Preparation method and application of cinnamyl aldehyde nanoparticles

Abstract

The invention discloses a preparation method and application of cinnamyl aldehyde nanoparticles, and the preparation method is characterized in that cinnamyl aldehyde is used as a functional component, zein is used as a carrier, sodium caseinate is used as a stabilizer, and pectin is used as a stabilizer and an adhesive to prepare the cinnamyl aldehyde nanoparticles. Experiments prove that the cinnamyl aldehyde nano-particles prepared by the invention can significantly improve the stability of cinnamyl aldehyde, enhance the antibacterial activity of cinnamyl aldehyde to staphylococcus aureus, and provide more choices for development and utilization of cinnamyl aldehyde.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for preparing cinnamaldehyde nanoparticles and their applications. Background Technology

[0002] Staphylococcus aureus, a Gram-positive hemolytic bacterium, can cause refractory mastitis and chronic infections, making it difficult to eradicate. Common antibacterial drugs include antibiotics and bacteriophage preparations, but long-term use of antibiotics easily leads to drug-resistant strains, and the rate of bacterial evolution is relatively faster than the development speed of bacteriophage preparations. Therefore, there is an urgent need to find a new type of antibacterial drug.

[0003] Traditional Chinese medicine (TCM) originates from natural plants and possesses advantages such as low toxicity, multiple targets, and low resistance to drug resistance. Cinnamon, a TCM herb, is the dried bark of the cinnamon tree (Cinnamon spp.), a plant in the Lauraceae family, and has been used both as food and medicine since ancient times. Cinnamaldehyde (CA) is its main active ingredient (molecular weight 132.16), exhibiting multiple pharmacological activities including antibacterial and anti-inflammatory effects. Due to its lipophilic properties, it can disrupt cell membrane structure, inducing the extracellular leakage of intracellular substances, thereby inhibiting Staphylococcus aureus and Escherichia coli. However, because cinnamaldehyde has a small molecular weight, it is relatively sensitive to external environmental factors such as oxygen, temperature, pH, and light. Furthermore, cinnamaldehyde is extremely insoluble in water but soluble in organic solvents such as ethanol and ethyl acetate, which significantly limits its application in food processing and pharmaceutical fields.

[0004] Studies have found that the efficacy, activity, and stability of traditional Chinese medicine can be improved through nanodelivery systems, such as nanoparticles, liposomes, microcapsules, and emulsions. Among these, nanoparticles refer to colloidal dispersion systems with particle sizes between 1 and 1000 nm, which can be used to encapsulate active substances and have advantages such as excellent stability and good biocompatibility. Currently, there are still few reports on the preparation of cinnamaldehyde nanoparticles, and even fewer studies on the antibacterial activity of cinnamaldehyde nanoparticles, making it difficult to provide more options and development ideas for the further promotion and application of cinnamaldehyde. Summary of the Invention

[0005] This invention discloses a method for preparing cinnamaldehyde nanoparticles and their applications, which can significantly improve the stability of cinnamaldehyde and enhance its antibacterial activity, providing more options for the development and utilization of cinnamaldehyde.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of this invention provides a method for preparing cinnamaldehyde nanoparticles, using cinnamaldehyde as the active ingredient, zein as the carrier, sodium caseinate as the stabilizer, and pectin as the stabilizer and binder to prepare the cinnamaldehyde nanoparticles.

[0008] Preferably, the preparation method of cinnamaldehyde nanoparticles includes the following steps:

[0009] (1) Sodium caseinate and pectin were dissolved in ultrapure water to obtain sodium caseinate solution and pectin stock solution, respectively;

[0010] (2) Dissolve zein in ethanol solution to obtain zein-ethanol solution, add cinnamaldehyde to zein-ethanol solution to obtain cinnamaldehyde-zein-ethanol solution;

[0011] (3) Add sodium caseinate solution and pectin stock solution to cinnamaldehyde-zein-ethanol solution, stir or homogenize, and adjust the pH of the system to 5.8-6.8 to obtain a mixed solution;

[0012] (4) The mixture is heated to 60-85°C to obtain a nanoparticle dispersion. The temperature of the nanoparticle dispersion is then reduced to room temperature and freeze-dried to obtain cinnamaldehyde nanoparticles.

[0013] Preferably, the sodium caseinate solution has a mass concentration of 0.05-0.25%;

[0014] And / or, the pectin stock solution has a pectin concentration of 0.05-0.25% by mass.

[0015] More preferably, the sodium caseinate solution has a mass concentration of 0.05-0.15%;

[0016] And / or, the pectin stock solution has a pectin concentration of 0.05-0.15% by mass.

[0017] Preferably, in the zein-ethanol solution, the concentration of zein is 2-20 mg / mL;

[0018] And / or, in the cinnamaldehyde-zein-ethanol solution, the concentration of cinnamaldehyde is 2-10 mg / mL;

[0019] And / or, the volume concentration of the ethanol is 70-90%.

[0020] More preferably, the concentration of cinnamaldehyde in the cinnamaldehyde-zein-ethanol solution is 4-8 mg / mL.

[0021] Preferably, in step (1), during the preparation of sodium caseinate solution and / or pectin stock solution, mechanical or magnetic stirring is performed for 6-18 hours;

[0022] And / or, in step (2), during the preparation of zein-ethanol solution and / or the preparation of cinnamaldehyde-zein-ethanol solution, mechanical or magnetic stirring is performed for 30-120 min.

[0023] Preferably, in step (2), the pH can be adjusted using an inorganic base and / or an inorganic acid; more preferably, the inorganic base is sodium hydroxide and the inorganic acid is hydrochloric acid.

[0024] Preferably, in step (2), the process of adding cinnamaldehyde to prepare cinnamaldehyde-zein-ethanol solution is carried out under light-protected conditions.

[0025] Preferably, in step (3), the mixture is stirred or homogenized for 30-180 min;

[0026] And / or, in step (4), maintain the temperature for 30-180 min after heating.

[0027] Preferably, in step (3), the volume ratio of cinnamaldehyde-zein-ethanol solution, sodium caseinate solution, and pectin stock solution is (1-2):(1-2):(1-2).

[0028] Preferably, in step (3), the freeze-drying time is 12-24 hours.

[0029] Preferably, in step (3), the process of preparing the mixture is carried out under light-protected conditions.

[0030] Preferably, in step (4), the preparation of the nano-dispersion is carried out under light-protected conditions.

[0031] The stirring described above can be performed using mechanical stirring or magnetic stirring. A second aspect of this invention provides cinnamaldehyde nanoparticles prepared by the method described above.

[0032] A third aspect of the present invention provides the application of the above-described cinnamaldehyde nanoparticles in inhibiting Staphylococcus aureus.

[0033] Furthermore, it has applications in inhibiting the formation of Staphylococcus aureus biofilms.

[0034] The present invention has the following advantages:

[0035] (1) In this invention, cinnamaldehyde is selected as the active ingredient, zein is selected as the carrier, sodium caseinate is selected as the stabilizer, pectin is selected as the stabilizer and binder, and cinnamaldehyde-loaded nanoparticles are prepared by pH-heat induction method. This preparation method is green and safe, has high biocompatibility, and is simple to operate and low in cost, which is conducive to large-scale scale-up and promotion.

[0036] (2) The cinnamaldehyde nanoparticles prepared by the present invention were verified by scanning electron microscopy and Fourier transform infrared spectroscopy. The cinnamaldehyde nanoparticles have good stability, especially in terms of thermal stability, pH stability and light stability, which is beneficial for long-term storage.

[0037] (3) The antibacterial test verified that, compared with free cinnamaldehyde, the minimum inhibitory concentration of cinnamaldehyde nanoparticles against Staphylococcus aureus was reduced by half, from 0.625 mg / mL to 0.3125 mg / mL; at the same time, cinnamaldehyde nanoparticles also have a significantly better ability than free cinnamaldehyde to destroy the ultramorphic structure of Staphylococcus aureus and inhibit the formation of Staphylococcus aureus biofilm. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the preparation process of cinnamaldehyde nanoparticles.

[0039] Figure 2 The following are the particle size, PDI and Zeta potential diagrams of cinnamaldehyde nanoparticles prepared with different sodium caseinate concentrations in Example 1; (A) The bar graph is a schematic diagram of particle size, and the curve graph is a schematic diagram of PDI; (B) Zeta potential diagram.

[0040] Figure 3 The following are the particle size, PDI and Zeta potential diagrams of cinnamaldehyde nanoparticles prepared with different sodium caseinate concentrations in Example 2; (A) The bar graph is a schematic diagram of particle size, and the curve graph is a schematic diagram of PDI; (B) Zeta potential diagram.

[0041] Figure 4 The graphs show the particle size, PDI, and Zeta potential of cinnamaldehyde nanoparticles prepared with different sodium caseinate concentrations in Example 3; (A) The bar graph is a schematic diagram of particle size, and the curve graph is a schematic diagram of PDI; (B) Zeta potential graph.

[0042] Figure 5 This is a scanning electron microscope image of the cinnamaldehyde nanoparticles prepared in Example 4.

[0043] Figure 6 This is the Fourier transform infrared spectrum of the cinnamaldehyde nanoparticles prepared in Example 4.

[0044] Figure 7 Here is a schematic diagram of the thermal stability of the cinnamaldehyde nanoparticles prepared in Example 4 at 80°C (0-120 min); (A) The bar graph is a schematic diagram of particle size, and the curve graph is a schematic diagram of PDI; (B) Zeta potential graph.

[0045] Figure 8Here is a schematic diagram of the pH stability of the cinnamaldehyde nanoparticles prepared in Example 4; (A) The bar graph is a schematic diagram of particle size, and the curve graph is a schematic diagram of PDI; (B) Zeta potential graph.

[0046] Figure 9 This is a comparison chart of the photostability of cinnamaldehyde nanoparticles prepared in Example 4 and free cinnamaldehyde.

[0047] Figure 10 This is a schematic diagram showing the effect of cinnamaldehyde nanoparticles and free cinnamaldehyde prepared in Example 4 on the ultrastructure of Staphylococcus aureus; A. Blank control; B. 1 / 2×MIC free cinnamaldehyde; C. 1×MIC free cinnamaldehyde; D. 2×MIC free cinnamaldehyde; E. 1 / 2×MIC cinnamaldehyde nanoparticles; F. 1×MIC cinnamaldehyde nanoparticles; G. 2×MIC cinnamaldehyde nanoparticles.

[0048] Figure 11 This is a comparison chart of the ability of cinnamaldehyde nanoparticles prepared in Example 4 and free cinnamaldehyde to inhibit biofilm formation in Staphylococcus aureus. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0050] Example 1

[0051] A method for preparing cinnamaldehyde nanoparticles, combined with Figure 1 As shown, it includes the following steps:

[0052] (1) Sodium caseinate was dissolved in ultrapure water to obtain sodium caseinate solutions with concentrations of 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), and 0.25% (w / v), respectively; pectin was dissolved in ultrapure water to obtain a pectin stock solution with a concentration of 0.15% (w / v). During the preparation of sodium caseinate solutions and pectin stock solutions, the mixture was continuously stirred magnetically at 700 rpm / min for 12 h at room temperature.

[0053] (2) Dissolve zein in 80% ethanol solution to make the concentration of zein 10 mg / mL, and stir continuously at 700 rpm / min magnetically for 30 min at room temperature to obtain zein-ethanol solution; add cinnamaldehyde to the above zein-ethanol solution to make the concentration of cinnamaldehyde 6 mg / mL, and stir continuously at 700 rpm / min magnetically for 30 min at room temperature in the dark to obtain cinnamaldehyde-zein-ethanol solution.

[0054] (3) Add sodium caseinate solution and pectin stock solution to cinnamaldehyde-zein-ethanol solution. The volume ratio of cinnamaldehyde-zein-ethanol solution, sodium caseinate solution and pectin stock solution is 1:1:1. Stir continuously with magnetic force at 700 rpm / min for 1 h at room temperature in the dark. Then, adjust the pH of the system to 6.2 to obtain a mixed solution.

[0055] (4) Heat the mixture in a water bath to 80°C and maintain the temperature for 1 hour to obtain a nano-dispersion. Cool it to room temperature and freeze-dry it under vacuum for 24 hours to obtain cinnamaldehyde nanoparticles.

[0056] Example 2

[0057] A method for preparing cinnamaldehyde nanoparticles includes the following steps:

[0058] (1) Sodium caseinate was dissolved in ultrapure water to obtain a sodium caseinate solution with a concentration of 0.15% (w / v); pectin was dissolved in ultrapure water to obtain pectin stock solutions with concentrations of 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), and 0.25% (w / v), respectively. During the preparation of sodium caseinate solution and pectin stock solution, the mixture was continuously stirred magnetically at 700 rpm / min for 12 h at room temperature.

[0059] (2) Dissolve zein in 80% ethanol solution to make the concentration of zein 10 mg / mL, and stir continuously at 700 rpm / min magnetically for 30 min at room temperature to obtain zein-ethanol solution; add cinnamaldehyde to the above zein-ethanol solution to make the concentration of cinnamaldehyde 6 mg / mL, and stir continuously at 700 rpm / min magnetically for 30 min at room temperature in the dark to obtain cinnamaldehyde-zein-ethanol solution.

[0060] (3) Add sodium caseinate solution and pectin stock solution to cinnamaldehyde-zein-ethanol solution. The volume ratio of cinnamaldehyde-zein-ethanol solution, sodium caseinate solution and pectin stock solution is 1:1:1. Stir continuously with magnetic force at 700 rpm / min for 1 h at room temperature in the dark. Then, adjust the pH of the system to 6.2 to obtain a mixed solution.

[0061] (4) Heat the mixture in a water bath to 80°C and maintain the temperature for 1 hour to obtain a nano-dispersion. Cool it to room temperature and freeze-dry it under vacuum for 24 hours to obtain cinnamaldehyde nanoparticles.

[0062] Example 3

[0063] A method for preparing cinnamaldehyde nanoparticles includes the following steps:

[0064] (1) Sodium caseinate was dissolved in ultrapure water to obtain a sodium caseinate solution with a concentration of 0.1% (w / v); pectin was dissolved in ultrapure water to obtain a pectin stock solution with a concentration of 0.15% (w / v). During the preparation of sodium caseinate solution and pectin stock solution, the mixture was continuously stirred magnetically at 700 rpm / min for 12 h at room temperature.

[0065] (2) Dissolve zein in 80% ethanol solution to make the concentration of zein 10 mg / mL, and stir continuously at 700 rpm / min for 30 min at room temperature to obtain zein-ethanol solution; add cinnamaldehyde to the above zein-ethanol solution to make the concentration of cinnamaldehyde 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL and 10 mg / mL respectively, and stir continuously at 700 rpm / min for 30 min at room temperature in the dark to obtain cinnamaldehyde-zein-ethanol solution.

[0066] (3) Add sodium caseinate solution and pectin stock solution to cinnamaldehyde-zein-ethanol solution. The volume ratio of cinnamaldehyde-zein-ethanol solution, sodium caseinate solution and pectin stock solution is 1:1:1. Stir continuously with magnetic force at 700 rpm / min for 1 h at room temperature in the dark. Then, adjust the pH of the system to 6.2 to obtain a mixed solution.

[0067] (4) Heat the mixture in a water bath to 80°C and maintain the temperature for 1 hour to obtain a nano-dispersion. Cool it to room temperature and freeze-dry it under vacuum for 24 hours to obtain cinnamaldehyde nanoparticles.

[0068] Example 4

[0069] A method for preparing cinnamaldehyde nanoparticles includes the following steps:

[0070] (1) Sodium caseinate was dissolved in ultrapure water to obtain a sodium caseinate solution with a concentration of 0.10% (w / v); pectin was dissolved in ultrapure water to obtain a pectin stock solution with a concentration of 0.15% (w / v). During the preparation of sodium caseinate solution and pectin stock solution, the mixture was continuously stirred magnetically at 700 rpm / min for 12 h at room temperature.

[0071] (2) Dissolve zein in 80% ethanol solution to make the concentration of zein 10 mg / mL, and stir continuously at 700 rpm / min magnetically for 30 min at room temperature to obtain zein-ethanol solution; add cinnamaldehyde to the above zein-ethanol solution to make the concentration of cinnamaldehyde 8 mg / mL, and stir continuously at 700 rpm / min magnetically for 30 min at room temperature in the dark to obtain cinnamaldehyde-zein-ethanol solution.

[0072] (3) Add sodium caseinate solution and pectin stock solution to cinnamaldehyde-zein-ethanol solution. The volume ratio of cinnamaldehyde-zein-ethanol solution, sodium caseinate solution and pectin stock solution is 1:1:1. Stir continuously with magnetic force at 700 rpm / min for 1 h at room temperature in the dark. Then adjust the pH to 6.2 to obtain the mixture.

[0073] (4) Heat the mixture in a water bath to 80°C and maintain the temperature for 1 hour to obtain a nano-dispersion. Cool it to room temperature and freeze-dry it under vacuum for 24 hours to obtain cinnamaldehyde nanoparticles.

[0074] Comparative Example 1

[0075] Comparative Example 1 is basically the same as Example 4, except that zein is replaced with phytosterol.

[0076] Comparative Example 2

[0077] A method for preparing cinnamaldehyde nanoparticles includes the following steps:

[0078] (1) Chitosan was dissolved in ultrapure water (with an appropriate amount of acetic acid added to dissolve it) to obtain a chitosan solution with a concentration of 0.125% (w / v). During the preparation process, the solution was continuously stirred magnetically at 700 rpm / min for 12 h at room temperature.

[0079] (2) Dissolve zein in 80% ethanol solution to make the concentration of zein 10 mg / mL, and stir continuously at 700 rpm / min magnetically for 30 min at room temperature to obtain zein-ethanol solution; add cinnamaldehyde to the above zein-ethanol solution to make the concentration of cinnamaldehyde 8 mg / mL, and stir continuously at 700 rpm / min magnetically for 30 min at room temperature in the dark to obtain cinnamaldehyde-zein-ethanol solution.

[0080] (3) Add chitosan solution to cinnamaldehyde-zein-ethanol solution. The volume ratio of cinnamaldehyde-zein-ethanol solution to chitosan solution is 1:2. Stir continuously with magnetic force at 700 rpm / min for 1 h at room temperature in the dark. Then, adjust the pH to 6.2 to obtain a mixture.

[0081] (4) Heat the mixture in a water bath to 80°C and maintain the temperature for 1 hour to obtain a nano-dispersion. Cool it to room temperature and freeze-dry it under vacuum for 24 hours to obtain cinnamaldehyde nanoparticles.

[0082] Product Test Implementation Examples

[0083] 1. Particle size, PDI, and zeta potential determination

[0084] 1.1 Determination Method

[0085] The particle size, PDI, and zeta potential of the cinnamaldehyde nanoparticles in Examples 1-4 were measured using a multi-angle particle size analyzer and a high-sensitivity zeta potential analyzer. All samples were diluted 10-fold with ultrapure water, and all measurements were performed at 25°C.

[0086] All experiments were performed in triplicate, and results are expressed as mean ± standard deviation. SPSS 27 software was used to analyze significance levels (p < 0.05). Different uppercase and lowercase letters indicate significant differences at the p < 0.05 level after Duncan's multiple comparison test; the same letters indicate no significant differences.

[0087] 1.2 Results

[0088] Example 1 used sodium caseinate solutions with concentrations of 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), and 0.25% (w / v) to finally prepare cinnamaldehyde nanoparticles, which, combined with... Figure 2 As shown, the particle sizes are 334.28±8.78 nm, 352.46±0.02 nm, 381.7±10.18 nm, 410.68±8.86 nm, and 426.33±8.88 nm, respectively; the PDI values ​​are 0.27±0.01, 0.22±2.5, 0.23±0.02, 0.21±0.02, and 0.24±0.01, respectively; and the Zeta potentials are -29.69±2.33 mV, -33.44±2.5 mV, -34.17±3.28 mV, -32.85±1.61 mV, and -31.75±0.96 mV, respectively.

[0089] Cinnamaldehyde nanoparticles with suitable particle size and PDI were obtained under sodium caseinate solution concentrations of 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), and 0.25% (w / v). In particular, when the sodium caseinate solution concentrations were 0.05% (w / v), 0.1% (w / v), and 0.15% (w / v), the particle size and PDI were smaller, and the absolute value of the Zeta potential was stable.

[0090] Example 2 used pectin stock solutions with concentrations of 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), and 0.25% (w / v) to finally prepare cinnamaldehyde nanoparticles, combined with... Figure 3 As shown, the particle sizes are 346.77±7.61 nm, 357.05±7.99 nm, 360.27±11.43 nm, 364.5±9.77 nm, and 388.2±4.19 nm, respectively; the PDI values ​​are 0.25±0.01, 0.21±0.01, 0.21±0.01, 0.25±0.02, and 0.29±0.01, respectively; and the Zeta potentials are -30.09±1.88 mV, -34.57±1.87 mV, -32.67±0.62 mV, -33.91±2.24 mV, and -29.57±1.6 mV, respectively.

[0091] Cinnamaldehyde nanoparticles with suitable particle size and PDI were obtained under pectin stock solution concentrations of 0.05% (w / v), 0.1% (w / v), 0.15% (w / v), 0.2% (w / v), and 0.25% (w / v). In particular, when the pectin stock solution concentrations were 0.05% (w / v), 0.1% (w / v), and 0.15% (w / v), the particle size and PDI were smaller, and the absolute value of the Zeta potential was stable.

[0092] Example 3: Cinnamaldehyde concentrations of 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, and 10 mg / mL were used to prepare cinnamaldehyde nanoparticles, which were then combined with... Figure 4As shown, the particle sizes are 336.64±7 nm, 342.28±7.02 nm, 361.65±3.12 nm, 363.34±8.21 nm, and 387.66±4.61 nm, respectively; the PDI values ​​are 0.27±0.01, 0.21±0.01, 0.20±0.02, 0.21±0.02, and 0.27±0.01, respectively; and the Zeta potentials are -28.44±1.8 mV, -33.36±1.16 mV, -32.66±0.85 mV, -32.62±1.51 mV, and -33.85±1.61 mV, respectively.

[0093] Cinnamaldehyde nanoparticles with suitable particle size and PDI were obtained at concentrations of 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL and 10 mg / mL. In particular, at concentrations of 4 mg / mL, 6 mg / mL and 8 mg / mL, the particle size and PDI were small and the absolute value of the Zeta potential was stable.

[0094] The cinnamaldehyde nanoparticles obtained in Example 4 have a particle size, PDI, and Zeta potential of 317.4±12.15 nm, 0.2±0.02, and -35.37±2.29 mV, respectively.

[0095] 2. Characterization tests of the cinnamaldehyde nanoparticles prepared in Example 4

[0096] 2.1 Microscopic morphological observation

[0097] 2.1.1 Method

[0098] Take a small amount of freeze-dried sample and sprinkle it evenly on the conductive adhesive. Use a bulb syringe to blow away any sample that does not adhere. Spray gold for about 120 seconds and observe the microstructure of the cinnamaldehyde nanoparticles using an emission scanning electron microscope (SU8220, HITACHI, Tokyo, Japan).

[0099] 2.1.2 Results

[0100] The results are as follows Figure 5 The cinnamaldehyde nanoparticles prepared by the pH-heat-induced method are spherical or nearly spherical, but have slight agglomeration, with a particle size of 300-400 nm, which is consistent with the results obtained by multi-angle particle size and high-sensitivity Zeta potential analysis.

[0101] 2.2 Infrared Spectroscopy Analysis

[0102] 2.2.1 Method

[0103] The preparation method of empty nanoparticles includes the following steps: (1) Sodium caseinate is dissolved in ultrapure water to obtain a sodium caseinate solution with a concentration of 0.1% (w / v); pectin is dissolved in ultrapure water to obtain a pectin stock solution with a concentration of 0.15% (w / v). During the preparation of sodium caseinate solution and pectin stock solution, the mixture is continuously stirred magnetically at 700 rpm / min for 12 h at room temperature. (2) Zein is dissolved in 80% ethanol solution to make the concentration of zein 10 mg / mL, and the mixture is continuously stirred magnetically at 700 rpm / min for 30 min at room temperature to obtain a zein-ethanol solution. (3) Add sodium caseinate and pectin stock solution to zein-ethanol solution. The volume ratio of zein-ethanol solution, sodium caseinate solution and pectin stock solution is 1:1:1. Stir continuously with magnetic force at 700 rpm / min for 1 h at room temperature in the dark. Then, adjust the pH of the system to 6.2, heat to 80°C in a water bath and maintain the temperature for 1 h to obtain nano-dispersion. Cool to room temperature and freeze dry under vacuum for 24 h to obtain empty nanoparticles.

[0104] The freeze-dried sample was mixed with potassium bromide and ground thoroughly in an agate mortar at a ratio of 1:100. The mixture was then compressed into tablets, and Fourier transform infrared spectroscopy was used to obtain the infrared spectra of zein, sodium caseinate (NaCas), pectin, cinnamaldehyde (CA), empty nanoparticles (ZNP / NPs), and cinnamaldehyde nanoparticles (ZNP / CANPs). The results are as follows: Figure 6 As shown, the ZNP / NPs and ZNP / CANPs amide I bands shift towards a lower wavenumber of 1623.3 cm⁻¹ due to hydrogen bonding. -1 and 1627.9cm -1 Moving. 1029.8cm -1 and 1031.1cm -1 This is due to the enhanced effect of pectin combined with sodium caseinate. The absence of characteristic peaks for CA in the ZNP / CANPs spectrum indicates that CA was successfully encapsulated and exists in an amorphous state within the nanoparticles.

[0105] 3. Stability test of cinnamaldehyde nanoparticles prepared in Example 4

[0106] 3.1 Thermal stability test

[0107] 3.1.1 Test Method

[0108] The thermal stability of cinnamaldehyde nanoparticles was evaluated by heating the nanoparticle dispersion (the nanodispersion obtained in step (4)) at 80 °C for 0, 20, 40, 60, 80, 100 and 120 min, respectively, and then returning it to room temperature. The particle size, PDI and Zeta potential of the samples with different treatments were measured for evaluation.

[0109] 3.1.2 Results

[0110] The results are as follows Figure 7 As shown, heating at 80℃ for 0-80 min resulted in no significant changes in particle size and PDI; however, significant changes occurred at 100 min and 120 min. The absolute value of the Zeta potential exhibited a similar trend to that of particle size and PDI.

[0111] 3.2 pH stability test

[0112] 3.2.1 Test Method

[0113] The pH stability of cinnamaldehyde nanoparticles was determined by adjusting the pH of the nanoparticle dispersions to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, and maintaining these pH values ​​for 24 hours. The particle size, PDI, and Zeta potential of the different treated samples were measured for evaluation.

[0114] 3.2.2 Results

[0115] See results Figure 8 Between pH 3 and 8, the particle size of the nanoparticles did not change significantly with increasing pH, while their PDI tended to increase, but still did not exceed 0.3. At pH 9, the particle size and PDI of the nanoparticles increased significantly, and the absolute value of the Zeta potential was the largest. This may be because at this pH, all components are negatively charged, resulting in a higher level of negative charge density and enhanced electrostatic repulsion, thus leading to the largest absolute values ​​of particle size, PDI, and Zeta potential.

[0116] 3.3 Illumination Stability Test

[0117] 3.3.1 Test Method

[0118] The freshly prepared 30 mL nanoparticle dispersion, free cinnamaldehyde, and the nanoparticle dispersions prepared from Comparative Examples 1 and 2 (the nanodispersions obtained in step (4)) were placed in a petri dish and irradiated under a UV lamp for 3 h. Every 30 min, the nanoparticle dispersion or free cinnamaldehyde was taken out, and the retention rate of cinnamaldehyde in the cinnamaldehyde nanoparticles and free cinnamaldehyde was calculated.

[0119] 3.3.2 Results

[0120] As shown in Figure 9, after irradiation with ultraviolet light for 0–180 min, the retention rate of cinnamaldehyde in the nanoparticles decreased from 100% to 96.64%. The retention rate of free cinnamaldehyde decreased with increasing irradiation time, from 100% to 54.68%. This may be because cinnamaldehyde was effectively encapsulated in the nanoparticles, slowing down its decomposition under ultraviolet irradiation. The retention rates of cinnamaldehyde nanoparticles prepared in Comparative Examples 1 and 2 decreased from 100% to 85.71% and 82.13%, respectively. The cinnamaldehyde retention rate of Example 4 was also better than that of Comparative Examples 1 and 2.

[0121] 4. Antibacterial test of cinnamaldehyde nanoparticles prepared in Example 4

[0122] The Staphylococcus aureus used in this antibacterial test was Staphylococcus aureus derived from dairy buffalo.

[0123] 4.1 Minimum Inhibitory Concentration Test

[0124] 4.1.1 Test Method

[0125] The minimum inhibitory concentration (MIC) of cinnamaldehyde nanoparticles and free cinnamaldehyde against Staphylococcus aureus was determined using a microbroth dilution method. Cinnamaldehyde nanoparticles and free cinnamaldehyde were serially diluted with tryptone soybean broth and added to 96-well plates (100 μL per well), followed by an equal volume of 10... 6 CFU / mL bacterial suspension. Simultaneously, drug control wells and bacterial suspension control wells were set up; that is, 100 μL of drug and 10 μL of bacterial suspension were added to 100 μL of broth, respectively. 6 CFU / mL bacterial suspension. After preparation, the 96-well plate was incubated at 37℃ for 18 hours. After 18 hours, 2,3,5-triphenyltetrazolium chloride staining solution was added, and the results were observed after incubation for 30 minutes. The results were valid when the broth control wells were clear and the bacterial control wells turned red.

[0126] 4.1.2 Results

[0127] Table 1

[0128]

[0129] The results are shown in Table 1. The MIC of cinnamaldehyde nanoparticles was 0.3125 mg / mL, and the MIC of free cinnamaldehyde was 0.625 mg / mL (calculated based on the effective content of cinnamaldehyde). Compared with free cinnamaldehyde, the MIC of cinnamaldehyde nanoparticles was reduced by half.

[0130] 4.2 Effects of cinnamaldehyde nanoparticles on the cell morphology of Staphylococcus aureus

[0131] 4.2.1 Method

[0132] The bacterial cell precipitate obtained after centrifugation and washing of the bacterial suspension was resuspended and divided into 8 equal portions. Different concentrations of cinnamaldehyde nanoparticles, free cinnamaldehyde (1 / 2×MIC, 1×MIC, 2×MIC), and an equal volume of mixed surfactant were added to each portion. A blank control was also included. The samples were incubated overnight at 37℃ and 220 rpm, centrifuged at 3000 rpm for 10 min, washed three times with sterile PBS, and then fixed overnight at 4℃ with 2.5% glutaraldehyde. After three washes with sterile PBS, the samples were sequentially dehydrated with 30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100% ethanol, incubating for 12 min each time, followed by centrifugation at 3000 rpm for 10 min. Finally, 40 μL of 100% ethanol was added, and the entire amount was aspirated and dropped onto a slide. After natural evaporation and gold sputtering, all samples were placed under a microscope, and their placement was photographed and recorded. The samples were then observed and photographed under a scanning electron microscope.

[0133] 4.2.2 Results

[0134] The results are as follows Figure 10 Cinnamaldehyde nanoparticles and free cinnamaldehyde significantly altered the morphology of Staphylococcus aureus. In the blank control group, bacterial cells were intact spherical, plump, smooth, and unruptured. In the cinnamaldehyde nanoparticle group and the cinnamaldehyde group, bacterial shape changed, with many bacterial cells collapsing, shrinking, and even rupturing. Notably, the nanoparticles, at a lower concentration, destroyed Staphylococcus aureus while exhibiting the same effect as free cinnamaldehyde, demonstrating that encapsulating cinnamaldehyde in nanoparticles can enhance its antibacterial activity.

[0135] 4.3 Test on the formation of Staphylococcus aureus biofilm by cinnamaldehyde nanoparticles

[0136] 4.3.1 Method

[0137] Add 200 μL of 10 to the 96-well plate 6 CFU / mL Staphylococcus aureus bacterial suspension was incubated at 37°C for 36 h, and the supernatant was discarded. The suspension was washed five times with ultrapure water, and 200 μL each of 1 / 2×MIC, 1×MIC, and 2×MIC cinnamaldehyde nanoparticles, free cinnamaldehyde, and an equal volume of solvent were added, with a blank control included. The suspension was incubated at 37°C for 6 h. The suspension was washed five times with ultrapure water, and 200 μL of methanol was added for fixation for 15 min. The methanol was discarded, and the suspension was air-dried. 200 μL of 0.1% crystal violet was added, and the suspension was incubated at room temperature for 15 min. The crystal violet was discarded, and the suspension was washed five times with ultrapure water. After air-drying overnight, 200 μL of 30% acetic acid was added, and the suspension was incubated at 37°C for 1 h. The liquid in the wells was transferred to a new 96-well plate, the plate was zeroed with ultrapure water, and the absorbance was read at 570 nm.

[0138] 4.3.2 Results

[0139] The results are as follows Figure 11 As shown, Figure 11 In the symbol, * represents significance. One asterisk (*) indicates that the p-value is less than 0.05, meaning the result is significant at the 0.05 level; two asterisks (**) indicate that the p-value is less than 0.01, meaning the result is significant at the 0.01 level; ns indicates that there is no significant difference.

[0140] from Figure 11 Analysis of the results showed that cinnamaldehyde nanoparticles and free cinnamaldehyde significantly reduced the formation of SA07 biofilms. At 1×MIC and 2×MIC, the inhibitory effect of cinnamaldehyde nanoparticles was superior to that of free cinnamaldehyde. At 1 / 2×MIC and 0×MIC, there was no significant difference in their ability to inhibit biofilm formation. This demonstrates that cinnamaldehyde nanoparticles and free cinnamaldehyde have a significant ability to inhibit SA07 biofilm formation in a concentration-dependent manner.

[0141] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing cinnamaldehyde nanoparticles, characterized in that, Cinnamaldehyde nanoparticles were prepared using cinnamaldehyde as the active ingredient, zein as the carrier, sodium caseinate as the stabilizer, and pectin as the stabilizer and binder.

2. The method for preparing cinnamaldehyde nanoparticles according to claim 1, characterized in that, Includes the following steps: (1) Sodium caseinate and pectin were dissolved in water to obtain sodium caseinate solution and pectin stock solution, respectively; (2) Dissolve zein in ethanol solution to obtain zein-ethanol solution, add cinnamaldehyde to zein-ethanol solution to obtain cinnamaldehyde-zein-ethanol solution; (3) Add sodium caseinate solution and pectin stock solution to cinnamaldehyde-zein-ethanol solution, stir or homogenize, and adjust the pH of the system to 5.8-6.8 to obtain a mixed solution; (4) The mixture is heated to 60-85°C to obtain a nanoparticle dispersion. The temperature of the nanoparticle dispersion is then reduced to room temperature and freeze-dried to obtain cinnamaldehyde nanoparticles.

3. The method for preparing cinnamaldehyde nanoparticles according to claim 2, characterized in that, The sodium caseinate solution has a mass concentration of 0.05-0.25%; And / or, the pectin stock solution has a pectin concentration of 0.05-0.25% by mass.

4. The method for preparing cinnamaldehyde nanoparticles according to claim 2, characterized in that, In the zein-ethanol solution, the concentration of zein is 2-20 mg / mL; And / or, in the cinnamaldehyde-zein-ethanol solution, the concentration of cinnamaldehyde is 2-10 mg / mL; And / or, the volume concentration of the ethanol is 70-90%.

5. The method for preparing cinnamaldehyde nanoparticles according to claim 2, characterized in that, In step (1), during the preparation of sodium caseinate solution and / or pectin stock solution, mechanical or magnetic stirring is performed for 6-18 hours; And / or, in step (2), during the preparation of zein-ethanol solution and / or the preparation of cinnamaldehyde-zein-ethanol solution, mechanical or magnetic stirring is performed for 30-120 min.

6. The method for preparing cinnamaldehyde nanoparticles according to claim 2, characterized in that, In step (3), stir or homogenize for 30-180 minutes; And / or, in step (4), maintain the temperature for 30-180 min after heating.

7. The method for preparing cinnamaldehyde nanoparticles according to claim 2, characterized in that, In step (3), the volume ratio of cinnamaldehyde-zein-ethanol solution, sodium caseinate solution, and pectin stock solution is (1-2):(1-2):(1-2).

8. Cinnamaldehyde nanoparticles prepared by the method for preparing cinnamaldehyde nanoparticles according to any one of claims 1-7.

9. The application of the cinnamaldehyde nanoparticles according to claim 8 in inhibiting Staphylococcus aureus.

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