High-load curcumin-chitosan nanoparticle as well as preparation method and application thereof

Abstract

The invention discloses high-load curcumin-chitosan nanoparticles as well as a preparation method and application thereof, and belongs to the field of biological materials. According to the technical scheme, curcumin is wrapped by chitosan to form nanoparticles, and the mass ratio of curcumin to chitosan is 5: 1-1: 5; the preparation method comprises the following steps: preparing a curcumin aqueous solution with the pH value of 11.0-13.0; preparing a chitosan aqueous solution with the pH value of 1.4-2.8; dropwise adding the curcumin aqueous solution into the chitosan solution to obtain a mixed solution; the dripping rate is 0.8 to 1.2 mL / min. The invention further discloses the high-load curcumin-chitosan nano-particles prepared by the method and application of the high-load curcumin-chitosan nano-particles to wrapping of probiotics by using the nano-particles. The technical problem to be solved by the invention is that the requirements of common delivery, stable storage, stability in a gastric acid environment, precise release in intestinal tracts and the like of curcumin and probiotics cannot be met in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, and particularly relates to a highly loaded curcumin-chitosan nanoparticle, its preparation method, and its application. Background Technology

[0002] Curcumin is a natural polyphenolic compound isolated from turmeric, primarily derived from turmeric root nodules, roots, and rhizomes, among other traditional Chinese medicines. As a functional component, it possesses potential beneficial biological activities, such as anti-cancer, anti-inflammatory, and antioxidant effects. Therefore, it is not only a dietary supplement but also a new generation of multifunctional pharmaceuticals.

[0003] Probiotics are a type of bacteria that can colonize or temporarily colonize the human gut, forming a living flora that can produce beneficial effects on the human body.

[0004] Compared with single components, the combined use of curcumin and probiotics shows stronger activity and intestinal flora regulation. However, the combined use of the two has the following difficulties: (1) Curcumin needs to reach a certain concentration to exert its effect, but it is highly hydrophobic, has poor physicochemical stability, poor bioaccessibility when taken orally, and is difficult to stably reach the intestine; (2) Live probiotics are very susceptible to the influence of external environmental pressures and are easily lost during processing and gastrointestinal transportation.

[0005] Currently, although there are encapsulation technologies for curcumin or probiotics, existing technologies cannot meet the requirements of co-delivery of curcumin and probiotics, stable storage, stability in the acidic environment of the stomach, and precise release in the intestine.

[0006] Therefore, there is an urgent need for a method that can achieve the requirements of co-delivery of curcumin and probiotics, stable storage, stability in the gastric acid environment, and precise release in the intestine. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the technical problem to be solved by this invention is that existing technologies cannot achieve the requirements of co-delivery of curcumin and probiotics, stable storage, stability in the gastric acid environment, and precise release in the intestine. This invention proposes a high-load curcumin-chitosan nanoparticle, its preparation method, and its application.

[0008] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: A method for preparing highly loaded curcumin-chitosan nanoparticles, wherein curcumin is encapsulated by chitosan to form nanoparticles, and the mass ratio of curcumin to chitosan is 5:1 to 1:5; The preparation method includes the following steps: A curcumin aqueous solution with pH = 11.0–13.0 was added dropwise to a chitosan solution with pH = 1.4–2.8 to obtain a mixed solution; the dropping rate was 0.8–1.2 mL / min.

[0009] In some embodiments, the concentration of curcumin in the curcumin aqueous solution is 0.2–0.6 mg / mL, the concentration of chitosan in the chitosan aqueous solution is in the range of 0.04–3.0 mg / mL, and the volume ratio of curcumin aqueous solution to chitosan aqueous solution is 1:2.

[0010] In some embodiments, the curcumin aqueous solution needs to be stirred until the curcumin is completely dissolved, and the chitosan aqueous solution needs to be stirred until the chitosan is completely dissolved; during the dropwise addition process, the curcumin aqueous solution and the chitosan aqueous solution are stirred continuously.

[0011] In some embodiments, the preparation method further includes centrifuging the mixture to remove curcumin that has not formed nanoparticles.

[0012] In some embodiments, the centrifugation time is 10 to 30 minutes and the centrifugation rate is 3500 to 4500 rpm.

[0013] This invention also discloses a highly loaded curcumin-chitosan nanoparticle, obtained by the above-described preparation method; the nanoparticles exhibit a DPPH radical scavenging rate ≥67.58% and an ABTS radical scavenging rate ≥93.45%; when the solution environment of the nanoparticles is 2.0≤pH≤6.0, the PDI of the nanoparticles is ≤0.25, and the Zeta potential of the nanoparticles is >20mV; when the solution environment of the nanoparticles is 3.0≤pH≤6.0, the average particle size of the nanoparticles is <200nm; when the solution environment of the nanoparticles is 2.0≤pH<3.0, the average particle size of the nanoparticles is 200-220nm; after storage for 15 days, the average particle size of the nanoparticles is ≤200nm, the PDI of the nanoparticles is ≤0.25, and the Zeta potential of the nanoparticles is ≥40mV; the T of the nanoparticles... 1 / 2 The time was 36.10 days; after the nanoparticles were freeze-dried and then rehydrated, the particle size change of the nanoparticles was ≤15%, and the change in the zeta potential of the nanoparticles was ≤20%.

[0014] This invention also provides an application of highly loaded curcumin-chitosan nanoparticles for antibacterial activity. The nanoparticles can inhibit the growth of *Phytophthora festans*. The half-maximal inhibitory concentration (EC50) of the nanoparticles against *Phytophthora festans* is [not specified]. 50 The concentration was 0.16 mg / mL; the nanoparticles inhibited the growth of *Botrytis cinerea*; the half-maximal inhibitory concentration (EC50) of the nanoparticles against *Botrytis cinerea* was 0.16 mg / mL. 50 The concentration was 0.14 mg / mL.

[0015] This invention also provides an application of highly loaded curcumin-chitosan nanoparticles in encapsulating probiotics, wherein a solution of nanoparticles is mixed with a probiotic suspension; the nanoparticles encapsulate the probiotics in a solution state to obtain nano-coated probiotics.

[0016] In some preferred embodiments, the probiotic is Lactobacillus.

[0017] In some preferred embodiments, the concentration of nanoparticles is 0.2-0.6 mg / mL, the concentration of probiotics in the probiotic suspension is 10^9-10^11 CFU / mL, and the volume ratio of the nanoparticle solution to the probiotic suspension is 12:1-8:1.

[0018] In some embodiments, after treatment with simulated gastric acid for 2 hours, the survival rate of the nano-coated probiotics was ≥7.25 LogCFU / mL; after treatment at 65°C for 30 minutes, the survival rate was ≥7.14 LogCFU / mL; after freeze-drying, the survival rate was ≥10.21 LogCFU / mL; after exposure to Ciprofloxacin for 24 hours, the survival rate was ≥63.45%; and after exposure to Tobramycin, the survival rate was ≥63.45%. After 24 hours of exposure to levofloxacin, the survival rate of the nano-coated probiotics was ≥58.34%; after 24 hours of exposure to neomycin, the survival rate was ≥53.54%; after 24 hours of exposure to levofloxacin, the survival rate was ≥50.2%; after 24 hours of exposure to tervofloxacin, the survival rate was ≥41.91%; and after 24 hours of exposure to gentamycin, the survival rate was ≥33.92%.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The preparation method of high-load curcumin-chitosan nanoparticles of the present invention uses a pH-driven method, which can achieve self-assembly of curcumin-chitosan nanoparticles by simple pH adjustment. It does not require organic solvents and complex equipment, and has the advantages of low energy consumption, high safety and increased drug loading. 2. The curcumin nanoparticles prepared using the method of this invention exhibit good acid resistance, storage resistance, and freeze-drying and reconstitution properties; they also demonstrate high DPPH and ABTS free radical scavenging rates; and can inhibit pathogenic Phytophthora. Phytophthorainfestans It can also inhibit the growth of Botrytis cinerea; Botrytiscinerea The growth; 3. After encapsulating probiotics with curcumin nanoparticles in this invention, the resulting nano-coated probiotics are heat-resistant, freeze-resistant, antibiotic-resistant, and digestion-resistant. Furthermore, the resulting curcumin nanoparticle co-loaded probiotic system can achieve co-delivery of curcumin and probiotics, stable storage, stability in the gastric acid environment, and precise release in the intestine. Attached Figure Description

[0020] Figure 1 A sample photograph of the nanoparticles provided in Example 3 of this invention; Figure 2 This is a transmission electron microscope (TEM) image of the nanoparticles provided in Example 3 of the present invention. Figure 3 The Fourier transform infrared (FTIR) image of the nanoparticles provided in Embodiment 3 of the present invention. Figure 4 The X-ray diffraction pattern (XRD) of the nanoparticles provided in Example 3 of this invention. Figure 5 The bar chart shows the antioxidant effect test results of the embodiments and comparative examples of the present invention. Figure 6 Transmission electron microscopy (TEM) images of probiotics encapsulated by nanoparticles before and after the present invention embodiment. Figure 7 The particle size of the nanoparticles provided in Example 3 of this invention at different pH values; Figure 8 The nanoparticles provided in Example 3 of this invention are PDI at different pH values; Figure 9 The zeta potentials of the nanoparticles provided in Example 3 of this invention at different pH values; Figure 10 This is a statistical chart showing the variation of performance parameters of nanoparticles under different storage conditions, provided in an embodiment of the present invention. Figure 11 This is a statistical chart of the performance parameters of the lyophilized and reconstituted nanoparticles provided in the embodiments of the present invention; Figure 12 This is a schematic diagram illustrating the encapsulation effect of nanoparticles on probiotics provided in an embodiment of the present invention; Figure 13 The above are growth curves of probiotics before and after nanoparticle encapsulation provided in the embodiments of the present invention. Figure 14 This is a diagram illustrating the simulated digestion effect of nano-coated probiotics provided in an embodiment of the present invention. Figure 15 The diagram shows the stress resistance effect of the nano-coated probiotics provided in the embodiment of the present invention. Detailed Implementation

[0021] The technical solutions in specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0022] This invention provides a method for preparing highly loaded curcumin-chitosan nanoparticles with antibacterial and antioxidant activities, characterized in that curcumin is encapsulated by chitosan to form nanoparticles, and the mass ratio of curcumin to chitosan is 5:1 to 1:5; The preparation method includes the following steps: A curcumin aqueous solution with pH = 11.0–13.0 was added dropwise to a chitosan solution with pH = 1.4–2.8 to obtain a mixed solution; the dropping rate was 0.8–1.2 mL / min.

[0023] The present invention provides a method for preparing highly loaded curcumin-chitosan nanoparticles with antibacterial and antioxidant activities using a pH-driven approach. This method achieves self-assembly of curcumin-chitosan nanoparticles through simple pH adjustment, eliminating the need for organic solvents and complex equipment. It offers advantages such as low energy consumption, high safety, and increased drug loading. The formation principle of pH-driven curcumin-loaded nanoparticles involves lowering the pH of the strong alkaline solution in which curcumin is dissolved, thereby reprotonating the negatively charged hydroxyl groups of curcumin. Simultaneously, the hydrophobicity of curcumin in an aqueous environment increases, spontaneously avoiding polar environments and forming nanoparticles loaded in the wall material. When an acidic chitosan solution is added dropwise to an alkaline curcumin solution, the system undergoes a drastic pH change, causing curcumin to rapidly transform from a dissolved state into insoluble dispersed phase particles. Since the chitosan is not yet fully protonated at this point, the number of positively charged amino groups in the solution is limited, making it difficult for them to effectively adsorb onto the surface of the curcumin dispersed phase particles. This results in a lack of electrostatic repulsion between the dispersed phase particles, leading to easy collision and aggregation, forming unevenly sized aggregates or precipitates. Furthermore, the instantaneous high local concentration gradient generated at the acid-base interface leads to uneven dispersion of the system, causing some curcumin particles to crystallize before being coated with chitosan, thus reducing the encapsulation efficiency and drug loading. Simultaneously, the acidic addition of chitosan causes instability in the interfacial tension between the dispersed and continuous phases, making it difficult for nanoparticles in the system to form a stable hydrophobic core-hydrophilic shell structure. The pH range of the curcumin aqueous solution is 11.0–13.0, its function being to transform curcumin from a nearly insoluble state to a soluble and reactive state, thereby enabling the controllable and efficient preparation of nanoscale particles under changing conditions. If the pH is too low, curcumin cannot dissolve; if the pH is too high (e.g., >13), it may cause chemical degradation of curcumin. The pH range of the chitosan aqueous solution is 1.4–2.8, its function being to ensure complete dissolution of chitosan, strong positive charge, effective electrostatic recombination ability, and to provide a sufficiently acidic environment for the pH-driven process. The purpose of controlling the droplet acceleration rate is to create mild reaction conditions that allow molecules to self-assemble in an orderly manner, thereby obtaining curcumin-chitosan nanoparticles with good dispersibility, uniform particle size, and high drug loading and encapsulation efficiency.

[0024] It is understood that, based on actual circumstances, those skilled in the art can adjust the mass fraction of each component within the above range. For example, the mass ratio of curcumin to chitosan can also be 3:1, 4:1, 2:1, 3:2, 1:1, 1:2, 1:3, 1:4, or any point value within the above range.

[0025] In some preferred embodiments, the mass ratio of curcumin to chitosan is 5:1 to 1:5. Experimental results show that within this range, the average particle size of the nanoparticles is <200 nm, and the polydispersity index is <0.19.

[0026] In some preferred embodiments, the mass ratio of curcumin to chitosan is 1:1. The effect of the CS / Cur mass ratio on encapsulation efficiency (EE) and drug loading (LC) was investigated. As the CS / Cur mass ratio decreased from 1:1 to 1:5, the encapsulation efficiency significantly decreased (p<0.05), mainly due to insufficient chitosan content at lower chitosan ratios to encapsulate more curcumin. When the CS / Cur mass ratio increased from 1:1 to 5:1, there was no significant difference in encapsulation efficiency (p>0.05), indicating that a CS / Cur mass ratio of 1:1 already achieved a high encapsulation effect. With increasing CS / Cur mass ratio (1:5→5:1, w / w), the drug loading significantly decreased (p<0.05), a trend consistent with expectations. The increase in the total mass of the nanoparticles (denominator) was much greater than the increase in the mass of curcumin encapsulated within the particles. Overall, the system with a CS / Cur mass ratio of 1:1 performed best, exhibiting smaller particle size, lower PDI, higher encapsulation efficiency and drug loading, and no precipitation was observed in the sample.

[0027] In some embodiments, the concentration of curcumin in the curcumin aqueous solution is 0.2–0.6 mg / mL, the concentration of chitosan in the chitosan aqueous solution is in the range of 0.04–3.0 mg / mL, and the volume ratio of curcumin aqueous solution to chitosan aqueous solution is 1:2.

[0028] The curcumin concentration range was set to achieve high loading through concentration matching: a wide combination of curcumin concentrations (0.2–0.6 mg / mL) and chitosan concentrations (0.04–3.0 mg / mL) covers all possibilities for achieving a curcumin to chitosan mass ratio from 5:1 to 1:5. Curcumin has extremely low solubility in water. However, under strongly alkaline conditions, the phenolic hydroxyl groups of curcumin ionize, making it negatively charged, and its solubility increases significantly. A pH of 11.0–13.0 ensures that curcumin can fully dissolve and ionize, existing in a uniform molecular state in the aqueous solution, which is a prerequisite for subsequent uniform complexation with chitosan. Concentration range selection: Lower limit (0.2 mg / mL): Ensures sufficient curcumin in the solution to form nanoparticles with practical application value during subsequent complexation with chitosan (i.e., the drug loading cannot be too low). If the concentration is too low, the nanoparticle yield will be low, the loading will be insufficient, and its biological activity will be affected. Upper limit (0.6 mg / mL): Even under alkaline conditions, curcumin has a physical limit to its solubility. Excessive concentration may lead to precipitation, crystallization, or self-aggregation before or during addition, thereby compromising the uniformity and stability of nanoparticle formation. 0.6 mg / mL represents the highest achievable concentration while ensuring solution stability and reactivity.

[0029] The chitosan concentration range is set because under acidic conditions, the amino groups of chitosan protonate, making the entire molecular chain positively charged. A pH of 1.4–2.8 ensures the full extension and positive charge of the chitosan molecular chains, which is crucial for its ability to effectively capture and encapsulate negatively charged curcumin molecules through electrostatic attraction. Concentration range selection: Lower limit (0.04 mg / mL): Sufficient positively charged chitosan molecules are needed to completely neutralize and encapsulate the added curcumin. If the chitosan concentration is too low, it will not be able to completely encapsulate the curcumin, leading to the precipitation of free curcumin, forming irregular precipitates rather than nanoparticles, resulting in a very low encapsulation rate. Upper limit (3.0 mg / mL): If the chitosan concentration is too high, the solution viscosity will be too high, and the molecular chains will be too crowded, potentially leading to: binding with curcumin too quickly and aggressively, forming large, uneven aggregates; and self-entanglement, affecting the particle size distribution and dispersion stability of the nanoparticles. 3.0 mg / mL is a critical concentration that ensures good encapsulation while avoiding the aforementioned adverse effects.

[0030] A 1:2 volume ratio of the two solutions is key to achieving a slow and controllable nucleation and growth process. Advantages: Uniform nucleation: Each incoming curcumin molecule or small aggregate is rapidly surrounded by an excess of chitosan molecules, forming an independent nucleation site. This is beneficial for generating small-sized, narrowly distributed nanoparticles. Prevention of curcumin self-aggregation: If added in reverse (chitosan to curcumin), or in a ratio close to 1:1, it may lead to excessively high local concentrations, making curcumin molecules more likely to aggregate and form large crystals or precipitates, rather than being effectively isolated and encapsulated by chitosan. Achieving high encapsulation efficiency: The excess chitosan environment ensures that almost all added curcumin can find positively charged partners to bind with, thus being encapsulated within the nanoparticles, maximizing encapsulation efficiency and loading.

[0031] It is understood that, based on actual circumstances, those skilled in the art can adjust the mass fractions of each component within the above range. For example, the concentration of curcumin in the curcumin aqueous solution can also be 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, or any value within the above range. Similarly, the concentration range of chitosan in the chitosan aqueous solution can also be 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, or 0. 1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, or any point value within the above range.

[0032] In some embodiments, the curcumin aqueous solution needs to be stirred until the curcumin is completely dissolved, and the chitosan aqueous solution needs to be stirred until the chitosan is completely dissolved; during the dropwise addition, the curcumin aqueous solution and the chitosan aqueous solution are continuously stirred separately. The purpose of continuous stirring is to prevent local pH abrupt changes in the system, promote the uniformity of acid-base mixing, and avoid the instantaneous precipitation or formation of large particle agglomerates of curcumin due to local supersaturation. Simultaneously, stirring maintains the dynamic equilibrium of the system, allowing newly formed nanoparticles to disperse promptly, preventing mutual adhesion, and ensuring the uniformity of particle size and the stability of the system. Furthermore, continuous stirring helps curcumin molecules to fully contact the positively charged amino groups of chitosan, promoting electrostatic adsorption and hydrophobic interactions, thereby improving the drug loading capacity and encapsulation efficiency of the nanoparticles.

[0033] In some embodiments, the preparation method further includes: centrifuging the mixture to remove curcumin that has not formed nanoparticles. After mixing the curcumin solution and chitosan solution in a 1:2 ratio, the solution is weakly acidic. Curcumin is insoluble in weakly acidic solutions and will form a precipitate. Centrifuging the mixture and collecting the supernatant removes the curcumin that has not formed nanoparticles from the mixture.

[0034] In some embodiments, the centrifugation time is 10 to 15 minutes and the centrifugation rate is 3500 to 4000 rpm.

[0035] This invention also discloses a highly loaded curcumin-chitosan nanoparticle with antibacterial and antioxidant activities, obtained by the above-described preparation method; the nanoparticles exhibit a DPPH radical scavenging rate ≥67.58% and an ABTS radical scavenging rate ≥93.45%; when the solution environment of the nanoparticles is 2.0≤pH≤6.0, the PDI of the nanoparticles is ≤0.25, and the Zeta potential of the nanoparticles is >20mV; when the solution environment of the nanoparticles is 3.0≤pH≤6.0, the average particle size of the nanoparticles is <200nm; when the solution environment of the nanoparticles is 2.0≤pH<3.0, the average particle size of the nanoparticles is 200-220nm; after storage for 15 days, the average particle size of the nanoparticles is ≤200nm, the PDI of the nanoparticles is ≤0.25, and the Zeta potential of the nanoparticles is ≥40mV; the T of the nanoparticles... 1 / 2 The time was 36.10 days; after the nanoparticles were freeze-dried and then rehydrated, the particle size change of the nanoparticles was ≤15%, and the change in the zeta potential of the nanoparticles was ≤20%.

[0036] This invention also provides an application of highly loaded curcumin-chitosan nanoparticles for antibacterial activity. The nanoparticles can inhibit the growth of *Phytophthora festans*. The half-maximal inhibitory concentration (EC50) of the nanoparticles against *Phytophthora festans* is [not specified]. 50 The concentration was 0.16 mg / mL; the nanoparticles inhibited the growth of *Botrytis cinerea*; the half-maximal inhibitory concentration (EC50) of the nanoparticles against *Botrytis cinerea* was 0.16 mg / mL. 50 The concentration was 0.14 mg / mL.

[0037] This invention also provides an application of highly loaded curcumin-chitosan nanoparticles in encapsulating probiotics, wherein a solution of nanoparticles is mixed with a probiotic suspension; the nanoparticles encapsulate the probiotics in a solution state to obtain nano-coated probiotics.

[0038] In existing technologies, encapsulating probiotics with nanoparticles often employs methods such as freeze-drying or antisolvent precipitation. Freeze-drying is cumbersome, energy-intensive, and can damage probiotics during the process. However, the curcumin-chitosan nanoparticles provided in this invention can achieve uniform encapsulation of probiotics even in solution. Figure 6 As can be seen, the surface of unencapsulated probiotics is clean and smooth. After encapsulation with curcumin-chitosan nanoparticles, a clear gray nanoshell forms on the surface of the probiotics, and these are individual single-cell coatings, not clustered together. This result confirms the successful preparation of the probiotic single-cell nanocoating.

[0039] Compared to using a single ingredient, the combination of curcumin and probiotics exhibits stronger bioactivity and gut microbiota regulation. However, the combined use of the two presents several challenges: curcumin is poorly soluble in water and has poor stability, making it difficult for probiotics to overcome the gastric acid barrier and enter the intestines. Furthermore, if curcumin and probiotics are used directly, their different solubilities (curcumin is extremely poorly soluble in water, while probiotics require water to thrive) and direct contact between curcumin and probiotics can potentially harm them. Therefore, achieving a synergistic effect between the two is difficult.

[0040] Chitosan, a natural cationic polymer, is considered a promising green biomaterial matrix due to its readily available, biodegradable, and highly bioactive properties. Obtained through the deacetylation of chitin, chitosan contains three main functional groups: an amino group at C2, a primary hydroxyl group at C3, and a secondary hydroxyl group at C6. The amino group is a unique functional group in chitosan, contributing its superior chemical reactivity and bioactivity compared to the acetyl group in chitin. Chitosan possesses non-toxic, highly adsorbent, biodegradable, highly bioactive, and targeted functional properties. The amino group's excellent chemical reactivity and bioactivity have led to its widespread use in various industrial applications, including food preservation and packaging.

[0041] Chitosan is a biodegradable polysaccharide with good biocompatibility and is considered a viable polymer for various pharmaceutical and food applications. Chitosan is prepared by the deacetylation of chitin; under acidic conditions, the amino groups on the chitosan molecule (…)… NH2) carries a positive charge ( NH3 + This process allows chitosan to become water-soluble. Due to its acid-resistant pH and positive charge, chitosan may electrostatically interact with negatively charged sialic acid present in mucus, a component of intestinal mucus, thereby improving bioavailability and promoting endocytosis. The amino groups (-NH2) in the chitosan molecule protonate and become positively charged (-NH3) in an acidic environment. + This allows the molecular chains to expand; in a neutral or alkaline environment, they deprotonate, causing the molecular chains to contract. This property can be used to achieve targeted release into the intestine.

[0042] Chitosan is a natural cationic polysaccharide obtained by deacetylation of chitin. Its unique amino group at the C2 position causes it to become positively charged under acidic conditions, making it water-soluble and giving it good chemical reactivity and biological activity. It is also non-toxic, biodegradable, and biocompatible. It can interact electrostatically with negatively charged sialic acid in intestinal mucus to enhance bioavailability, and can also achieve targeted release in the intestine by utilizing changes in environmental pH. It is currently used in food preservation packaging and in the fields of medicine and food.

[0043] In this invention, chitosan is used to encapsulate curcumin to form nanoparticles, which are then used to encapsulate probiotics, forming a uniform and complete nanoparticle attachment layer on the surface of the probiotics, resulting in nano-coated probiotics. Due to the high curcumin loading characteristic of curcumin-chitosan nanoparticles, the curcumin content is high after the formation of nano-coated probiotics, which can effectively synergize with the probiotics. Moreover, curcumin-chitosan nanoparticles have a protective effect on probiotics, giving the nano-coated probiotics better heat resistance, cold resistance, antibiotic resistance, and gastric digestion resistance.

[0044] In some preferred embodiments, the probiotic is Lactobacillus. Lactobacillus rhamnosus belongs to the Lactobacillus class and is classified as a third-level probiotic, making it one of the most important components of probiotics. Studies show that it has similar effects to some other probiotics, such as enhancing the host's intestinal resistance, regulating the microecological balance, treating diarrhea, providing antioxidant effects, and treating ulcers. It also strengthens the immune system and lowers blood pressure. Experiments have shown that Lactobacillus rhamnosus has strong survival ability, can survive in the intestines for a long time, has a relatively accurate predictive function for the occurrence of gastrointestinal diseases, and does not produce adverse reactions or side effects in humans.

[0045] In some preferred embodiments, the concentration of nanoparticles is 0.2-0.6 mg / mL, the concentration of probiotics in the probiotic suspension is 10^9-10^11 CFU / mL, and the volume ratio of the nanoparticle solution to the probiotic suspension is 12:1-8:1.

[0046] In some embodiments, after treatment with simulated gastric acid for 2 hours, the survival rate of the nano-coated probiotics was ≥7.25 LogCFU / mL; after treatment at 65°C for 30 minutes, the survival rate was ≥7.14 LogCFU / mL; after freeze-drying, the survival rate was ≥10.21 LogCFU / mL; after exposure to Ciprofloxacin for 24 hours, the survival rate was ≥63.45%; and after exposure to Tobramycin, the survival rate was ≥63.45%. After 24 hours of exposure to levofloxacin, the survival rate of the nano-coated probiotics was ≥58.34%; after 24 hours of exposure to neomycin, the survival rate was ≥53.54%; after 24 hours of exposure to levofloxacin, the survival rate was ≥50.2%; after 24 hours of exposure to tervofloxacin, the survival rate was ≥41.91%; and after 24 hours of exposure to gentamycin, the survival rate was ≥33.92%.

[0047] To more clearly and in detail describe the preparation method of highly loaded curcumin-chitosan nanoparticles with antibacterial and antioxidant activities provided in the embodiments of the present invention, the following description will be based on specific embodiments.

[0048] Example 1 Dissolve 0.4 mg / ml Cur in deionized water at pH 12.0. Add 0.04 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Then adjust the pH to 2.0 with HCl. Stir magnetically until both Cur and CS are completely dissolved. While stirring magnetically, add the Cur solution dropwise to the CS solution at a volume ratio of 1:2 to obtain NPs, with a dropping rate of 1 mL / min. Centrifuge for 15 min to remove unencapsulated Cur, and the resulting supernatant is the final nanoparticle.

[0049] Example 2 Dissolve 0.4 mg / ml Cur in deionized water at pH 11.6. Add 0.05 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until both Cur and CS are completely dissolved. While stirring magnetically, add the Cur solution dropwise to the CS solution at a volume ratio of 1:2 to obtain NPs, with a dropping rate of 1 mL / min. Centrifuge for 15 min to remove unencapsulated Cur, and the resulting supernatant is the final nanoparticle.

[0050] Example 3 Dissolve 0.6 mg / ml Cur in deionized water at pH 11.6. Add 0.15 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until both Cur and CS are completely dissolved. While stirring magnetically, add the Cur solution dropwise to the CS solution at a volume ratio of 1:2 to obtain NPs, with a dropping rate of 1 mL / min. Centrifuge for 10 min to remove unencapsulated Cur, and the resulting supernatant is the final nanoparticle.

[0051] Example 4 Dissolve 0.3 mg / ml Cur in deionized water at pH 11.6. Add 0.15 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until both Cur and CS are completely dissolved. While stirring magnetically, add the Cur solution dropwise to the CS solution at a volume ratio of 1:2 to obtain NPs, with a dropping rate of 1 mL / min. Centrifuge for 30 min to remove unencapsulated Cur, and the resulting supernatant is the final nanoparticle.

[0052] Example 5 Dissolve 0.5 mg / ml Cur in deionized water at pH 11.6. Add 0.5 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until both Cur and CS are completely dissolved. While stirring magnetically, add the Cur solution dropwise to the CS solution at a volume ratio of 1:2 to obtain NPs, with a dropping rate of 1 mL / min. Centrifuge for 20 min to remove unencapsulated Cur, and the resulting supernatant is the final nanoparticle.

[0053] Example 6 Dissolve 0.4 mg / ml Cur in deionized water at pH 11.6. Add 0.6 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until both Cur and CS are completely dissolved. While stirring magnetically, add the Cur solution dropwise to the CS solution at a volume ratio of 1:2 to obtain NPs, with a dropping rate of 1 mL / min. Centrifuge for 20 min to remove unencapsulated Cur, and the resulting supernatant is the final nanoparticle.

[0054] Example 7 Dissolve 0.4 mg / ml Cur in deionized water at pH 11.6. Add 0.8 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until both Cur and CS are completely dissolved. While stirring magnetically, add the Cur solution dropwise to the CS solution at a volume ratio of 1:2 to obtain NPs, with a dropping rate of 1 mL / min. Centrifuge for 10 min to remove unencapsulated Cur, and the resulting supernatant is the final nanoparticle.

[0055] Example 8 Dissolve 0.4 mg / ml Cur in deionized water at pH 11.6. Add 1.0 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Then adjust the pH to 2.2 with HCl. Stir magnetically until both Cur and CS are completely dissolved. While stirring magnetically, add the Cur solution dropwise to the CS solution at a volume ratio of 1:2 to obtain NPs, with a dropping rate of 1 mL / min. Centrifuge for 20 min to remove unencapsulated Cur, and the resulting supernatant is the final nanoparticle.

[0056] Comparative Example 1 Dissolve 0.6 mg / ml Cur in deionized water at pH 12.0. Add 0.15 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until both Cur and CS are completely dissolved. Mix the Cur and CS solutions directly at a volume ratio of 1:2. Centrifuge for 15 min to remove unencapsulated Cur.

[0057] Comparative Example 2 Dissolve 0.4 mg / ml Cur in deionized water at pH 12.0. Add 0.05 mg / ml CS to a 2% acetic acid aqueous solution and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until both Cur and CS are completely dissolved. Mix the Cur and CS solutions directly at a volume ratio of 1:2. Centrifuge for 15 min to remove unencapsulated Cur.

[0058] Comparative Example 3 Dissolve 0.6 mg / ml Cur in deionized water at pH 12.0. Add 0.15 mg / ml CS to the deionized water and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until Cur and CS are completely dissolved. While stirring magnetically, add the CS solution dropwise to the Cur solution to obtain NPs. Centrifuge for 15 min to remove unencapsulated Cur. The resulting supernatant is the final nanoparticle.

[0059] Comparative Example 4 Dissolve 0.4 mg / ml Cur in deionized water at pH 12.0. Add 0.05 mg / ml CS to the deionized water and stir magnetically until CS is completely dissolved. Adjust the pH to 2.0 with HCl. Stir magnetically until Cur and CS are completely dissolved. While stirring magnetically, add the CS solution dropwise to the Cur solution to obtain NPs. Centrifuge for 15 min to remove unencapsulated Cur. The resulting supernatant is the final nanoparticle.

[0060] Effect Evaluation 1: Nanoparticle Property Effect Test 1. Particle size detection The average particle size and polydispersity index of the nanoparticles prepared in Examples 1-8 and Comparative Examples 1-4 were measured using a Malvern nanoparticle size analyzer at 25°C, with an equilibration time of 60 s. Specific results are shown in Table 1.

[0061] 2. Encapsulation efficiency and loading rate determination The nanoparticles prepared in Examples 1-8 and Comparative Examples 1-4 were first diluted with 80% ethanol (v / v), and then ultrasonically broken up for 20 min. The results were measured using a 426 nm UV-Vis spectrophotometer. A curcumin standard solution was prepared, and a standard curve for curcumin was constructed using a 426 nm UV-Vis spectrophotometer. The measurement results were compared with the curcumin standard curve to obtain the curcumin concentration. The mass of curcumin in the nanoparticles was calculated. The encapsulation efficiency and loading rate of the nanoparticles were further calculated; the specific results are shown in Table 1.

[0062] The calculation formula is as follows: Formula 1: Encapsulation efficiency (%) = Mass of encapsulated curcumin (mg) / Total mass of curcumin (mg) * 100% Formula 2: Loading rate (%) = Mass of encapsulated curcumin (mg) / Total mass of nanoparticles (mg) * 100% Table 1. Average particle size, polydispersity index, encapsulation efficiency, and loading rate of nanoparticles in Examples 1-8 and Comparative Examples 1-4.

[0063] First, as shown in Table 1, the average particle size and polydispersity index (PDI) of the direct mixing preparation methods (Comparative Examples 1 and 2) are significantly different from those of the preparation method in this invention. The nanoparticles prepared by the method of this invention have smaller average particle sizes, with the smallest reaching 156.3 nm; nanoparticles with different mass ratios all exhibit a PDI of less than 0.25, indicating relatively uniform nanoparticles; and the nanoparticles prepared based on this method have higher encapsulation efficiency and loading rate.

[0064] Second, as shown in Table 1, compared with the preparation method in this invention, the method of "dropping CS solution into Cur solution" (Comparative Examples 3 and 4) results in chitosan instantly crosslinking with multiple curcumin molecules after being dropped into the solution, forming large aggregates. The nanoparticles are uneven in size, resulting in significantly reduced encapsulation efficiency and drug loading, poor stability, and a tendency to flocculate and precipitate.

[0065] Furthermore, Table 1 shows the effect of the CS / Cur mass ratio on encapsulation efficiency (EE) and drug loading (LC). As the CS / Cur mass ratio decreased from 1:1 to 1:5, the encapsulation efficiency decreased significantly (p<0.05), mainly due to insufficient chitosan content to encapsulate more curcumin at lower chitosan ratios. When the CS / Cur mass ratio increased from 1:1 to 5:1, there was no significant difference in encapsulation efficiency (p>0.05), indicating that a CS / Cur mass ratio of 1:1 could achieve a high encapsulation effect. As the CS / Cur mass ratio increased (1:5→5:1, w / w), the drug loading decreased significantly (p<0.05), a trend consistent with expectations. According to formula (2), the increase in the total mass of nanoparticles (denominator) was much greater than the increase in the mass of curcumin encapsulated in the particles.

[0066] In summary, the CS / Cur mass ratio of 1:1 performed best, exhibiting smaller particle size, lower PDI, higher encapsulation efficiency and drug loading, and no precipitation was observed in the samples (e.g. Figure 1 Therefore, this ratio was selected for subsequent experiments.

[0067] 3. Microstructural Detection by Transmission Electron Microscopy (TEM) The curcumin-chitosan nanoparticles prepared in Example 3 (curcumin:chitosan = 1:1) were analyzed using transmission electron microscopy. Specific results are shown in [link to results]. Figure 2 .

[0068] according to Figure 1 As can be seen from TEM, the chitosan nanoparticles loaded with curcumin form a spherical structure with a relatively uniform particle size.

[0069] 4. Fourier Transform Infrared Spectroscopy (FTIR) Detection Fourier transform infrared spectroscopy was used to detect the curcumin-chitosan nanoparticles prepared in Example 3 (curcumin:chitosan = 1:1). Specific results are shown in [link to results]. Figure 3 .

[0070] according to Figure 2 As can be seen, the FTIR spectrum shows that the characteristic absorption peaks of curcumin in the nanoparticles almost disappear; this is because curcumin has hydrogen bonds or hydrophobic interactions with polysaccharides, which restricts the stretching and bending of its chemical bonds. This phenomenon leads to the disappearance of most of the characteristic peaks of curcumin in the nanoparticles, indicating that curcumin has been successfully encapsulated in the nanoparticles.

[0071] 5. X-ray diffraction pattern detection (XRD) The curcumin-chitosan nanoparticles prepared in Example 3 (curcumin:chitosan = 1:1) were analyzed using X-ray diffraction (XRD). Specific results are shown in [link to XRD analysis]. Figure 4 .

[0072] according to Figure 3 As can be seen from the XRD pattern, after Cur is loaded onto CS to form nanoparticles, the characteristic peaks of CS and Cur disappear, indicating that Cur is transformed from a crystalline form to an amorphous structure after being embedded in nanoparticles.

[0073] Comparative Example 5 Curcumin was dissolved in an aqueous sodium hydroxide solution with a pH of 12 to prepare a curcumin solution.

[0074] Comparative Example 6 Shellac was dissolved in water at pH 12.0 at 50°C and the reaction was maintained for 30 minutes. After the shellac solution cooled to room temperature, curcumin (Cur, dissolved in water at pH 12.0) was added and stirred for 15 minutes until the curcumin was completely dissolved. The pH of the mixture was then adjusted to 7.0 to form nanoparticles. The resulting system was centrifuged at 3000g for 10 minutes to remove unencapsulated free curcumin.

[0075] Comparative Example 7 Curcumin (Cur, 0.4 mg / mL) was added to deionized water adjusted to pH 12.0 with 1M NaOH and stirred until completely dissolved. Different concentrations of propylene glycol alginate (PGA, 0.08, 0.2, 0.4, 0.8, 1.2, and 2.0 mg / mL) were added to deionized water and stirred until completely dissolved, then the pH was adjusted to 2.3 with 1.0M HCl. The PGA solution was added to the curcumin solution under stirring at 600 rpm to form nanoparticles (NPs, final pH approximately 7.0). The mixture was then centrifuged at 3000g for 10 minutes to remove unencapsulated free curcumin.

[0076] Comparative Example 8 Cellulose nanocrystal (CNC) powder was dissolved in deionized water by stirring and then sonicated for 10 minutes to obtain a CNC stock suspension. The pH of the CNC suspension was then adjusted to 2.2 using 1.0M HCl. The curcumin solution was prepared by dissolving curcumin in deionized water at pH 12.0, with stirring time controlled to within 15 minutes to avoid significant degradation of curcumin. The CNC suspension was then slowly poured into the continuously stirred curcumin solution to form nanoparticles (NPs). The resulting mixture was centrifuged at 3000g for 10 minutes to remove unencapsulated free curcumin.

[0077] Performance Evaluation 2: Nanoparticle Performance Test (a) Antioxidant performance test of nanoparticles 1. DPPH free radical scavenging rate determination: (1) Reagent preparation Preparation of DPPH stock solution: Accurately weigh 4 mg of DPPH powder, dissolve it in anhydrous ethanol, and prepare a DPPH stock solution with a mass concentration of 0.004%. The stock solution should be stored in a light-proof environment for later use.

[0078] Preparation of control standard: Select vitamin C (VC) with the same concentration as curcumin as the positive control for this experiment.

[0079] (2) Sample reaction operation Sample preparation: (1) Take the curcumin-chitosan nanoparticles prepared in Example 3 and prepare a solution with a curcumin concentration of 0.6 mg / ml as the test sample solution; (2) Take the samples from Comparative Examples 6-8 and prepare solutions with a curcumin concentration of 0.6 mg / ml as the test sample solutions; Take 0.5 mL of the test sample solution and add 4.5 mL of the above DPPH stock solution. Shake gently to mix evenly.

[0080] Place the mixed reaction solution at room temperature and in the dark, and let it stand for 30 minutes.

[0081] (3) Absorbance measurement and parallel experiments Blank control determination: Replace the test sample with 95% ethanol, follow the above steps, and measure its absorbance at a wavelength of 517 nm, which is recorded as A0.

[0082] Sample absorbance measurement: The absorbance of the sample solution after the reaction is completed is measured at the same wavelength (517nm) and recorded as A1.

[0083] The DPPH free radical scavenging rate can be calculated using the following formula: In the formula: A0 is the absorbance of the DPPH working solution; A1 is the absorbance of the sample to be tested.

[0084] Parallel experiment requirements: Each sample group must be repeated 3 times in parallel to ensure the reliability of the data.

[0085] For details, please see [link / details]. Figure 5 And Table 2.

[0086] 2. ABTS free radical scavenging rate determination: (1) Prepare a 7.4 mmol / L LABTS+ solution with 95% ethanol and a 2.6 mmol / L potassium persulfate K2S2O8 solution with distilled water. Mix the two solutions in equal volume ratio of 1:1 and place them at room temperature in the dark for 12 hours to allow them to react completely, thereby preparing the ABTS+ mother liquor. Dilute the above ABTS mother liquor with 95% ethanol to obtain an ABTS working solution with an absorbance of 0.7 ± 0.02 at a wavelength of 734 nm.

[0087] (2) Preparation of sample solutions: Prepare vitamin C with the same concentration as curcumin as a positive control group; take the curcumin-chitosan nanoparticles prepared in Example 3 and prepare a solution with a curcumin concentration of 0.6 mg / ml as the test sample solution; take the samples from Comparative Examples 5-8 and prepare solutions with a curcumin concentration of 0.6 mg / ml as the test sample solutions. (3) Accurately pipette 4.00 mL of ABTS+ solution, then pipette 1.00 mL of sample solution. After mixing thoroughly, place in a dark place at room temperature and allow to react for 6 minutes. Measure the absorbance at 734 nm. Calculate the scavenging rate of ABTS free radicals for different samples using the following formula: In the formula: A0 is the absorbance value when 1.00 mL of 95% ethanol is added to 1.00 mL of ABTS+ working solution; A1 is the absorbance value of 1.00 mL of sample to 4.00 mL of ABTS+ working solution. See the detailed results below. Figure 5 And Table 2.

[0088] Table 2 Antioxidant Performance Test Data

[0089] From Table 2 and Figure 5 It can be seen that, at the same curcumin concentration, the scavenging rate of free curcumin for ABTS was 16.99%, while the scavenging rate of CS / Cur nanoparticles for ABTS was as high as 93.45%, which was not significantly different from that of Vc (p>0.05). The scavenging rate of CS / Cur for DPPH free radicals was 67.58%, slightly lower than that for ABTS, but significantly higher than that for free curcumin (p<0.05). In summary, the free radical scavenging rate of CS / Cur nanoparticles for ABTS was higher than that for DPPH. The above experimental results indicate that coating curcumin with CS is an effective method to enhance its antioxidant activity. The coating effect of CS transforms the crystal structure of curcumin into an amorphous state, thereby improving the water solubility of curcumin and promoting the contact and reaction between the phenolic hydroxyl groups in curcumin and free radicals, thus enhancing its antioxidant effect.

[0090] Curcumin was synthesized into nanoparticles using a pH-driven method with shellac (S), propylene glycol alginate (PGA), and cellulose nanocrystals (CNC), respectively. The results showed that, at the same curcumin concentration, the antioxidant properties of shellac (S), propylene glycol alginate (PGA), cellulose nanocrystals (CNC), and curcumin nanoparticles were all lower than those of chitosan / curcumin nanoparticles. Compared with carriers such as shellac (S), propylene glycol alginate (PGA), and cellulose nanocrystals (CNC), chitosan-curcumin nanoparticles exhibited superior antioxidant properties. This advantage stems from the synergistic electron-donating effect between the amino and hydroxyl groups of chitosan and the phenolic hydroxyl groups of curcumin, as well as the enrichment of free radicals due to the positively charged nature of chitosan molecules. Simultaneously, chitosan significantly improved the dispersibility and water solubility of curcumin, and enhanced its photostability and oxidative stability through its hydrogen-bonded network structure, thereby strengthening the free radical scavenging rate and the persistence of antioxidant activity. This synergistic system exhibited higher scavenging rates in model reactions such as DPPH and ABTS, indicating that chitosan can significantly amplify the antioxidant capacity of curcumin in pH-driven nanosystems.

[0091] (II) Physicochemical stability of nanoparticles 1. pH stability The pH of the nanoparticles was adjusted to 2.0-7.0 using 1.0M HCl or NaOH to evaluate their pH stability. Figure 7 As shown, the average particle size of CS / Cur at pH 2.0 is slightly larger than that of the samples at pH 3.0-6.0. The particle size of freshly prepared CS / Cur remains stable below 200 nm in the pH 3.0-6.0 range. The PDI remains stable below 0.25 when pH 2.0-6.0. Figure 8 The Zeta potential values ​​in each group were all greater than 20mV. Figure 9 The results indicate that the CS / Cur system is stable within this pH range. At pH 7.0, the particle size increases rapidly, and the potential approaches zero, indicating that the electrostatic repulsion between nanoparticles weakens, making them easier to aggregate. Studies have shown that PGA nanoparticles loaded with curcumin exhibit higher pH stability than those at 4.0. Therefore, the chitosan-curcumin nanoparticles prepared in this study have greater application advantages and are more adaptable to strongly acidic environments.

[0092] 2. Storage stability The storage stability of NPs was investigated by storing them in glass vials at 4 °C and 25 °C for 15 days. These physical stability parameters were evaluated by measuring particle size, PDI, and Zeta surface potential. Based on previous studies, the chemical stability of Cur during storage was determined using first-order kinetic calculations [3]. The calculation formula is shown below:

[0093] Where k is the degradation rate constant, t1 / 2 is the half-life, C0 is the initial Cur concentration, and C is the Cur concentration on day t.

[0094] The average particle size of CS / Cur measured during 15 days of storage under different environments is as follows: Figure 10 As shown in Figure A, the average particle size of CS / Cur remained relatively stable for the first 5 days under both natural light and dark conditions at 25°C, but showed an increasing trend after day 10, with a significant increase in average particle size (p<0.05). The same was true for PDI. Figure 10 B). Under 4°C dark conditions, the average particle size and PDI of CS / Cur were relatively stable, with no significant differences among groups (p>0.05). From the Zeta potential ( Figure 10 C) It can also be seen that CS / Cur is more stable under dark conditions at 4°C, indicating that low-temperature storage is more beneficial. If the absolute value of the surface charge of nanoparticles is greater than 30mV, the colloidal dispersion is considered stable.

[0095] The degradation process of Cur follows a first-order kinetic model. For example... Figure 10 As shown in Table D, the T1 / 2 of free Cur under natural light at 25°C is only 2.83 days (Table 3). However, after encapsulation, the T1 / 2 of CS / Cur increases to 10.7 days, suggesting that encapsulation may slow down the degradation rate of Cur. To verify this hypothesis, measurements were taken at 25°C in the dark (… Figure 10 E) and 4°C darkness ( Figure 10 F) Degradation rate of Cur under certain conditions. The T1 / 2 of free Cur under dark conditions was higher than that under natural light conditions, indicating that light exposure is an important factor in the degradation of Cur during storage. The T1 / 2 of nanoparticles encapsulated under dark conditions at 4°C was as high as 36.1 days, indicating that low-temperature storage can effectively reduce the degradation of Cur.

[0096] Moreover, it can be seen that chitosan encapsulation can significantly improve storage stability.

[0097] Table 3. Degradation Kinetic Parameters

[0098] (III) Reconstitution of freeze-dried nanoparticles Fresh NPS were freeze-dried and then added to the original volume of deionized water, and stirred for 10 min. The appearance of the reconstituted NPS was photographed, and the average particle size, polydispersity index (PDI) and zeta surface potential of the NPS were characterized using a Malvern nanoparticle size analyzer at 25 °C, with an equilibration time of 60 s.

[0099] After freeze-drying, the solubilizing effect of the product is a key indicator for evaluating its feasibility as a solid dosage form. Compared to liquid nanoparticle products, powdered nanoparticle products exhibit significant advantages in transportation, storage, and use. Figure 11 As shown, we evaluated the redispersibility of lyophilized CS / Cur. No obvious aggregates were observed after dispersion, and there were no significant differences in particle size and zeta potential before and after lyophilization (p<0.05). The results indicate that self-assembled CS / Cur has good redispersibility.

[0100] Efficacy Evaluation 3: Bactericidal Activity Test 1. For pathogenic fungi (Phytophthora infestans) Phytophthorainfestans bactericidal activity assay (1) Phytophthora infestans ( Phytophthorainfestans The samples were derived from potato late blight infection samples, identified by ITS sequence and preserved on PDA slant medium (4℃).

[0101] (2) The colony growth rate method was used for determination.

[0102] The solutions of each sample (curcumin, chitosan, and curcumin-chitosan nanoparticles prepared in Example 3) were added to PDA medium and prepared to final concentrations of 0, 0.25, 0.50, 1.00 and 2.00 mg / mL, respectively.

[0103] Each plate was inoculated with a 4 mm diameter fungal hyphae block in the center and incubated at 28°C.

[0104] When the control group colonies covered the entire plate, the colony diameter was measured.

[0105] The antibacterial rate (IR,%) is calculated using the following formula:

[0106] IR=100× Dc: Colony diameter (mm) in the blank control group; Dt: Colony diameter (mm) in the drug-treated group.

[0107] Probabilistic regression analysis was performed using DPS software to calculate the half-maximal inhibitory concentration (EC50). 50 ).

[0108] (3) Results analysis: Curcumin-chitosan nanoparticles have an effect on pathogenic Phytophthora ( PhytophthorainfestansThe mycelial growth of Cur–CSNPs was significantly inhibited. At a concentration of 1.0 mg / mL, the mycelial growth inhibition rate in the Cur–CSNPs experimental group was approximately 85%, significantly higher than that in the pure curcumin group (approximately 60%) and the chitosan group (approximately 48%). Probabilistic regression calculations showed that the EC50 of Cur–CSNPs… 50 The concentration was 0.16 mg / mL, significantly lower than that of curcumin (0.23 mg / mL) and chitosan (0.52 mg / mL), indicating a synergistic effect between the two in the nanostructure. Microscopic observation showed that the surface of the fungal hyphae after treatment exhibited obvious shrinkage, breakage, and cytoplasmic extravasation, indicating that Cur–CS nanoparticles exert their antibacterial effect by disrupting cell membrane integrity.

[0109] 2. For Botrytis cinerea ( B.cinerea bactericidal activity assay (1) Botrytis cinerea ( B.cinerea The samples were derived from samples infected with gray mold of pepper, identified by ITS sequence and preserved on PDA slant medium (4℃).

[0110] (2) The colony growth rate method was used for determination.

[0111] The solutions of each sample (curcumin, chitosan, and curcumin-chitosan nanoparticles prepared in Example 3) were added to PDA medium and prepared to final concentrations of 0, 0.25, 0.50, 1.00 and 2.00 mg / mL, respectively.

[0112] Each plate was inoculated with a 4 mm diameter fungal hyphae block in the center and incubated at 28°C.

[0113] When the control group colonies covered the entire plate, the colony diameter was measured.

[0114] The antibacterial rate (IR,%) is calculated using the following formula:

[0115] IR=100× Dc: Colony diameter (mm) in the blank control group; Dt: Colony diameter (mm) in the drug-treated group.

[0116] Probabilistic regression analysis was performed using DPS software to calculate the half-maximal inhibitory concentration (EC50). 50 ).

[0117] (3) Results analysis: Cur–CSNPs on B.cinerea It exhibited stronger inhibitory activity. At a concentration of 1.0 mg / mL, the Cur–CSNPs experimental group showed stronger inhibitory activity. B.cinereaThe inhibition rate reached approximately 92%, significantly higher than that of the curcumin group (approximately 71%). Its EC50... 50 The value was 0.14 mg / mL, significantly lower than that of pure curcumin (0.26 mg / mL) and chitosan (0.43 mg / mL).

[0118] Based on the above experiments, the antibacterial mechanism of curcumin-chitosan nanoparticles, which exhibit excellent antibacterial effects, was analyzed: Synergistic effect of chitosan. Chitosan itself has a certain amino group hydrogen-donating capacity, which can form hydrogen bonds with the phenolic hydroxyl groups of curcumin, improving the stability of curcumin in the aqueous phase and preventing oxidative degradation. Nanoscale effect. The particle size of Cur–CSNPs is within 200 nm, resulting in a significantly increased specific surface area, promoting the contact reaction rate with free radicals, and thus exhibiting higher antioxidant activity. Correlation between antioxidant and antibacterial activity. The antioxidant properties of curcumin can slow down fungal-induced oxidative stress and inhibit ROS generation, thereby indirectly inhibiting the infection process of pathogenic fungi. Cur–CSNPs possess a dual protective effect of "chemical scavenging + biological barrier".

[0119] Performance Evaluation 4: Evaluation of the performance of nano-coated probiotics The LGG suspension was mixed with 60% glycerol at a ratio of 1:1 (v / v) and stored at -80°C. The glycerol-preserved LGG strain was then incubated in MRS broth at a concentration of 2% (v / v) under anaerobic conditions at 37°C for 24 hours to produce LGG inoculum. The culture was then centrifuged at 4000 rpm and 4°C for 10 minutes. After centrifugation, the supernatant was discarded, and the precipitate was washed twice with 0.85% (w / v) sterile physiological saline to prepare a bacterial suspension.

[0120] LGG (Lactobacillus rhamnosus) cells were serially diluted with 0.85% (w / v) physiological saline, then mixed with MRS agar using the pour plate method and incubated anaerobically at 37°C for 48 h. Colony forming units (CFU) were then counted. The prepared LGG cells were subsequently used in the encapsulation process.

[0121] The bacterial suspension prepared above was mixed with nanoparticles at a ratio of 1:10 (v / v) at room temperature (avoiding vigorous shaking). The mixture was then centrifuged at 4000 rpm for 10 min, and excess embedding solution was discarded. Next, the mixture was washed twice in 0.85% sterile physiological saline to remove excess nanoparticles, yielding nano-coated probiotics, named LGG@nps. A portion of the final LGG@nps was used for subsequent experiments, while the other portion was pre-frozen at -80℃ and then freeze-dried to obtain lyophilized LGG@nps samples for further experiments.

[0122] (a) Detection of Zeta potential The zeta potential of ordinary LGG and LGG@nps after different embedding times was measured using dynamic light scattering (DLS) technology. The samples were ultrasonically dispersed in ultrapure water (0.05%, w / v) and then analyzed at 25°C.

[0123] from Figure 12 As can be seen from A, with increasing encapsulation time, LGG adsorbs more and more CS / Cur, and the color of the bacterial suspension changes from light to dark. Successful adsorption is determined by measuring the zeta potential. Figure 12 (B) The free LGG surface has a negative charge of -2.37 mV. After mixing the free LGG with CS / Cur, the potential changes from negative to positive, indicating that the nanoparticles were successfully adsorbed onto the LGG surface. The deepest color was observed after 60 min of embedding, and there was no significant difference in potential between 60 min and 90 min (p>0.05). This slight deviation in zeta potential may be attributed to the thinness of the nanoparticle coating, indicating an electrostatic interaction between LGG and the nanoparticles. Therefore, it is speculated that LGG may reach saturation after 60 min of embedding.

[0124] (ii) TEM detection Bacterial suspensions were dropped onto a 400-mesh copper mesh and dried. TEM images of ordinary LGG or embedded LGG@nps suspensions were observed at 80 kV. After thoroughly mixing the bacterial suspensions, a small amount of ordinary LGG or embedded LGG@nps suspension was dropped onto a 400-mesh copper mesh and dried.

[0125] The results are as follows Figure 6 As can be seen, the surface of unencapsulated probiotics is clean and smooth. After encapsulation with curcumin-chitosan nanoparticles, a clear gray nanoshell forms on the surface of the probiotics, and it is a single-cell coating rather than an aggregate. This result confirms the successful preparation of the probiotic single-cell nanocoating.

[0126] (III) Growth curve detection LGG or encapsulated LGG@nps were introduced into MRS medium, and the optical density (OD600) of the culture was measured at a wavelength of 600 nm every 30 min under specific culture conditions using a growth curve measuring instrument.

[0127] To investigate whether the presence of a CS / Cur coating on the LGG surface significantly affects LGG growth and reproduction, the absorbance value (OD600) at a wavelength of 600 nm was recorded. The OD600 value was positively correlated with the bacterial concentration; a higher OD600 value indicated a higher bacterial concentration. Uncoated and coated LGGs showed similar growth curves. Figure 13 This result indicates that when LGG cells are embedded in CS / Cur, they maintain their ability to grow and reproduce, exhibiting distinct lag phases, exponential growth phases, and stationary phases. However, the lag phase of ordinary LGG cells is shorter than that of coated LGG cells. Coated LGG cells showed a certain degree of growth and reproduction delay compared to uncoated LGG cells. This may be because the presence of the coating reduces the efficiency of material and energy exchange between the LGG and its external environment. In the final stage, the number of coated LGG cells was comparable to that of uncoated cells, indicating that coated LGG cells can carry out normal growth, reproduction, and metabolism.

[0128] (iv) Viability testing 1. Tolerance to simulated digestion Simulated gastric juice consisted of 2 g / L sodium chloride and 3 g / L pepsin, with the pH adjusted to 2.0. Simulated intestinal juice (SIF) was prepared by adding 10 g / L bile salts and 1 g / L pancreatin to 20 mM phosphate-buffered saline (PBS), with a final pH of 7.0. The sample and simulated gastric juice were mixed at a 1:9 (v / v) ratio and incubated with shaking at 37°C for 2 hours. The gastric digestate from the sample was then transferred and mixed with the SIF. After 2 hours of intestinal digestion, the viability of LGGs was determined.

[0129] In vitro curcumin release experiments were conducted in a simulated gastrointestinal environment. Curcumin content was expressed as relative concentration (RC%), obtained by dividing the measured curcumin content by the actual amount of curcumin added. Samples were taken and measured every 30 minutes. The sample and simulated gastric juice were mixed at a ratio of 1:9 (v / v) and incubated with shaking at 37°C for 2 hours. The sample was then subjected to intestinal digestion using a simulated bile salt solution (0.6% w / v, pH 6.8) under the same temperature and shaking conditions for 2 hours. The sample and simulated gastric juice were mixed, and the mixture was placed in a shaker at 37°C for 1 hour of digestion. After gastric digestion was complete, 250 mg of bile salts and 10 mg of trypsin were added, and simulated intestinal digestion was continued for another 2 hours.

[0130] To exert their health benefits, probiotics must be able to survive in the gastrointestinal environment and maintain a high concentration within the gut. Therefore, the survival of LGG under simulated gastrointestinal conditions in vitro was investigated, and the results are as follows: Figure 14 As shown in Figure A, the free LGG bacteria showed a viable count of 5.39 Logs after 2 hours of treatment with simulated gastric fluid, while the encapsulated group only lost 2.78 Logs, retaining 7.25 Logs. After treatment with simulated intestinal fluid, all free LGG bacteria died, and no activity was detected, while the LGG encapsulated in CS / Cur bacteria still showed a viable count of 4.43 Logs after 2 hours of treatment with simulated intestinal fluid. This indicates that the nanocoating has a protective effect on probiotics, protecting LGG from the harmful effects of acidic conditions.

[0131] Under simulated gastrointestinal conditions, the change of Cur's release curve over time is as follows: Figure 14 As shown in Figure B, during the 2-hour SGF digestion phase, free Cur was rapidly released at a rate of 54.48%, while the Cur release rate from nanoparticles significantly decreased to 31.6% (p<0.05). After SIF digestion, both free and encapsulated Cur exhibited sustained release characteristics, although the release amount of encapsulated Cur was significantly lower than that of free Cur. The significantly reduced release rate of encapsulated Cur indicates controlled release. This may be related to the hydrogen bonding and strong hydrophobic interactions between Cur and the delivery carrier. However, for LGG@nps, the Cur release rate was 22.2% during the SGF phase, while the final Cur release rate was 69.68% during the SIF phase, significantly higher than that of free Cur and nanoparticles (p<0.05). This may be because the electrostatic interactions and spatial repulsion provided by the combined use of probiotics and nanoparticles reduced nanoparticle aggregation under simulated gastrointestinal conditions, resulting in more uniform nanoparticle dispersion and facilitating the release of more Cur from the nanoparticles. The results show that nanoparticles coated with CS can effectively delay the release of Cur by forming a protective layer, thereby preventing Cur from being hydrolyzed by pepsin, improving its stability in the gastric environment, and promoting the transport of Cur to the small intestine.

[0132] 2. Heat resistance The effect on the heat resistance of probiotics was tested by heating ordinary LGG and LGG@nps at 72°C for 15 seconds, 72°C for 3 minutes, or 65°C for 30 minutes, respectively. After heating, the test tubes were quickly placed in an ice bath for cooling. Viable cell counts were then performed on ordinary LGG and LGG@nps.

[0133] High temperatures are detrimental to the survival of probiotics, making it difficult to directly add them to foods that require heating. Pasteurization is a crucial heat treatment step used in the manufacturing process of many types of food and beverages to extend shelf life. Therefore, heat treatment was applied to coated LGG@nps and free LGG, and different heat treatment results were observed. Figure 15 As shown in Figure A, after treatment at 72°C for 15 seconds, the CFU of LGG decreased significantly (p<0.05), while there was no significant difference in LGG@nps before and after treatment (p>0.05). Treatment at 72°C for 3 minutes and at 65°C for 30 minutes resulted in a decrease in viability for all samples. LGG showed a decrease of 2.82 and 5.83 log values, respectively, while LGG@nps showed decreases of 1.98 and 2.99 log values, respectively. This indicates that unencapsulated LGG is highly sensitive to heat inactivation, and encapsulation provides better protection for LGG cells.

[0134] 3. Freeze-drying tolerance After being pre-frozen at -80°C overnight, the samples were freeze-dried and then vacuum-dried in a freeze dryer for 60 hours. After the process, viable cell counts were performed on ordinary LGG and LGG@nps.

[0135] During freeze-drying, probiotic cells face various stresses such as mechanical stress and abiotic pressures, leading to damage to cell membrane integrity, fluidity, and sensitive protein structures, resulting in physiological dysfunction and even cell death. Studying the impact of freeze-drying on probiotic survival rates is crucial for ensuring the stability, efficacy, and quality of probiotic products during storage and distribution. Figure 15 B shows the cell survival of LGG and LGG@nps before and after freeze-drying. Compared with the initial quantity, the reduction of free LGG after freeze-drying was 1.04 log, while that of LGG@nps was only 0.43 log, indicating that the nanocoating has a protective effect on LGG and can prevent ice crystals from damaging cells to a certain extent.

[0136] 4. Antibiotic resistance To assess the tolerance of LGG to six antibiotics (Ciprofloxacin, Tobramycin, Neomycin, Levofloxacin, Norfloxacin, and Gentamycin), the six antibiotics were serially diluted twofold, ranging from 62.5 μg / mL to 4000 μg / mL, and added to 96-well plates. After mixing an equal volume of bacterial suspension and antibiotic solution in each well, the 96-well plates were incubated at 37°C for 24 hours. A volume of bacterial suspension from each well was inoculated onto MRS agar plates, and after 24 hours of incubation, the minimum inhibitory concentration (MBC) was determined by observing the colony count (CFU) on the agar plates. Ordinary LGG or LGG@nps was added to the antibiotic at its respective MBC concentration, followed by MRS medium. Vortex mixing was performed after each addition. The mixture was incubated at 37°C for 24 hours. The collected bacteria were then inoculated onto MRS agar plates, and viable cell counts were performed on both standard LGG and LGG@nps to assess their viability.

[0137] Antibiotics are among the most widely used drugs globally, and their usage continues to increase dramatically. However, antibiotic treatment itself often weakens the effects of probiotics. We investigated the ability of this nano-coated probiotic to resist antibiotic effects. After nanoparticle treatment, LGG or LGG@nps were exposed to six clinically relevant antibiotics (Ciprofloxacin, Tobramycin, Neomycin, Levofloxacin, Norfloxacin, Gentamycin) for 24 hours, all at concentrations exceeding their respective minimum bactericidal concentrations (MBC, Table 4). The results showed ( Figure 15 (C) Nanoparticle-treated LGG@nps can tolerate higher antibiotic concentrations, with survival rates ranging from a high of 63.45% to a low of 33.92%, while ordinary LGG completely stops growing. Nanoparticle-encapsulated LGG can grow in fresh MRS medium, while ordinary LGG cannot grow under the same conditions.

[0138] Table 4. Minimum bactericidal concentrations (MBC) of six different antibiotics against Lactobacillus rhamnosus (LGG)

[0139] Note: All data were measured at least three times. The mean and standard error were determined. Data analysis was performed using the ANOVA procedure in SPSS. At the 95% level (… p Differences were considered statistically significant at values ​​<0.05. The graphs were generated using Origin 2022 software.

Claims

1. A method for preparing highly loaded curcumin-chitosan nanoparticles, characterized in that, Curcumin is encapsulated by chitosan to form nanoparticles, wherein the mass ratio of curcumin to chitosan is 5:1 to 1:

5. The preparation method includes the following steps: A curcumin aqueous solution with pH 11.0–13.0 was added dropwise to a chitosan solution with pH 1.4–2.8 to obtain a mixed solution; the dropping rate was 0.8–1.2 mL / min.

2. The preparation method according to claim 1, characterized in that, The curcumin concentration in the curcumin aqueous solution is 0.2–0.6 mg / mL, the chitosan concentration in the chitosan aqueous solution is 0.04–3.0 mg / mL, and the volume ratio of the curcumin aqueous solution to the chitosan aqueous solution is 1:

2.

3. The preparation method according to claim 1, characterized in that, The curcumin aqueous solution needs to be stirred until the curcumin is completely dissolved, and the chitosan aqueous solution needs to be stirred until the chitosan is completely dissolved; during the dropwise addition process, the curcumin aqueous solution and the chitosan aqueous solution are stirred continuously respectively.

4. The preparation method according to claim 1, characterized in that, The preparation method further includes centrifuging the mixture to remove curcumin that has not formed nanoparticles.

5. A highly loaded curcumin-chitosan nanoparticle with antibacterial and antioxidant activity, characterized in that, The nanoparticles are prepared by any one of claims 1-4; the nanoparticles have a DPPH radical scavenging rate of ≥67.58% and an ABTS radical scavenging rate of ≥93.45%. When the solution environment in which the nanoparticles are located is 2.0≤pH≤6.0, the PDI of the nanoparticles is ≤0.25 and the Zeta potential of the nanoparticles is >20mV; When the solution environment in which the nanoparticles are located is 3.0 ≤ pH ≤ 6.0, the average particle size of the nanoparticles is < 200 nm; when the solution environment in which the nanoparticles are located is 2.0 ≤ pH < 3.0, the average particle size of the nanoparticles is 200-220 nm. After being stored for 15 days, the nanoparticles have an average particle size ≤200nm, a PDI ≤0.25, and a Zeta potential ≥40mV. The nanoparticles' T 1 / 2 It takes 36.10 days; The nanoparticles are freeze-dried and then rehydrated, with the particle size change ≤15% and the zeta potential change ≤20%.

6. The application of the high-load curcumin-chitosan nanoparticles for antibacterial activity according to claim 5, characterized in that, The nanoparticles can inhibit pathogenic Phytophthora. Phytophthorainfestans The growth of the nanoparticles; the half-maximal inhibitory concentration (EC50) of the nanoparticles against pathogenic Phytophthora infestans. 50 It is 0.16 mg / mL; The nanoparticles can inhibit *Botrytis cinerea*. Botrytiscinerea The growth of the nanoparticles; the half-maximal inhibitory concentration (EC50) of the nanoparticles against *Botrytis cinerea*. 50 The concentration was 0.14 mg / mL.

7. The application of the highly loaded curcumin-chitosan nanoparticles according to claim 5 in the encapsulation of probiotics, characterized in that, The solution of the nanoparticles is mixed with a probiotic suspension; the nanoparticles encapsulate the probiotics in the solution state to obtain nano-coated probiotics.

8. The application according to claim 7, characterized in that, The probiotic is Lactobacillus.

9. The application according to claim 7, characterized in that, The concentration of the nanoparticles is 0.2-0.6 mg / mL, the concentration of probiotics in the probiotic suspension is 10^9-10^11 CFU / mL, and the volume ratio of the nanoparticle solution to the probiotic suspension is 12:1-8:

1.

10. The application according to claim 7, characterized in that, After treatment with simulated gastric acid for 2 hours, the survival rate of the nano-coated probiotics was ≥7.25 LogCFU / mL; after treatment at 65℃ for 30 minutes, the survival rate was ≥7.14 LogCFU / mL; after freeze-drying, the survival rate was ≥10.21 LogCFU / mL; and after exposure to Ciprofloxacin for 24 hours, the survival rate was ≥63.45%. After exposure to Tobramycin for 24 hours, the survival rate of the nano-coated probiotics was ≥58.34%; after exposure to Neomycin for 24 hours, the survival rate was ≥53.54%; after exposure to Levofloxacin for 24 hours, the survival rate was ≥50.2%; and after exposure to Norfloxacin for 24 hours, the survival rate was ≥41.91%. After the nano-coated probiotics were exposed to a Gentamycin environment for 24 hours, the survival rate of the nano-coated probiotics was ≥33.92%.

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