM map of CaCO3 in Comparative Example 2 of the present invention;

[0036] Figure 12 It is the AFM map of CNC2 / Ca1 in Example 4 of the present invention;

[0037] Figure 13 It is the AFM map of CNC2 / Ca2 in Example 2 of the present invention;

[0038] Figure 14 It is the AFM map of CNC2 / Ca3 in Example 5 of the present invention;

[0039] Figure 15 It is the elastic modulus diagram of CNC2 / Ca in Examples 4, 2 and 5 of the present invention;

[0040] Figure 16 It is the TEM map of CNC / Ca composite, CNC and CaCO3 in Examples 1 to 5 of the present invention;

[0041] Figure 17 It is the TSI of CNC / Ca-PE, CNC-PE and CaCO3-PE in Examples 1 to 5 of the present invention;

[0042] Figure 18 It is the storage time schematic diagram of CNC / Ca-PE, CNC-PE and CaCO3-PE in Examples 1 to 5 of the present invention;

[0043] Figure 19 It is the microscopic image of CNC / Ca-PE, CNC-PE and CaCO3-PE at pH = 2, 7 and 12 in Examples 1 to 5 of the present invention;

[0044] Figure 20 It is the Cryo-SEM image of CNC2 / Ca-PE and CNC-PE in Examples 4, 2 and 5 of the present invention;

[0045] Figure 21Cryo-SEM images of CNC2 / Ca-PE and CNC-PE at pH = 2 in Example 4, Example 2 and Example 5 of the present invention;

[0046] Figure 22 Release curves of CNC2 / Ca-PE and CNC-PE in 20% ethanol in Example 4, Example 2 and Example 5 of the present invention;

[0047] Figure 23 Release curves of CNC2 / Ca-PE and CNC-PE in pH = 2 HCl in Example 4, Example 2 and Example 5 of the present invention;

[0048] Figure 24 Release curves of CNC2 / Ca-PE and CNC-PE in pH = 6.8 PBS solution in Example 4, Example 2 and Example 5 of the present invention;

[0049] Figure 25 Microscopic images of CNC2 / Ca-PE and CNC-PE in Example 4, Example 2 and Example 5 of the present invention;

[0050] Figure 26 Microscopic images of simulated gastric digestion of CNC2 / Ca-PE and CNC-PE in Example 4, Example 2 and Example 5 of the present invention;

[0051] Figure 27 Microscopic images of simulated intestinal digestion of CNC2 / Ca-PE and CNC-PE in Example 4, Example 2 and Example 5 of the present invention;

[0052] Figure 28 Bioaccessibility of CNC2 / Ca-PE and CNC-PE after simulated gastrointestinal digestion in Example 4, Example 2 and Example 5 of the present invention;

[0053] Figure 29 Antibacterial activities of CNC / Ca-PE and CNC-PE against Escherichia coli in Examples 1 to 5 of the present invention;

[0054] Figure 30 Antibacterial activities of CNC / Ca-PE and CNC-PE against Staphylococcus aureus in Examples 1 to 5 of the present invention. Detailed implementation mode

[0055] The present invention is as Figure 1 shown in FIGS. 1 to 30, and a preparation method of an acid-responsive emulsion includes the following steps:

[0056] S1. Prepare volatile oil;

[0057] S2. Preparation of CNC suspension: Disperse microcrystalline cellulose in sulfuric acid, stir in a water bath, and then terminate the reaction; Centrifuge the reaction product to separate the precipitate; Dialyze the reaction product to displace the excess sulfuric acid to obtain a CNC suspension with a constant pH.

[0058] S3. Preparation of CNC-CaCO3 composite particles: Put Ca(OH)2 into pure water, stir at room temperature to obtain a Ca(OH)2 solution; Add the CNC suspension and stir at room temperature to mix them evenly; Then, introduce CO2 gas at a uniform flow rate and stir at room temperature to obtain CNC-CaCO3 composite particles.

[0059] S4. Preparation of composite emulsion: Use volatile oil as the oil phase, take the CNC-CaCO3 composite particles as the water phase, drop the oil phase into the water phase containing the CNC-CaCO3 composite particles, shear and homogenize to obtain a composite emulsion.

[0060] In this S2, disperse microcrystalline cellulose in 20 times the amount of sulfuric acid with a mass fraction of 64%, stir in a water bath at 45 °C and 400 rpm for 45 min, and then add 10 times the amount of pure water to terminate the reaction; Centrifuge the reaction product at 4 °C and 5000 rpm for 10 min to separate the precipitate.

[0061] In this S2, encapsulate the reaction product in a regenerated cellulose dialysis bag for dialysis to displace the excess sulfuric acid, and dialyze for 5 days to obtain a CNC suspension with a constant pH.

[0062] In this S3, put Ca(OH)2 into pure water, stir with a magnetic stirrer at 500 rpm for 10 min at room temperature to obtain a Ca(OH)2 solution with a mass fraction of 0.07%-0.14%.

[0063] In this S3, add the CNC suspension, stir with a magnetic stirrer at 500 rpm for 10 min at room temperature to mix them evenly; Then, introduce CO2 gas at a uniform flow rate for 10 min, and stir with a magnetic stirrer at 500 rpm for 10-12 h at room temperature to obtain CNC-CaCO3 composite particles.

[0064] In this S4, the volatile oil includes tea oil and Mosla chinensis Maxim. volatile oil. The specific preparation process of the Mosla chinensis Maxim. volatile oil is as follows: Weigh the Mosla chinensis Maxim. medicinal materials, add 6-8 times the amount of water, distill and extract for 3-5 h, separate the water layer and the volatile oil layer, and add anhydrous sodium sulfate to the volatile oil layer for dehydration to obtain the Mosla chinensis Maxim. volatile oil.

[0065] In this S4, shear at 22000 rpm for 5-10 min.

[0066] The homogenization conditions in S4 are as follows: homogenize in a high-pressure homogenizer at pressures of 300 bar and 500 bar for 10 - 30 min each.

[0067] The CNC-CaCO3 composite particles in S3 include CNC / Ca2 composite particles and CNC2 / Ca composite particles.

[0068] The composite emulsion in S4 includes CNC / Ca2 composite emulsion and CNC2 / Ca composite emulsion.

[0069] Example 1: A method for preparing an acid-responsive emulsion, comprising the following steps:

[0070] S1. Weigh Mosla chinensis Maxim. herbs, put them into a round-bottom flask, add 6 times the amount of water, distill and extract for 3 h, stop heating, open the valve, separate the water layer and the volatile oil layer, add anhydrous sodium sulfate to the volatile oil layer for dehydration, and obtain Mosla chinensis Maxim. volatile oil.

[0071] S2. Prepare a CNC suspension. Disperse MCC in 64 wt% sulfuric acid at 20 times (m / v), stir in a water bath at 45 °C and 400 rpm for 45 min, and then add 10 times the amount of pure water to terminate the reaction. The reaction product is centrifuged at 4 °C and 5000 rpm for 10 min, and the reaction product is encapsulated in a regenerated cellulose dialysis bag (12,000 Kda) for dialysis treatment to replace the excess acid. Dialyze for 5 days to obtain a CNC suspension with a constant pH. Dilute the CNC suspension to 0.4% (m / v).

[0072] S3. Preparation of CNC1 / Ca2 composite particles. Add 0.0700 g of Ca(OH)2 to 55.22 mL of pure water, and under normal temperature conditions, stir with a magnetic stirrer at 500 rpm for 10 min to obtain a 0.1% Ca(OH)2 solution. Take 14.78 mL of the 0.4% CNC suspension prepared in S2, and make their total volume 70 mL. Under normal temperature conditions, stir with a magnetic stirrer at 500 rpm for 10 min to mix them evenly. Then, introduce CO2 gas at a uniform flow rate for 10 min, and under normal temperature conditions, stir with a magnetic stirrer at 500 rpm overnight to obtain a CNC1 / Ca2 complex.

[0073] S4. Preparation of the emulsion. Mix 16.8 mL of camellia oil and 4.2 mL of Mosla chinensis Maxim. volatile oil evenly as the oil phase, and take 49 mL of the CNC1 / Ca2 complex prepared in S3 as the water phase. Slowly drip the oil phase into the water phase containing the CNC1 / Ca2 complex, shear at 22000 rpm for 5 min, and then transfer it to a high-pressure homogenizer at pressures of 300 bar and 500 bar for homogenization for 21 min each, and obtain an emulsion stabilized by the CNC1 / Ca2 complex.

[0074] Example 2: A preparation method of an acid-responsive emulsion, comprising the following steps:

[0075] S1. Weigh the Mosla chinensis Maxim. herbs, put them into a round-bottom flask, add 6 times the amount of water, distill and extract for 3 h, stop heating, open the valve, separate the water layer and the volatile oil layer, add anhydrous sodium sulfate to the volatile oil layer for dehydration to obtain Mosla chinensis Maxim. volatile oil.

[0076] S2. Prepare a CNC suspension. Disperse MCC in 64 wt% sulfuric acid at 20 times (m / v), stir in a water bath at 45 °C and 400 rpm for 45 min, and then add 10 times the amount of pure water to terminate the reaction. The reaction product is centrifuged at 4 °C and 5000 rpm for 10 min, and the reaction product is encapsulated in a regenerated cellulose dialysis bag (12,000 Kda) for dialysis treatment to replace the excess acid. Dialyze for 5 days to obtain a CNC suspension with a constant pH. Dilute the CNC suspension to 0.7% (m / v).

[0077] S3. Preparation of CNC2 / Ca2 composite particles. Add 0.0705 g of Ca(OH)2 to 45.37 mL of pure water, and stir at 500 rpm with a magnetic stirrer for 10 min at room temperature to obtain a 0.1% Ca(OH)2 solution. Take 24.63 mL of the 0.7% CNC suspension prepared in S2, and make the volume of the two 70 mL. Stir evenly at 500 rpm with a magnetic stirrer for 10 min at room temperature. Then introduce CO2 gas at a uniform flow rate for 10 min, and stir at 500 rpm with a magnetic stirrer overnight at room temperature to obtain a CNC2 / Ca2 composite.

[0078] S4. Preparation of the emulsion. Mix 16.8 mL of camellia oil and 4.2 mL of Mosla chinensis Maxim. volatile oil evenly as the oil phase, and take 49 mL of the CNC2 / Ca2 composite prepared in S3 as the water phase. Slowly drip the oil phase into the water phase containing the CNC2 / Ca2 composite, shear at 22000 rpm for 5 min, and then transfer it to a high-pressure homogenizer under a pressure of 300 bar and 500 bar for homogenization for 21 min each to obtain an emulsion stabilized by the CNC2 / Ca2 composite.

[0079] Example 3: A preparation method of an acid-responsive emulsion, comprising the following steps:

[0080] S1. Weigh the Mosla chinensis Maxim. herbs, put them into a round-bottom flask, add 6 times the amount of water, distill and extract for 3 h, stop heating, open the valve, separate the water layer and the volatile oil layer, add anhydrous sodium sulfate to the volatile oil layer for dehydration to obtain Mosla chinensis Maxim. volatile oil.

[0081] S2. Prepare CNC suspension. Disperse MCC in 20 times (mass / volume) of 64 wt% sulfuric acid. Stir in a water bath at 45 °C and 400 rpm for 45 min. Then add 10 times of pure water to terminate the reaction. The reaction product is centrifuged at 4 °C and 5000 rpm for 10 min. The reaction product is encapsulated in a regenerated cellulose dialysis bag (12,000 Kda) for dialysis to replace excess acid. Dialysis for 5 days to obtain a CNC suspension with a constant pH. Dilute the CNC suspension to 1% (m / v).

[0082] Preparation of S3 and CNC3 / Ca2 composite particles: 0.0705 g of Ca(OH)2 was added to 35.52 mL of pure water. At room temperature, a magnetic stirrer was used to stir at 500 rpm for 10 min to obtain a 0.1% Ca(OH)2 solution. 34.48 mL of the 1% CNC suspension prepared by S2 was taken to make the volume of the two 70 mL. At room temperature, a magnetic stirrer was used to stir at 500 rpm for 10 min to mix the two evenly. Then, CO2 gas was introduced at a uniform flow rate for 10 min. At room temperature, a magnetic stirrer was used to stir at 500 rpm overnight to obtain a CNC3 / Ca2 complex.

[0083] S4, preparation of emulsion, 16.8 mL tea oil and 4.2 mL Elsholtzia elata volatile oil were mixed evenly as the oil phase, and 49 mL of CNC3 / Ca2 complex prepared in S3 was taken as the water phase. The oil phase was slowly dripped into the water phase containing the CNC3 / Ca2 complex, sheared at 22000 rpm for 5 min, and then transferred to a high-pressure homogenizer at a pressure of 300 bar and 500 bar for homogenization for 21 min each, to obtain a stable emulsion of CNC3 / Ca2 complex.

[0084] Example 4: A method for preparing an acid-responsive emulsion, comprising the following steps:

[0085] S1. Weigh the medicinal material of Elsholtzia jiangruta, put it into a round-bottom flask, add 6 times the amount of water, distill and extract for 3 hours, stop heating, open the valve, separate the water layer and the volatile oil layer, add anhydrous sodium sulfate to the volatile oil layer for dehydration, and obtain Elsholtzia jiangruta volatile oil.

[0086] S2. Prepare CNC suspension. Disperse MCC in 20 times (m / v) 64 wt% sulfuric acid. Stir in a water bath at 45 °C and 400 rpm for 45 min. Then add 10 times pure water to terminate the reaction. The reaction product is centrifuged at 4 °C and 5000 rpm for 10 min. The reaction product is encapsulated in a regenerated cellulose dialysis bag (12,000 Kda) for dialysis to replace excess acid. Dialysis for 5 days to obtain a CNC suspension with a constant pH. Dilute the CNC suspension to 0.7% (m / v).

[0087] Preparation of S3 and CNC2 / Ca1 composite particles: 0.0507 g of Ca(OH)2 was added to 45.37 mL of pure water. At room temperature, a magnetic stirrer was used to stir at 500 rpm for 10 min to obtain a 0.07% Ca(OH)2 solution. 24.63 mL of the 0.7% CNC suspension prepared in S2 was taken to make the volume of the two 70 mL. At room temperature, a magnetic stirrer was used to stir at 500 rpm for 10 min to mix the two evenly. Then, CO2 gas was introduced at a uniform flow rate for 10 min. At room temperature, a magnetic stirrer was used to stir at 500 rpm overnight to obtain a CNC2 / Ca1 complex.

[0088] S4, preparation of emulsion, 16.8 mL tea oil and 4.2 mL Elsholtzia elata volatile oil were mixed evenly as the oil phase, and 49 mL of CNC2 / Ca1 complex prepared in S3 was taken as the water phase. The oil phase was slowly dripped into the water phase containing the CNC2 / Ca1 complex, sheared at 22000 rpm for 5 min, and then transferred to a high-pressure homogenizer at a pressure of 300 bar and 500 bar for homogenization for 21 min each, to obtain a stable emulsion of CNC2 / Ca1 complex.

[0089] Example 5: A method for preparing an acid-responsive emulsion, comprising the following steps:

[0090] S1. Weigh the medicinal material of Elsholtzia jiangruta, put it into a round-bottom flask, add 6 times the amount of water, distill and extract for 3 hours, stop heating, open the valve, separate the water layer and the volatile oil layer, add anhydrous sodium sulfate to the volatile oil layer for dehydration, and obtain Elsholtzia jiangruta volatile oil.

[0091] S2. Prepare CNC suspension. Disperse MCC in 20 times (m / v) 64 wt% sulfuric acid. Stir in a water bath at 45 °C and 400 rpm for 45 min. Then add 10 times pure water to terminate the reaction. The reaction product is centrifuged at 4 °C and 5000 rpm for 10 min. The reaction product is encapsulated in a regenerated cellulose dialysis bag (12,000 Kda) for dialysis to replace excess acid. Dialysis for 5 days to obtain a CNC suspension with a constant pH. Dilute the CNC suspension to 0.7% (m / v).

[0092] S3. Preparation of CNC2 / Ca3 composite particles: 0.1001 g of Ca(OH)2 was added to 45.37 mL of pure water. Under normal temperature conditions, it was stirred for 10 min at 500 rpm using a magnetic stirrer to obtain a 0.14% Ca(OH)2 solution. 24.63 mL of the 0.7% CNC suspension prepared in S2 was taken, and the total volume of the two was made to be 70 mL. Under normal temperature conditions, it was stirred for 10 min at 500 rpm using a magnetic stirrer to mix them evenly. Then, CO2 gas was introduced at a uniform flow rate for 10 min. Under normal temperature conditions, it was stirred overnight at 500 rpm using a magnetic stirrer to obtain the CNC2 / Ca3 composite.

[0093] S4. Preparation of the emulsion: 16.8 mL of camellia oil and 4.2 mL of Mosla chinensis Maxim. volatile oil were mixed evenly as the oil phase, and 49 mL of the CNC2 / Ca3 composite prepared in S3 was taken as the water phase. The oil phase was slowly dropped into the water phase containing the CNC2 / Ca3 composite, sheared at 22000 rpm for 5 min, and then transferred to a high-pressure homogenizer under pressures of 300 bar and 500 bar for homogenization for 21 min each, to obtain an emulsion stabilized by the CNC2 / Ca3 composite.

[0094] Comparative Example 1: A preparation method of a CNC emulsion without adding CaCO3, comprising the following steps:

[0095] S1. Weigh the Mosla chinensis Maxim. medicinal materials, put them into a round-bottom flask, add 6 times the amount of water, distill and extract for 3 h, stop heating, open the valve, separate the water layer and the volatile oil layer, and add anhydrous sodium sulfate to the volatile oil layer for dehydration to obtain Mosla chinensis Maxim. volatile oil.

[0096] S2. Preparation of a 0.7% CNC suspension: MCC was dispersed in 64 wt% sulfuric acid at 20 times (m / v), stirred in a water bath at 45 °C and 400 rpm for 45 min, and then 10 times the amount of pure water was added to terminate the reaction. The reaction product was centrifuged at 4 °C and 5000 rpm for 10 min, and the reaction product was encapsulated in a regenerated cellulose dialysis bag (12,000 Kda) for dialysis treatment to replace the excess acid. After dialysis for 5 days, a CNC suspension with a constant pH was obtained. The CNC suspension was diluted to 0.7% (m / v).

[0097] S3. Preparation of a CNC emulsion: 16.8 mL and 4.2 mL of Mosla chinensis Maxim. volatile oil were mixed evenly as the oil phase, and 49 mL of the CNC suspension prepared in S2 was taken as the water phase. The oil phase was slowly dropped into the CNC suspension, sheared at 22000 rpm for 5 min, and then transferred to a high-pressure homogenizer under pressures of 300 bar and 500 bar for homogenization for 21 min each, to obtain a CNC-stabilized emulsion.

[0098] Comparative Example 2: A preparation method of CaCO3 emulsion without CNC, comprising the following steps:

[0099] S1. Weigh the Mosla chinensis Maxim. herbs, put them into a round-bottom flask, add 6 times the amount of water, distill and extract for 3 h, stop heating, open the valve, separate the water layer and the volatile oil layer, add anhydrous sodium sulfate to the volatile oil layer for dehydration to obtain Mosla chinensis Maxim. volatile oil.

[0100] S2. Preparation of CaCO3 particles: Add 0.1001 g of Ca(OH)2 to 70 mL of pure water. Under normal temperature conditions, use a magnetic stirrer to stir at 500 rpm for 10 min to obtain a 0.1% Ca(OH)2 solution, and then introduce CO2 gas at a uniform flow rate for 10 min. Under normal temperature conditions, use a magnetic stirrer at 500 rpm to complete the reaction to obtain CaCO3 particles.

[0101] S3. Preparation of CaCO3 emulsion: Take 16.8 mL of camellia oil and 4.2 mL of Mosla chinensis Maxim. volatile oil, mix them evenly as the oil phase, and take 49 mL of the CaCO3 suspension prepared in S2 as the water phase. Slowly drop the oil phase into the CaCO3 suspension, shear at 22000 rpm for 5 min, and then transfer it to a high-pressure homogenizer under pressures of 300 bar and 500 bar for homogenization for 21 min each to obtain a CaCO3-stabilized emulsion.

[0102] Test Example 1: Determination of the infrared spectrum of the composite:

[0103] Using a Fourier transform infrared spectrometer under the conditions of a wavelength of 4000 - 400 cm-1, a resolution of 4 cm-1, and a cumulative scanning number of 16, the CNC / Ca nanocomposites, CNC, and CaCO3 prepared in Examples 1 to 5 were respectively mixed and pressed into tablets with potassium bromide for infrared spectral analysis. The ratio of potassium bromide to the sample was 100:1 (mg / mg). The infrared spectra of CNC1 / Ca2, CNC2 / Ca2, CNC3 / Ca2, CNC2 / Ca1, CNC2 / Ca3, CNC, and CaCO3 in Examples 1 to 5 were measured, and the results are shown in Figure 1 .

[0104] Result analysis: As can be seen from Figure 1 , in the CNC / Ca nanocomposites in Examples 1 to 5, the original peaks of the original CNC and CaCO3 were retained. The characteristic peak positions of CNC were 3338 cm -1 and 2902 cm -1 , which were attributed to the -OH and C-H stretching vibrations in the cellulose molecule. The CaCO3 particles were at 1424 cm -1 , 875 cm -1 and 713 cm-1 There is an absorption peak. The calcite-type CaCO3 crystal has an absorption peak at 1424 cm -1 The V3 characteristic absorption peak at is the antisymmetric stretching vibration of C-O. The absorption peak at 875 cm -1 is the V2 absorption peak of the calcite CaCO3 crystal and the out-of-plane deformation vibration peak of CO3 2- The absorption peak at 713 cm -1 is the V4 absorption peak of the calcite CaCO3 crystal, which is related to the bending vibration of the C-O bond. Therefore, the CaCO3 particles produced in this study are calcite-type crystals. In the FT-IR spectrum of the CNC / Ca nanocomposite particles, due to the relatively high content of CaCO3 in the nanocomposite particles, significant V2 characteristic absorption peaks of the CaCO3 particles appeared at around 875 cm -1 for CNC1 / Ca2, CNC2 / Ca2, and CNC2 / Ca3. After being prepared into the CNC / Ca nanocomposite particles in Examples 1 to 5, the characteristic peaks of the single components were retained, indicating that the process of preparing the CNC / Ca nanocomposites with different ratios of CNC and CaCO3 belongs to a physical process.

[0105] Test Example 2: X-ray Photoelectron Spectroscopy Analysis

[0106] The CNC / Ca nanocomposites, CNC, and CaCO3 prepared in Examples 1 to 5 were measured using an XPS spectrometer. Using Avantage software, under the conditions of 12 kV, Al Kα irradiation (hv = 1486.6 eV), beam spot 400 μm, pass energy 50 eV, and step size 0.1 eV, the XPS spectra of C, O, and Ca atoms on the sample surface were obtained. The XPS spectra of CNC1 / Ca2, CNC2 / Ca2, CNC3 / Ca2, CNC2 / Ca1, CNC2 / Ca3, CNC, and CaCO3 in Examples 1 to 5 were measured, and the results are shown in Figures 2 - 8 and Table 1.

[0107] Table 1: Atomic Densities of CNC / Ca Nanocomposites, CNC, and CaCO3 in Examples 1 to 5

[0108]

[0109] Result Analysis: As can be seen from Figure 2 , the XPS spectrum of CNC shows characteristic peaks of carbon (C) and oxygen (O) elements. The C peak appears at 284 - 286 eV, and the O peak appears at 531 - 532 eV, which are mainly related to hydroxyl (-OH), aldehyde (-CHO), and carboxyl (-COOH). Since CaCO3 consists of Ca² + and CO3² -Composition, XPS shows characteristic peaks corresponding to the elements carbon (C), oxygen (O), and calcium (Ca). The CNC / Ca nanocomposite particles exhibit typical peaks of C 1s, Ca 2p, and O 1s at approximately 286 eV, 347 - 348 eV, and 533 eV, respectively. The atomic densities of CNC, CaCO3, and CNC / Ca nanocomposite particles are shown in Table 1. CNC is composed of D - glucose units (C6H 10 O5)n and shows high - density C (57.44%) and O (42.56%) signals. The C element accounts for 40.40% of the CaCO3 particles, which is caused by the background noise from the carbon support film of the copper grid. Therefore, the C signal can be ignored. The relative density of atoms is related to the relative content of surface elements of the composite particles. Due to the relatively low content of CaCO3, the Ca atomic densities of the CNC2 / Ca1 and CNC3 / Ca2 nanocomposites are 0.69% and 0.79%, respectively. The relative content of CaCO3 in the CNC2 / Ca3 nanocomposite is the highest, but the relative content of the element Ca is low (0.71%). This may be because most of the CaCO3 is wrapped by CNC, resulting in only a small amount of Ca element detected on the surface of the material. XPS analysis can also determine the nature of the bonds involved on the material surface through deconvolution. In this work, the C 1s in CNC and CNC / Ca nanocomposite particles was deconvoluted, and the results are as Figures 3 - 8 shown. The C 1s peak was decomposed into three peaks of C - O - C, C - C, and O - C = O, indicating non - covalent bond interactions between the surface groups of CNC and CaCO3.

[0110] Test Example 3: X - ray diffraction measurement:

[0111] Using an X - ray diffractometer, under the conditions of a current of 40 mA, a voltage of 40 kV, a scanning speed of 2° / min, and a scanning range of 10° - 80°, the CNC / Ca nanocomposites with different ratios in Examples 1 to 5, as well as CNC and CaCO3, were characterized by diffraction using Cu Kα radiation. The XRD spectra of CNC1 / Ca2, CNC2 / Ca2, CNC3 / Ca2, CNC2 / Ca1, CNC2 / Ca3, CNC, and CaCO3 in Examples 1 to 5 were measured, and the results are shown in Figure 9 .

[0112] Result analysis: From Figure 9It can be seen that all CNC / Ca nanocomposite particles retain the diffraction peaks of CNC. CNC has obvious characteristic peaks near 14.8°, 16.6°, 22.7° and 34.5°, corresponding to the crystal planes (1-10), (110), (200) and (004) of cellulose Iβ respectively. A typical calcite polycrystalline peak was found at 29.3° in the XRD pattern of CaCO3. In addition, the characteristic peaks of CaCO3 at 23.05°, 29.39°, 35.98°, 39.42°, 43.16°, 47.5°, 48.50° and 57.40° correspond to Ca. In the CNC / Ca nanocomposite particles with different ratios, as the relative content of CNC decreases, the diffraction peaks of the cellulose I crystal structure at 16.0° and 22.0° decrease. This phenomenon is attributed to the increase in the proportion of composite particles in the crystalline and amorphous regions of the amorphous structure of CaCO3. In addition, the diffraction peaks of CaCO3 appear in the XRD patterns of all CNC / Ca nanocomposite particles, but the intensity of the crystallization peaks of CaCO3 particles decreases, which may be caused by the influence of CNC on the growth and crystallization transformation of CaCO3 particles.

[0113] Test Example 4: AFM measurement of the composite:

[0114] Using AFM of a high-resolution scanning probe microscope, the CNC2 / Ca composites with different mass ratios prepared in Example 4, Example 2 and Example 5, as well as the suspensions of CNC and CaCO3 were dropped on a mica sheet and naturally dried at 25 °C for two hours, and the apparent morphology and elastic modulus of the particles were observed through the capture mode. The AFM images of CNC2 / Ca1, CNC2 / Ca2, CNC2 / Ca3, CNC and CaCO3 in Example 4, Example 2 and Example 5 were measured, and the results are shown in Figures 10 - 15 .

[0115] Result analysis: Figures 10 - 14 It can be seen that the surface roughness of CNC and CaCO3 are 2.60 nm and 3.26 nm respectively. The surface roughness of CNC2 / Ca1, CNC2 / Ca2 and CNC2 / Ca3 are 4.41, 4.55 and 5.27 nm respectively, indicating that the surface roughness of the nanocomposite particles formed by CaCO3 and CNC with different masses is significantly enhanced. As the content of CaCO3 increases, the surface roughness of the CNC / Ca nanocomposite gradually increases. The rough surface provides a larger surface area to enhance the adsorption at the oil-water interface, which helps to maintain the stability of the emulsion. From Figure 15It can be seen that the elastic moduli of CNC, CaCO3, and the elastic CNC and CaCO3 in the CNC2 / Ca nanocomposite particles are (5.34 ± 0.1) GPa and (6.68 ± 0.11) GPa, respectively. Both CNC and CaCO3 are highly rigid materials. The elastic moduli of CNC2 / Ca1, CNC2 / Ca2, and CNC2 / Ca3 are (4.93 ± 0.38), (4.06 ± 0.24), and (3.33 ± 0.17) GPa, respectively. The smaller the elastic modulus, the stronger the deformation ability. The significant deformability of the particles can endow them with additional degrees of freedom at the interface to cover a larger interface area, resist extrusion, and increase the adsorption capacity. The nanocomposite particles have a smaller elastic modulus, indicating that it is easier to form a three-dimensional lattice structure under high-pressure homogenization conditions.

[0116] Test Example 5: TEM determination of the composite:

[0117] Using a transmission electron microscope, the CNC / Ca composites with different mass ratios prepared in Examples 1 to 5, as well as the suspensions of CNC and CaCO3, were precipitated on a Cu grid and stained with uranyl acetate. The samples were naturally dried at room temperature and then imaged using an electron microscope at an acceleration voltage of 80 - 120 kV. The TEM images of CNC1 / Ca2, CNC2 / Ca2, CNC3 / Ca2, CNC2 / Ca1, CNC2 / Ca3, CNC, and CaCO3 in Examples 1 to 5 were measured, and the results are shown in Figure 16 .

[0118] Result analysis: As Figure 16 can be seen, CNC is a needle-like structure at the nanoscale due to the removal of the amorphous region. The CaCO3 particles are in a cubic crystal structure, which is consistent with the FT-IR and XRD results. In the TEM image of the CNC / Ca nanocomposite particles, the square CaCO3 does not appear in the field of view. Instead, round CaCO3 crystal nuclei are observed. Although CNC does not affect the nucleation of CaCO3, it affects the growth and structure of the CaCO3 grains, which is consistent with the XRD results. Due to the electrostatic adsorption between negatively charged CNC and Ca 2+ , CNC is easily adsorbed onto the primary nuclei of CaCO3, resulting in the inhibition of the growth of CaCO3 grains into nanoscale primary grains with a smooth spherical or irregular surface. The electrostatic adsorption between CNC and Ca 2+The strong complexation between them leads to the formation of smooth spherical CaCO3 particles in the system, about 300 nm, and CNC is adsorbed around the surface of CaCO3 particles. The spherical CaCO3 in the nanoparticles can be arranged relatively closely in oil and water to form a more stable interfacial film, which is beneficial to the stability of the emulsion. The morphology of CaCO3 in the CNC2 / Ca3 system is inconsistent with that of other samples. CNC is entangled on the surface of CaCO3 particles, which is consistent with the XPS results. This may be due to the relatively high concentration of Ca in the system 2+ interacting on the surface of CNC to form CaCO3 microcrystals rather than CaCO3 particles, resulting in irregularly shaped and larger-sized CaCO3 particles.

[0119] Test Example 6: Long-term stability analysis of emulsion

[0120] Use a Turbiscan Laboratory Stability Analyzer to record the stability of the emulsion. Put about 20 mL of freshly prepared emulsion into a bottle and scan it from bottom to top every 10 min for 1 d at 25 °C, and record the Turbiscan Stability Index (TSI). Fresh CNC-PE and CNC / Ca-PE were stored at room temperature for 0, 15, 30, 60, 90, and 120 days respectively, and the appearance changes of the emulsion were photographed to evaluate the long-term stability of the emulsion. The results are shown in Figure 17 and Figure 18 .

[0121] Result analysis: As can be seen from Figure 17 , in Comparative Example 1, the TSI of CNC-PE increased rapidly within 5 h, indicating that a single CNC is not sufficient to improve the stability of the emulsion. In Comparative Example 2, the TSI value of CaCO3-PE showed an upward trend within 24 h. Adding a small amount of CaCO3 can effectively improve the stability of CNC-PE, reduce the TSI of the emulsion, and effectively slow down the migration speed of emulsion droplets. Adjusting the ratio of CNC to CaCO3 further enhances the stability.

[0122] The results are as shown in Figure 18It can be seen that in Comparative Example 1, CNC-PE exhibited weak emulsification and emulsion stability, which was due to the weak interfacial adsorption of CNC in the continuous phase and strong electrostatic repulsion. The protective ability of CaCO3 particles against EO was weak, so the newly prepared Comparative Example 2 showed oil phase separation. During the storage period of 30 d, CaCO3-PE showed a layering phenomenon. Due to the too large initial particle size of CaCO3 or the weak adsorption energy on the oil-water interface, stable adsorption could not occur on the surface of the emulsion droplets formed after homogenization. In Examples 1 to 5, when CNC was combined with CaCO3, the emulsifying ability of the CNC / Ca nanocomposite particles and the long-term stability of the stabilized emulsion were significantly improved. CNC1 / Ca2-PE and CNC2 / Ca3-PE showed obvious oil phase exudation with the extension of storage time. However, CNC2 / Ca2-PE, CNC3 / Ca2-PE, and CNC2 / Ca1-PE did not show layering and oil phase exudation phenomena during long-term storage (120 d) at room temperature, which might be due to the complexation of CNC with Ca ions in a certain mass ratio, enhancing the fixation of the interfacial layer. A suitable particle size of the nanocomposite particles was beneficial to the enhanced construction of the three-dimensional network in the emulsion continuous phase.

[0123] Test Example 7: pH responsiveness of emulsion

[0124] The structural behaviors of CNC and CNC / Ca-PE in different pH environments were observed, and the pH responsiveness of the emulsions was investigated. The pH of CNC and CNC / Ca-PE was adjusted to 2, 7, and 12 with 0.1 mol / L HCl and NaOH solutions. After storing at room temperature for 24 h, the microscopic morphology of the emulsion was recorded using a DS-Fi2 microscope equipped with NIS elements, and the results are shown in Figure 19 .

[0125] Cryo-SEM was used to observe CNC2 / Ca1-PE, CNC2 / Ca2-PE, and CNC2 / Ca3-PE in Examples 4, 2, and 5 and CNC-PE in Comparative Example 1 that were freshly prepared and treated at pH = 2. Briefly, the emulsion samples were rapidly frozen in liquid nitrogen at -140 °C for 30 s. The frozen samples were transferred to the preparation chamber through a cryotransfer system (Quorum PP3000T, UK), and sublimation gold coating was carried out under vacuum. The apparent morphology of the emulsion was observed at -90 °C and imaged at 15,000X, and the results are shown in Figure 20 and Figure 21 .

[0126] Result analysis: As shown in Figure 19It can be seen that at pH = 7 and pH = 12, the droplets of CNC / Ca-PE, CNC-PE, and CaCO3-PE in Examples 1 to 5 are very small. This may be due to the strong electrostatic and steric repulsion of CNC / Ca-PE, CNC-PE, and CaCO3-PE under neutral and alkaline conditions, effectively preventing the aggregation of emulsion droplets. Under acidic conditions (pH = 2), due to the relatively weak stability of Comparative Example 1, there is a slight tendency for some weak droplets to increase, resulting in the flocculation of some weak droplets. Under acidic conditions, the microstructure of the emulsions stabilized by CNC / Ca nanocomposite particles in Comparative Example 2 and Examples 1 to 5 showed different changes. Due to the disintegration of the CaCO3 particles on the surface of the emulsion droplets, the oil phase was completely exposed to the water phase, and the emulsion droplets in Comparative Example 2 almost disappeared, indicating that CaCO3-PE has a strong acid-responsive behavior. In Examples 1 to 5, under acidic conditions, due to the disappearance of the CaCO3 particles on the surface of the droplets, the interfacial film became thinner or had cavities, and the droplets were significantly aggregated and the size increased. This result shows that CNC / Ca-PE has a certain responsiveness, and this behavior is attributed to the effect of pH on the CaCO3 particles.

[0127] To further explore the effect of acidic environment on the structure of CNC / Ca-PE emulsion droplets, Cryo-SEM was used to observe the interfacial details of the emulsion droplets at pH = 2 to reveal the acid-responsive behavior mechanism of the emulsion. From Figure 20 it can be seen that the surfaces of the droplets of all emulsion samples were wrapped by a dense interfacial layer responsible for maintaining and stabilizing the emulsion before acid treatment. In Comparative Example 1, obvious fibrous structures were visible on the surface of the spherical CNC-PE droplets. In Examples 4 and 2, there were disordered chain-like connections between the tiny droplets of CNC2 / Ca1-PE and CNC2 / Ca2-PE, which may be due to the fact that during the emulsification process, a large amount of CNC complexed with Ca 2+ to form a chain-like network. This chain-like network plays an important role in restricting the migration of droplets and preventing droplet aggregation. In Example 5, the droplets of CNC2 / Ca3-PE were larger and there was no network connection between the droplets. This is because the CNC2 / Ca3 nanocomposite particles adsorbed on the surface of the oil droplets, restricting the contact with the particles in the system, resulting in insufficient attractive force to form a network.

[0128] After acid treatment, the surface structural characteristics of the emulsion droplets changed from Figure 21It can be seen that in Comparative Example 1, the adsorbed cellulose structure on the surface of the CNC-PE droplets was lost, but the interfacial film structure was intact and the droplet size did not change significantly. After acid treatment of CNC / Ca-PE in Examples 4, 2, and 5, the droplet diameter increased and the droplet surface was broken. Among them, CNC2 / Ca1-PE and CNC2 / Ca2-PE showed droplet aggregation, larger droplets, droplet fracture, and disappearance of the network structure. This is because there is no CaCO3 in the emulsion system under acidic conditions, resulting in the weakening of the interfacial film and network structure in the emulsion. The droplet size of CNC2 / Ca3-PE changed insignificantly, and only slight fractures appeared on the droplet surface, which is related to the structure of the CNC2 / Ca3 nanocomposite. For the CNC2 / Ca3 nanocomposite particles, CNC tightly wrapped around the CaCO3 particles, preventing the attack of hydrochloric acid, resulting in only some CaCO3 particles not being covered by CNC and being damaged by acid.

[0129] Test Example 8: In vitro release of emulsion

[0130] The dialysis method was used to determine the in vitro release characteristics of CNC / Ca-PE. The CNC2 / Ca1, CNC2 / Ca2, and CNC2 / Ca3 nanocomposites in Examples 4, 2, and 5 and the CNC-stabilized emulsion in Comparative Example 1 were used to determine the emulsion release. Briefly, 0.2 mL of the emulsion was placed in a dialysis bag (0.5 - 1 kDa) and rotated at 200 rpm at 37 °C, and incubated in 20% ethanol (containing 0.1% Tween 80), 0.1 mL HCl (containing 0.1% Tween 80), and pH = 6.8 PBS buffer (containing 0.1% Tween 80), respectively. At 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12 h, 3 mL of the sample was taken out from the culture medium and replaced with 3 mL of pre-warmed culture medium. The EO content in the culture medium at different time points of culture was measured at 277 nm using a Shimadzu UV-2600 spectrophotometer in Japan. The cumulative release percentage of EO was calculated, and all samples were analyzed 3 times. The results are shown in Figures 22 - 24 .

[0131] The results are shown by Figures 22 - 24It can be seen that in the ethanol medium, the cumulative release rate of CNC-PE in Comparative Example 1 was 71.47±0.77%, while in Examples 4, 2, and 5, the cumulative release rates of CNC2 / Ca1-PE, CNC2 / Ca2-PE, and CNC2 / Ca3-PE were 70.82±4.25%, 63.71±2.97%, and 69.62±2.59%, respectively. Comparative Example 1 showed a faster release rate, while in Examples 1 to 5, the CNC / Ca nano-composite stable emulsion slowed down the release of EO from the emulsion to varying degrees in the alcohol medium. Therefore, the CNC / Ca nano-composite particles improved the stability of the emulsion and made the emulsion release slower in the alcohol medium. This slow release rate may help reduce drug leakage of EO during blood circulation.

[0132] In the acidic medium, the cumulative release rate of CNC-PE in Comparative Example 1 was 51.51±11.31%, while in Examples 4, 2, and 5, the cumulative release rates of CNC2 / Ca1-PE, CNC2 / Ca2-PE, and CNC2 / Ca3-PE were 60.16±5.49%, 61.39±1.30%, and 62.81±2.38%, respectively. The emulsions stabilized by the CNC / Ca nano-composite particles in Examples 4, 2, and 5 had a faster release rate than CNC-PE in Comparative Example 1, which was caused by the demulsification effect of the CNC / Ca-PE emulsion under the stimulation of the acidic environment. In the PH=6.8 PBS buffer solution, the cumulative release rate of CNC-PE in Comparative Example 1 was 56.16±2.46%, while in Examples 4, 2, and 5, the cumulative release rates of CNC2 / Ca1-PE, CNC2 / Ca2-PE, and CNC2 / Ca3-PE were 53.37±1.90%, 46.48±1.09%, and 49.87±3.66%, respectively. The in vitro release results showed that compared with the ethanol and acidic media, less EO was released from the CNC / Ca-PE in the PH=6.8 PBS buffer solution.

[0133] Test Example 9: Emulsion Simulating Gastrointestinal Release

[0134] A simulated gastrointestinal digestive system was constructed to study the potential gastrointestinal digestive fate of EO delivery samples (CNC2 / Ca1-PE, CNC2 / Ca2-PE, CNC2 / Ca3-PE, and CNC-PE in Example 4, Example 2, and Example 5). The emulsion samples were sequentially placed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) for digestion. Simulated gastric digestion: The emulsion was mixed with SGF at a ratio of 1:1 (v / v), and the pH was adjusted to 2 with 0.1 mL of HCl solution, and then incubated at 37 °C and 150 rpm for 1 h. The simulated reaction was terminated by adjusting the pH to 7 with 0.1 mL of NaOH solution, and the microstructure of the emulsion droplets digested in simulated gastric fluid was photographed. During simulated intestinal digestion, the emulsion digested in SGF was mixed with SIF at a ratio of 1:1 (v / v), and the pH was adjusted to 7 again. The resulting mixture was incubated at 37 °C and 150 rpm for 2 h while maintaining the pH at 7. The final morphology of the emulsion droplets after gastrointestinal digestion was photographed. Finally, the remaining EO in the emulsion was calculated by simulating intestinal digestion with a UV-visible spectrophotometer. The bioaccessibility of EO in the emulsion was calculated as shown in Equation (2):

[0135] Bioavailability (%) = (Concentration of EO after digestion) / (Concentration of EO before digestion) * 100

[0136] Table 2: Bioaccessibility of CNC2 / Ca-PE and CNC-PE after simulated gastrointestinal digestion in Example 4, Example 2, and Example 5

[0137]

[0138] Results analysis, from Figure 25 and Figure 26 it can be seen that compared with the freshly prepared CNC-PE in Comparative Example 1, the emulsion droplets after gastric acid digestion were severely aggregated due to structural fragmentation. This is because the adsorption ability of CNC at the interface is limited and the balance of electrostatic interactions in the system is broken, resulting in the aggregation of CNC-PE droplets during the gastric digestion stage. The CNC / Ca-PE droplets aggregated in the gastric phase. In the gastric phase, the dissolution of CaCO3 particles disrupted the integrity of the outer shell protecting the oil droplets. The Ca 2+ obtained by acid hydrolysis of CaCO3 is an effective method to provide Ca 2+ in human biological processes. From Figure 27 it can be seen that in the intestinal stage, the number of CNC-PE and CNC / Ca-PE emulsion droplets decreased significantly because the intestinal fluid diluted the emulsion again, accelerating the secondary destruction of the droplets damaged in the gastric stage. In addition, bile salt ions and lipase in the simulated intestinal fluid will compete with the nanocomposite particles at the oil-water interface, resulting in the removal of the nanocomposite particles from the oil-water interface.

[0139] From Table 2 andFigure 28 It can be seen that the bioaccessibilities of CNC-PE, CNC2 / Ca1-PE, CNC2 / Ca2-PE, and CNC2 / Ca3-PE are 74.83±0.89%, 48.85±4.82%, 59.11±4.44%, and 36.06±0.97%, respectively. Compared with CNC2 / Ca1-PE, CNC2 / Ca2-PE, and CNC2 / Ca3-PE, CNC-PE has a high bioavailability, indicating that CNC-PE can better protect EO from gastrointestinal degradation, which may be related to its stability in the gastrointestinal tract. The low bioaccessibility of CNC / Ca-PE indicates that most of the EO is rapidly absorbed or metabolized in the gastrointestinal stage. In summary, this study demonstrates that CNC / Ca-PE has better long-term stability and acid-responsive release, and is a reliable gastrointestinal-targeted release preparation.

[0140] Test Example 10: Antibacterial Activity

[0141] The antibacterial activity of the emulsion was investigated by the disc diffusion method. The antibacterial activities of CNC / Ca-PE, CNC-PE, and CaCO3-PE in Examples 1 to 5 were evaluated using Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). After isolation, single colonies were selected and inoculated into liquid sterile LB medium for incubation. 0.2 mL of the bacterial suspension (about 106 CFU / mL) was carefully and evenly spread on the sterile solid medium and incubated at 37 °C for 0.5 h. Sterile antibiotic discs (6 mm) loaded with 40 μL of the emulsion and 10 μL of levofloxacin (25 μg / mL) were placed on the medium, respectively, and incubated at 37 °C for 24 h to measure the inhibition zone. Each experimental group included 1 levofloxacin group and 3 emulsion sample groups, as shown in Figure 29 、 Figure 30 and Table 3.

[0142] Table 3: Encapsulation efficiency of CNC / Ca-PE and CNC-PE in Examples 1 to 5 and inhibition zones against Escherichia coli and Staphylococcus aureus

[0143]

[0144] According to Figure 29 、 Figure 30As can be seen from Table 3, in Comparative Example 1, CNC-PE showed a smaller inhibition zone, indicating that the growth of Escherichia coli and Staphylococcus aureus was inhibited to a certain extent. In Comparative Example 2, CaCO3-PE had no obvious effect on the growth of Escherichia coli and Staphylococcus aureus, and only a small amount of EO was encapsulated. The size of the inhibition zone of emulsions stabilized by different ratios of CNC / Ca was related to their viscosity, EE, and release rate. CNC2 / Ca1-PE, CNC2 / Ca2-PE, and CNC3 / Ca2-PE had larger inhibition zones against Escherichia coli and Staphylococcus aureus, mainly due to the higher encapsulation amount of EO, which was consistent with the results of EE. In addition, the change in the inhibition zone might be related to the interaction between the nanocomposite and EO. According to the cryo-scanning electron microscopy images of CNC / Ca-PE, the network structure between the droplets of CNC2 / Ca1-PE and CNC2 / Ca2-PE led to a more tortuous release path for the EO antibacterial active ingredient. And CNC2 / Ca1-PE and CNC2 / Ca2-PE had a higher EE, gradually releasing more EO and forming an obvious inhibition zone. In addition, the emulsions loaded with EO had a stronger inhibitory effect on the growth of Escherichia coli than on Staphylococcus aureus, which might be due to the additional hydrophobicity of the outer membrane to Escherichia coli and the better affinity of Escherichia coli to carvacrol. The EO emulsion loaded with CNC had a stronger inhibitory effect on Escherichia coli than on Staphylococcus aureus. The cell wall of Staphylococcus aureus might be denser, thus hindering the penetration of the EO component. And the inhibition zones of CNC2 / Ca1-PE and CNC2 / Ca2-PE against Staphylococcus aureus were larger than those against Escherichia coli, showing a stronger inhibitory effect on Staphylococcus aureus. This was because a certain concentration of Ca 2+ rapidly disrupted the phospholipid vesicles of the cell membrane of Staphylococcus aureus, but could not affect Escherichia coli. However, Ca 2+ The CNC2 / Ca3-PE with the highest relative concentration did not show an inhibition zone. It might be due to the special structure of the CNC2 / Ca3 complex, and the encapsulation of CaCO3 particles by CNC prevented Ca 2+ from contacting the bacterial cell membrane.

[0145] The design focus of the present invention is that different mass ratios of CNC / Ca nanocomposite particles are successfully prepared in this study; CNC affects the growth and shape of CaCO3 crystals through electrostatic and complexation effects, thereby generating nanoscale composite particles as PE stabilizers. Compared with CNC-PE and CaCO3-PE, CNC / Ca-PE has excellent stability. In addition, all CNC / Ca-PEs remain stable for up to 4 months except for CNC1 / Ca2-PE and CNC2 / Ca3-PE. Due to the participation of CaCO3 in the system, CNC / Ca-PE exhibits pH-responsive behavior, manifested as an increase in emulsion particle size, destruction of the shell layer and three-dimensional structure, and rapid release in acidic media. The CNC / Ca-PE nanocomposite particles loaded with EO exhibit good antibacterial activity, and their emulsifying properties and structure affect the antibacterial activity of the composite particles. CNC / Ca-PE has a high drug loading capacity and is expected to become a pH-responsive oral drug carrier.

[0146] The above are only the preferred embodiments of the present invention, and do not impose any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing an acid-responsive emulsion, characterized in that: The steps include: S1, preparing volatile oil; S2. preparing a CNC suspension: dispersing microcrystalline cellulose in sulfuric acid, stirring in a water bath, and then terminating the reaction; centrifuging the reaction product to separate the precipitate; dialyzing the reaction product to displace excess sulfuric acid to obtain a CNC suspension with a constant pH; S3, preparation of CNC-CaCO3 composite particles: put Ca(OH)2 into pure water, stir at room temperature to obtain Ca(OH)2 solution; add CNC suspension, stir at room temperature to make the two mixed evenly; then introduce CO2 gas at a uniform flow rate, stir at room temperature to obtain CNC-CaCO3 composite particles; S4, preparing a composite emulsion: using volatile oil as the oil phase, taking CNC-CaCO3 composite particles as the water phase, dropping the oil phase into the water phase containing the CNC-CaCO3 composite particles, shearing, homogenizing, and obtaining a composite emulsion; In S2, microcrystalline cellulose was dispersed in 20 times the mass fraction of 64% sulfuric acid, stirred in a water bath at 45°C and 400 rpm for 45 min, and then 10 times the mass fraction of pure water was added to terminate the reaction; the reaction product was centrifuged at 4°C and 5000 rpm for 10 min to separate the precipitate; In S2, the reaction product is encapsulated in a regenerated cellulose dialysis bag and dialyzed to displace excess sulfuric acid, and the CNC suspension with a constant pH is obtained by dialysis for 5 days; In S3, Ca(OH)2 is placed in pure water and stirred at room temperature using a magnetic stirrer at 500 rpm for 10 min to obtain a Ca(OH)2 solution with a mass fraction of 0.07%-0.14%; CNC suspension was added to S3, and stirred at 500 rpm on a magnetic stirrer for 10 min at room temperature to make the two mixed evenly; then CO2 gas was introduced at a uniform flow rate for 10 min, and stirred at 500 rpm on a magnetic stirrer for 10-12 h at room temperature to obtain CNC-CaCO3 composite particles.

2. The method for preparing an acid-responsive emulsion according to claim 1, characterized in that: The volatile oil in S4 includes tea oil and Elsholtzia jiangruta volatile oil. The specific preparation process of the Elsholtzia jiangruta volatile oil is as follows: weigh Elsholtzia jiangruta medicinal materials, add 6-8 times of water, distill and extract for 3-5 hours, separate the water layer and the volatile oil layer, add anhydrous sodium sulfate to the volatile oil layer for dehydration, and obtain Elsholtzia jiangruta volatile oil.

3. The method for preparing an acid-responsive emulsion according to claim 1, characterized in that: The S4 was sheared at 22000 rpm for 5-10 min.

4. The method for preparing an acid-responsive emulsion according to claim 1, characterized in that: The homogenization conditions in S4 are: homogenization in a high-pressure homogenizer at a pressure of 300 bar and 500 bar for 10-30 min respectively.

5. The method for preparing an acid-responsive emulsion according to claim 1, characterized in that: The CNC-CaCO3 composite particles in S3 include CNC / Ca2 composite particles and CNC2 / Ca composite particles.

6. The method for preparing an acid-responsive emulsion according to claim 1, characterized in that: The composite emulsion in S4 includes CNC / Ca2 composite emulsion and CNC2 / Ca composite emulsion.

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