Anthocyanin nanoparticles and methods of making the same

Abstract

The application provides a cyanine nanoparticle, which is prepared by wrapping cyanine with lactoferrin and sodium carboxymethyl cellulose to form a nanoparticle, wherein the weight ratio of lactoferrin, sodium carboxymethyl cellulose and cyanine is 12.8-19.2 parts of lactoferrin, 2.56-3.84 parts of sodium carboxymethyl cellulose and 12.8-19.2 parts of cyanine. The application also provides a preparation method and application of the cyanine nanoparticle. In order to avoid structural changes of cyanine in the preparation process, the application adopts a green and safe self-assembly method, and adjusts the synthesis conditions without adding any chemical coupling agent and chemical emulsifier. The application is a simple, safe and environmentally-friendly synthesis method, provides a cyanine nutritional additive with more stable properties and safe components for the market, and provides a basis and technical support for developing foods and beverages containing stable cyanine.

Description

Technical Field

[0001] This invention relates to anthocyanin nanoparticles and their preparation method. Background Technology

[0002] Anthocyanins (ACN) are water-soluble flavonoid pigments commonly used in the food industry as food colorings or nutritional additives. They have been reported to possess various biological activities, including antioxidant, anti-inflammatory, and anticancer properties. However, due to their electron-deficient structure, they are highly susceptible to attack by reactive oxygen species and free electrons, leading to poor stability and easy degradation during processing. Changes in pH, light, heat, enzyme activity, and contact with oxygen and metal ions during processing, storage, or cooking can all cause oxidative discoloration and reduced biological activity of anthocyanins, significantly limiting their commercial application as natural pigments and nutritional additives.

[0003] Currently, there is a considerable amount of research literature addressing the stability of anthocyanins. For example, some studies suggest converting anthocyanins into acylated anthocyanins through chemical reactions to improve their stability. For instance, using succinic acid, methyl salicylate, methyl benzoate, etc., as acyl donors, and catalyzing with a bio-enzyme, can significantly improve the thermal stability, photostability, and resistance to heavy metal ions of anthocyanins (Patent Application No.: CN202111352413.2, Invention Title: A Bio-enzyme Complex and its Application in Plant Extraction). However, since the solvents for acylation reactions are often mixtures of various organic reagents, anthocyanin samples stabilized by acylation must be completely detoxified or proven safe for consumption before being applied to food; they cannot be directly used as food additives. Patent application number 202210609647.9, invention title: "A Method for Improving Anthocyanin Stability," discloses a method for improving anthocyanin stability using peach gum polysaccharide, comprising the following steps: washing and drying peach gum to constant weight and then pulverizing it; using distilled water as a solvent, fully swelling the peach gum; adding a certain amount of sodium hydroxide to hydrolyze it; adjusting the pH to neutral; concentrating the solution; and adding ethanol to fully precipitate the peach gum polysaccharide. Then, the peach gum polysaccharide and anthocyanin are thoroughly mechanically mixed to obtain a peach gum polysaccharide-anthocyanin complex. The solubility, resistance to heavy metal ions, thermal stability, and alkali resistance of the complex are then measured. Due to the encapsulation of anthocyanins by the gum arabic polysaccharide in the mixture, the short-term dissolution rate of the complex (within 120 min) is significantly improved compared to anthocyanins. However, the complex strength is not high, and the storage stability needs further improvement. (Xu Qing, et al., Research Progress on Factors Affecting Anthocyanin Stability and Improvement Methods, Food Research and Development, April 2020, Vol. 41, No. 7). Anthocyanins can form complexes with organic macromolecules such as proteins, cyclodextrins, and polysaccharides through interactions such as hydrogen bonding, electrostatic effects, and hydrophobic effects, thereby improving their thermal and storage stability. Therefore, it is feasible to use macromolecules such as proteins to self-assemble with anthocyanins to form complexes. However, most current research focuses on binary complexes formed by anthocyanins and single macromolecules, with a lack of research on ternary complexes. Wu's research found that mannose protein can form a complex with anthocyanins through hydrophobic interactions, increasing the thermal stability of anthocyanins (pH 7.0, heat treatment at 80℃ or 126℃ for 30 min) by 4 to 5 times. However, mannose protein itself is prone to aggregation, and the average size of its synthesized complex is 279.4 nm. It also exhibits significant thermal aggregation behavior when heated, which is not conducive to maintaining the stable properties of anthocyanin nanoparticles during high-temperature processing.

[0004] Current literature on anthocyanin stability research largely focuses on anthocyanin extraction from plants, with limited coverage of methods to modify stability through formulation. Recent advancements in nanotechnology have provided feasible methods for preparing anthocyanin nanoparticles, such as enhancing physicochemical stability, bioavailability, and bioactivity through active or passive binding with macromolecules. Embedding anthocyanins into nanoparticle structures can, to some extent, separate them from the external environment, mitigating their reaction with environmental factors and thus stabilizing them. The functional properties of nanoparticles, such as size, encapsulation efficiency, and stability, are influenced by the type of wall material and the synthesis method. Currently, there are no published studies on anthocyanin nanoparticles that can simultaneously address anthocyanin stability and achieve high encapsulation rates. Summary of the Invention

[0005] This invention provides anthocyanin nanoparticles. This invention also provides a method for preparing these anthocyanin nanoparticles.

[0006] This invention provides anthocyanin nanoparticles, which are prepared by encapsulating anthocyanins with lactoferrin and sodium carboxymethyl cellulose to form nanoparticles, wherein the weight ratio of lactoferrin, sodium carboxymethyl cellulose, and anthocyanins is:

[0007] Lactoferrin 12.8-19.2 parts, sodium carboxymethyl cellulose 2.56-3.84 parts, anthocyanins 12.8-19.2 parts.

[0008] More preferably, the weight ratio of lactoferrin, sodium carboxymethyl cellulose, and anthocyanins is:

[0009] Lactoferrin 16 parts, sodium carboxymethyl cellulose 3.2 parts, anthocyanins 16 parts.

[0010] The degree of substitution (DS) of the sodium carboxymethyl cellulose is 0.7-1.2. More preferably, the degree of substitution (DS) of the sodium carboxymethyl cellulose is 0.9.

[0011] The anthocyanin nanoparticles of this invention are obtained by adding anthocyanins to a sodium carboxymethyl cellulose dispersion solution, stirring, then adding a lactoferrin solution and stirring again.

[0012] This invention provides a method for preparing the aforementioned anthocyanin nanoparticles, comprising the following steps:

[0013] a. Dissolve anthocyanins in 0.2M pH 3.0 citrate buffer solution, place on a magnetic stirrer and stir continuously at room temperature for 1 hour. Store the resulting anthocyanin solution away from light.

[0014] b. Prepare aqueous solutions of lactoferrin and sodium carboxymethyl cellulose respectively, and stir magnetically for 1 hour at 75℃ and 40℃ respectively, then adjust the pH to 5.0 with dilute hydrochloric acid.

[0015] c. Take the sodium carboxymethyl cellulose dispersion solution into a capped brown bottle and stir it at a constant speed on a magnetic stirrer. Use a syringe to sample the anthocyanin solution from step a and add it drop by drop into the bottle. Stir for 30 minutes to ensure uniform mixing, and then adjust the pH value to 5.0.

[0016] d. Take lactoferrin solution, add it dropwise to the bottle, and stir continuously on a magnetic stirrer for 24 hours to obtain a nanoparticle mixed solution;

[0017] e. Filtration: The mixed solution from step d is ultrafiltered at 1000×g for 20 min using an ultracentrifugal filter with a nominal molecular weight limit of 30kDa to remove unencapsulated free anthocyanins; then filtered through a filter membrane with a pore size of 0.45μm to remove impurities, thus obtaining anthocyanin nanoparticle dispersion.

[0018] Lactoferrin (LF), used in this invention, is a natural protein found in milk. As a glycoprotein belonging to the transferrin family, it is a good and safe drug nanocarrier and is also commonly used in the food industry to prepare nanoparticles encapsulating bioactive small molecules. When nanoparticles are prepared based on lactoferrin using mild methods, relatively safe products can usually be obtained without involving any chemical reactions. Notably, lactoferrin has an isoelectric point (pI≈8.5) higher than most other proteins (pI≈5), and can form nanocomposites through electrostatic interactions when coexisting with other negatively charged macromolecules in aqueous solution. Sodium carboxymethyl cellulose (CMC) is an anionic polymer typically prepared by reacting natural cellulose with caustic alkali and monochloroacetic acid. It can combine with other biological substances to form polymers with excellent encapsulation capabilities, thus also making it a good nanoparticle wall material.

[0019] This invention selects lactoferrin and sodium carboxymethyl cellulose (CMC) to combine under mild reaction conditions. Anthocyanins are encapsulated under the electrostatic attraction between the negatively charged CMC and positively charged lactoferrin molecules to form stable nanoparticles. The reaction conditions, including pH, degree of substitution of CMC, order of feedstock loading, CMC treatment temperature, and lactoferrin treatment temperature, were investigated and optimized to obtain smaller and more stable anthocyanin nanoparticles. The microstructure, infrared spectral information, and stability of the anthocyanin nanoparticles were then characterized.

[0020] To avoid structural changes in anthocyanins during formulation, this invention employs a green and safe self-assembly method, adjusting synthesis conditions without adding any chemical coupling agents or emulsifiers. This invention provides a simple, safe, and environmentally friendly synthesis method, offering the market a more stable and safer anthocyanin nutritional additive, and providing a theoretical basis and technical support for the development of foods and beverages containing stable anthocyanins. Attached Figure Description

[0021] Figure 1 Flowchart of the preparation process of anthocyanin nanoparticle dispersion containing lactoferrin and sodium carboxymethyl cellulose;

[0022] Figure 2 The ζ-potential values ​​of lactoferrin (LF) and sodium carboxymethyl cellulose (CMC) at different pH values ​​and the SEI values ​​between them;

[0023] Figure 3 Fourier transform infrared spectra of the raw materials and nanoparticle samples prepared by this method;

[0024] Figure 4 Scanning electron microscope images of blank nanoparticles (left) and nanoparticle samples prepared according to this method (right);

[0025] Figure 5 Results of nanoparticle stability experiments: (A) Particle size change trend after heating in a water bath at 90℃ for 3 hours; (B) Particle size change trend after dilution by different factors; (C) Particle size change trend after continuous storage for 15 days.

[0026] Figure 6 Results of anthocyanin retention stability experiments: (A) Trend of anthocyanin retention rate at different pH levels; (B) Trend of anthocyanin retention rate over time under light exposure at 4℃; (C) Trend of anthocyanin retention rate over time under light-proof storage at 4℃; (E) Trend of anthocyanin retention rate over time under light-proof storage at 25℃. Detailed Implementation

[0027] Example 1: Preparation method of anthocyanin nanoparticles of the present invention

[0028] The preparation process of anthocyanin nanoparticles encapsulated with lactoferrin and sodium carboxymethyl cellulose is described as follows: Figure 1As shown. Anthocyanins were dissolved in 0.2M pH 3.0 citrate buffer and continuously stirred on a magnetic stirrer at room temperature for 1 hour to obtain a 4 mg / mL anthocyanin solution, which was stored at 4°C protected from light until use. Aqueous solutions of 1.6 mg / mL lactoferrin (LF) and 0.32 mg / mL sodium carboxymethyl cellulose (CMC) were prepared and magnetically stirred at 75°C and 40°C for 1 hour, respectively. The pH of the LF and CMC dispersions was immediately adjusted to 5.0 with dilute hydrochloric acid. To avoid oxidative decomposition of the anthocyanin samples during preparation, the dispersion of the packaging material should be cooled to room temperature and protected from light before adding anthocyanins. 10 mL of CMC dispersion was placed in a capped brown bottle and stirred at a constant speed on a magnetic stirrer. 4 mL of anthocyanin solution was accurately sampled using a syringe and added dropwise to the bottle. The bottle was capped and continuously stirred for 30 minutes to ensure thorough mixing and reaction. Subsequently, the pH of the reaction system was adjusted to 5.0 using sodium hydroxide solution to achieve the optimal reaction conditions for LF. Afterward, 10 mL of LF solution was accurately sampled using a syringe and added dropwise to the bottle. The bottle was capped and continuously stirred on a magnetic stirrer for 24 hours to obtain a nanoparticle mixed solution. Unencapsulated free anthocyanins in the mixed sample were removed by ultrafiltration at 1000×g for 20 min using an ultracentrifuge filter with a nominal molecular weight limit of 30 kDa. Larger impurity particles were removed by filtration through a 0.45 μm pore size filter membrane. The final sample was the nanoparticle dispersion, which was stored in the dark at 4°C for further research.

[0029] Example 2: Process investigation experiment on the preparation method of anthocyanin nanoparticles of the present invention

[0030] Before optimizing the synthesis process, the preparation process of anthocyanin nanoparticles encapsulated with lactoferrin and sodium carboxymethyl cellulose is briefly described as follows:

[0031] Take 10 mL of CMC dispersion solution into a capped brown bottle and stir at a constant speed on a magnetic stirrer. Accurately sample 10 mL of LF solution using a syringe and add it dropwise to the bottle. Cap the bottle and stir continuously for 1 h to allow the solution to mix and react. Then, cool at room temperature for 30 min to prevent the subsequent addition of anthocyanins to the system from thermal decomposition. After this, accurately sample 4 mL of anthocyanin solution using a syringe and add it dropwise to the bottle. Cap the bottle and stir continuously at room temperature for 30 min. Then, adjust the pH of the reaction system to 5.0 with sodium hydroxide solution to achieve the optimal reaction conditions for LF, and continue stirring on a magnetic stirrer for 24 h to obtain a nanoparticle mixed solution. Unencapsulated free anthocyanins in the mixed sample were removed by ultrafiltration at 2700×g for 20 min using an ultracentrifuge filter with a nominal molecular weight limit of 30 kDa. Larger impurity particles were removed by filtration through a filter membrane with a pore size of 0.45 μm. The final sample was a nanoparticle dispersion, which was stored in the dark at 4°C for further research.

[0032] 1. Selection of reaction pH

[0033] like Figure 2 As shown in the broken-line graph, the zeta potentials of lactoferrin and sodium carboxymethyl cellulose (SCMC) in aqueous solutions with different pH values ​​were measured. The concept of electrostatic interaction strength (SEI value) was introduced to provide a more intuitive method for quantifying the electrostatic force between lactoferrin and SCMC. Both lactoferrin and SCMC were prepared as 1 mg / mL aqueous solutions. Following the nanoparticle preparation process, the solutions were heated and stirred at 75°C and 40°C, respectively, and the pH was adjusted to 3, 4, 5, 6, and 7. The SEI value was calculated based on the zeta potentials of lactoferrin and SCMC at different degrees of substitution (DS) at the same pH; the value is the product of the absolute values ​​of the two solutions.

[0034] Within the studied pH range, as the solution became increasingly alkaline, the zeta potential of lactoferrin gradually decreased from 37.77±1.44 mV to 16.80±1.18 mV; however, it consistently exhibited a positive charge. Sodium carboxymethyl cellulose carried a negative charge over a relatively wide pH range, and the zeta potential varied slightly depending on the degree of substitution. The degree of substitution is directly related to the number of charged groups attached to the sodium carboxymethyl cellulose molecule, thereby increasing the charge density of the polysaccharide. This, in turn, leads to polysaccharides with higher degrees of substitution exhibiting stronger charge properties in aqueous solutions.

[0035] Due to the low charge density of sodium carboxymethyl cellulose, the SEI value decreases to its lowest point (approximately 285.02 to 448.07 mV) under acidic conditions, especially when the pH value is close to 3. 2At neutral pH (pH 7), lactoferrin approaches its isoelectric point, and its net charge gradually approaches zero, which also reduces its SEI value. Therefore, a moderate pH value can improve the charge-carrying properties of proteins and polysaccharides when selecting a reaction system. In this experiment, the electrostatic interaction strength between lactoferrin and sodium carboxymethyl cellulose (CMC) with three different DS values ​​all peaked at pH 5, which is considered the ideal reaction condition for the formation of nanocomplexes between CMC and LF.

[0036] 2. Selection of sodium carboxymethyl cellulose with different degrees of substitution

[0037] Nanoparticles were prepared using three types of sodium carboxymethyl cellulose as raw materials according to the above process. The average particle size and polydispersity index (PDI) were determined by dynamic light scattering (DLS), the zeta potential was determined by electrophoresis, and the anthocyanin content was determined by HPLC-DAD to calculate the anthocyanin encapsulation efficiency. The results are shown in Table 1.

[0038] Table 1 Performance indicators of nanoparticles prepared from sodium carboxymethyl cellulose with different degrees of substitution

[0039]

[0040] Different letters in the same column indicate significant differences (P<0.05).

[0041] It can be inferred that sodium carboxymethyl cellulose (CMC) with a higher degree of substitution generates more electrostatic repulsion between CMC molecular chains, thereby inhibiting the formation of uniformly sized nanoparticles. However, due to its higher charge density, CMC with a DS of 1.2 can improve the particle properties of nanoparticles, resulting in smaller and more stable nanoparticles. However, because anthocyanins are more hydrophilic, a larger specific surface area increases the probability of free anthocyanins detaching from the particles. Therefore, the encapsulation effect of nanoparticles prepared with CMC with a DS of 1.2 is not ideal. As the NPs particle size increases, the specific surface area in the solution decreases, which is also unfavorable for anthocyanin adsorption. Therefore, the anthocyanin encapsulation efficiency of CMC with a DS of 0.7 is also relatively low. Furthermore, its average particle size and PDI are slightly larger, and the particle structure is relatively loose, failing to tightly and stably fix small molecules within the particles. A moderate DS value can provide higher anthocyanin encapsulation efficiency, and the sample exhibits good particle properties. In conclusion, CMC with a DS of 0.9 is more suitable for the preparation of nanoparticles.

[0042] 3. Selection of loading order for different raw materials

[0043] In the experiment, four different nanoparticle samples were prepared according to the above-mentioned experimental conditions. The order of abbreviations in the sample names represents the loading order. In order to reflect the effect of different addition orders on the loading effect of anthocyanins (ACN), we set two ultrafiltration conditions of 1000×g and 2700×g for comparison. The performance indicators of the obtained nanoparticles are shown in Table 2.

[0044] Table 2 Performance indicators of nanoparticles prepared under different raw material loading sequences

[0045]

[0046] Different letters in the same column indicate significant differences (P<0.05).

[0047] The addition of ACN, LF, and CMC sequences can directly affect the interactions of these materials, thereby influencing the structural properties of the nanocomplexes. In group A samples, sodium carboxymethyl cellulose and lactoferrin, with opposite charges, initially bind electrostatically to form larger particles. Once the CMC-LF complex has formed, anthocyanins cannot overcome the electrostatic repulsion with lactoferrin on the particle surface and can only be adsorbed to a limited extent on the CMC molecular branches, resulting in low encapsulation efficiency. When larger mechanical forces are used to remove small molecule particles from the dispersion, the particle size of group A samples decreases, while the anthocyanin encapsulation efficiency drops to 65.73%. These changes can be attributed to the separation of anthocyanin molecules adsorbed on the outer surface of the particles under stronger forces. In contrast, the loading sequence in group B is more scientifically sound. The positively charged anthocyanins and the negatively charged sodium carboxymethyl cellulose react with each other first, allowing the nanoparticles to encapsulate more anthocyanin molecules at the structural center, resulting in the highest anthocyanin encapsulation efficiency. After being coated with a positively charged protein layer, a relatively compact particle structure is formed. This structure can resist changes caused by strong external forces, so the particle size does not change significantly when the centrifugal force is increased. For the sample in group C, both the protein and anthocyanins are positively charged and are uniformly dispersed in the aqueous solution through electrostatic repulsion. Even with the addition of sodium carboxymethyl cellulose, it is difficult to form particles of uniform size, resulting in the highest PDI value and the lowest encapsulation efficiency. The particle size results of group D are similar to those of group A, which is also not conducive to the formation of stable nanoparticles. In summary, the loading order of adding anthocyanins to sodium carboxymethyl cellulose before adding lactoferrin is more conducive to the formation of nanoparticles. At the same time, adjusting the centrifugal force of ultrafiltration to 1000×g reduces the consumption of anthocyanins during sample preparation.

[0048] 3. Selection of treatment temperature for sodium carboxymethyl cellulose

[0049] Four points between room temperature and 80℃ were selected as factors for investigation, and the performance indicators of the prepared nanoparticles are shown in Table 3.

[0050] Table 3 Performance indicators of sodium carboxymethyl cellulose nanoparticles prepared by different temperature treatments

[0051]

[0052] Different letters in the same column indicate significant differences (P<0.05).

[0053] Appropriate heating can enhance the charge of sodium carboxymethyl cellulose (CMC), but the charge decreases when the temperature reaches 80℃. Generally, the critical temperature of CMC is between 80℃ and 100℃. If the temperature exceeds this range, the adhesion and solubility of CMC will decrease sharply, or even become completely ineffective. Therefore, the zeta potential of the prepared nanoparticles is also affected, with the absolute charge value showing a trend of first increasing and then decreasing. However, the change in the treatment temperature of sodium carboxymethyl cellulose did not affect the encapsulation efficiency of anthocyanins, and there was no significant difference between the groups. This shows that heating does not change the binding strength between anthocyanins and lactoferrin. Therefore, in subsequent processes, we chose 40℃ as the treatment temperature for sodium carboxymethyl cellulose to obtain smaller nanoparticles with less energy consumption.

[0054] Regarding the selection of treatment temperature for lactoferrin, since the two denaturation temperatures of bovine lactoferrin under neutral pH conditions are 60.4℃ (for N-leaf) and 89.1℃ (for C-leaf), we selected four temperature gradients—45℃, 60℃ (near the N-leaf denaturation temperature), 75℃, and 90℃ (near the C-leaf denaturation temperature)—to treat LF. The particle size results and anthocyanin encapsulation efficiency of the prepared samples are shown in Table 4.

[0055] Table 4 Performance indicators of nanoparticles prepared from lactoferrin treated at different temperatures

[0056]

[0057] Different letters in the same column indicate significant differences (P<0.05).

[0058] Under the influence of the two denaturation temperatures of lactoferrin, the nanoparticle dispersion system exhibits the largest particle size (246.3±4.3 nm) near the first denaturation temperature of 60℃, followed by the second denaturation temperature of 90℃ (176.3±1.1 nm). At this temperature, the N and C lobes are unfolding, and the disordered and relatively relaxed protein conformation is not conducive to nanoparticle formation; therefore, the encapsulation efficiency of anthocyanins is significantly lower at both temperatures. The nanoparticle sample obtained at 45℃ has an excessively low absolute potential and a slightly high PDI, and is therefore not considered. At 75℃, a relatively stable and uniform nanoparticle dispersion system can be formed between the two denaturation temperatures, exhibiting higher encapsulation efficiency. Therefore, the final selected LF treatment temperature is 75℃.

[0059] Example 3: Characterization experiment of anthocyanin nanoparticles of the present invention

[0060] Anthocyanin nanoparticles encapsulated with lactoferrin and sodium carboxymethyl cellulose were prepared according to the optimized process. The infrared spectrum and microstructure of the final product were characterized to determine whether nanoparticles were formed.

[0061] Figure 3 Fourier transform infrared spectra of lactoferrin (LF), sodium carboxymethyl cellulose (CMC), anthocyanins (ACN), blank nanoparticles (CMC+LF), and nanoparticle samples (NPs) prepared according to this method. For lactoferrin, the amide A band (3500-3200 cm⁻¹) is shown. -1 The identification location is at 3302cm. -1 The amide I band (1625-1750 cm) -1 ), Amide II band (1475-1575cm) -1 ), Amide III band (1225-1425cm) -1 The peaks correspond to 1655, 1536, and 1391 cm⁻¹, respectively. -1 Compared to LF, the amide A band of the nanoparticle sample showed a significant red shift, while the amide I and amide III bands shifted, and the characteristic peak of the amide II band disappeared. This indicates that the amide bond conformation and intramolecular hydrogen bonds of LF changed after nanoparticle formation. When CMC forms a conjugate with LF, the OH stretching vibration of sodium carboxymethyl cellulose changes from 3426 cm⁻¹. -1 Transferred to 3414cm -1 This revealed the formation of hydrogen bonds between the two. Furthermore, at 1628 cm⁻¹... -1 The carboxyl absorption peak at 1438 cm⁻¹ and the absorption peak at 1438 cm⁻¹ -1 The buckling signal at the point was masked by the influence of the LF signal, which also confirms the presence of static electricity in the contact. Anthocyanins at 1283 cm⁻¹ -1 The typical absorption band of flavonoids at this location corresponds to the stretching of the pyran ring, at 1343 cm⁻¹. -1 The absorption band at these locations corresponds to the CO angle distortion of phenol. After anthocyanins were encapsulated in nanoparticles, these two characteristic peaks weakened or even disappeared, indicating that anthocyanins were successfully encapsulated by the nanoparticles and successfully embedded inside the particles.

[0062] The microstructure and dispersion state of nanoparticles are used Figure 4The SEM images are shown in the figure. Sodium carboxymethyl cellulose (CMC) has a chain-like structure, which becomes spongy after freeze-drying. Therefore, when the blank CMC+LF nanoparticle sample is observed under SEM, the fibrous structure of CMC+LF forms a complex three-dimensional electrostatic entanglement structure with lactoferrin, exhibiting a large microscopic size. When an excess of anthocyanin is added to the reaction system, it strongly interferes with the electrostatic complexation between CMC+LF and lactoferrin. This effect leads to the disintegration of the network structure of the LF+CMC complex, forming small, dense, and relatively dispersed nanoparticles. From the SEM images, the nanoparticles prepared according to this method have a near-spherical morphology, a smooth and uniform surface, and no visible pores or cracks, indicating that these particles have a certain degree of rigidity.

[0063] Example 4: Stability experiment of nanoparticles prepared in this invention

[0064] The particle stability of nanoparticles Figure 5 It indicates. In Figure 5 In (A), the average particle size of nanoparticles increased significantly under high temperature treatment at 90℃, but the particle size remained small within a certain range and remained dynamically stable during long-term heating. After continuous heating for 3 hours, the particle size of the sample recovered to some extent, indicating that the nanoparticle sample had good thermal stability under high temperature treatment at 90℃. Figure 5 In (B), the nanoparticles maintain a stable particle size within a 20-fold dilution. However, as the dilution factor increases to a certain extent, the increase in particle size is related to the weakening of inter-component interactions at low concentrations, resulting in a sharp increase in particle size. This indicates that the nanoparticles exhibit good dilution stability within a 20-fold dilution volume. Figure 5 As shown in (C), the nanoparticles maintained a relatively stable particle size and dispersion within the first 9 days of the experiment. From day 12 onwards, a significant increase in size was observed. Despite the increase in particle size, the nanoparticles still exhibited an excellent size of 218.8 nm on day 15, indicating good storage stability during the analysis.

[0065] Figure 6 The effects of pH, light, and temperature on anthocyanin retention were evaluated to understand the environmental limitations of nanoparticle applications. Figure 6 (A) indicates that pH has a significant impact on the stability of anthocyanins, and within a relatively wide pH range of 3-8, nanoparticles generally have a positive effect on the retention rate of anthocyanins. Figure 6(B) It can be seen that under light irradiation, the anthocyanin retention rate of nanoparticles and free anthocyanins decreases over time, but the anthocyanin retention rate of the nanoparticle sample is consistently higher than that of the free anthocyanin sample. Therefore, the formation of nanoparticles has a certain photoprotective effect on anthocyanins. Storage temperature has a significant impact on the stability of anthocyanins. Figure 6 (C) Figure 6 (D) shows the anthocyanin retention rates of the two samples stored at 4℃ and 25℃, respectively. After 20 days, at 4℃ and 25℃, the anthocyanin retention rates of the nanoparticles remained above 90.4% and 72.7%, respectively, reflecting that the stable electrostatic interaction between the core and wall materials in the composite structure can effectively protect anthocyanins. Overall, the anthocyanin retention rate of the nanoparticle sample was consistently higher than that of the free anthocyanin sample, indicating that the nanocarrier prepared by this method can indeed reduce the degradation of anthocyanins by environmental factors.

Claims

1. Anthocyanin nanoparticles, characterized in that: Anthocyanins were encapsulated with lactoferrin and sodium carboxymethyl cellulose to prepare nanoparticles, wherein the weight ratio of lactoferrin, sodium carboxymethyl cellulose, and anthocyanins was: Lactoferrin 12.8-19.2 parts, sodium carboxymethyl cellulose 2.56-3.84 parts, anthocyanins 12.8-19.2 parts; the preparation method includes the following steps: a. Dissolve anthocyanins in 0.2 M pH 3.0 citrate buffer solution, place on a magnetic stirrer and stir continuously at room temperature for 1 h. Store the resulting anthocyanin solution away from light. b. Prepare aqueous solutions of lactoferrin and sodium carboxymethyl cellulose respectively, and stir magnetically for 1 hour at 75℃ and 40℃ respectively, then adjust the pH to 5.0 with dilute hydrochloric acid. c. Take the sodium carboxymethyl cellulose dispersion solution into a capped brown bottle and stir it at a constant speed on a magnetic stirrer. Use a syringe to sample the anthocyanin solution from step a and add it drop by drop into the bottle. Stir for 30 minutes to ensure uniform mixing, and then adjust the pH value to 5.

0. d. Take lactoferrin solution, add it dropwise to the bottle, and stir continuously on a magnetic stirrer for 24 h to obtain a nanoparticle mixed solution; e. Filtration: The mixed solution from step d is ultrafiltered at 1000 × g for 20 min using an ultracentrifugal filter with a nominal molecular weight limit of 30 kDa to remove unencapsulated free anthocyanins; then filtered through a filter membrane with a pore size of 0.45 μm to remove impurities, thus obtaining anthocyanin nanoparticle dispersion. The degree of substitution (DS) of the sodium carboxymethyl cellulose is 0.

9.

2. The anthocyanin nanoparticles according to claim 1, characterized in that: The weight ratio of lactoferrin, sodium carboxymethyl cellulose, and anthocyanins is: Lactoferrin 16 parts, sodium carboxymethyl cellulose 3.2 parts, anthocyanins 16 parts.

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