A nanocomposite carrier, a preparation method and application thereof

Abstract

This invention provides a nanocomposite carrier, its preparation method, and its applications. The nanocomposite carrier is formed by the self-assembly of chitosan oligosaccharide-phenolic acid copolymer, blueberry anthocyanins, and tremella oligosaccharides. The preparation method includes: reacting chitosan oligosaccharides with phenolic acid compounds to obtain a copolymer; mixing the copolymer with blueberry anthocyanins under acidic conditions, adjusting the pH, adding an aqueous solution of tremella oligosaccharides, and then homogenizing and drying under high pressure to obtain the final product. This invention constructs a pH-responsive intestinal targeted delivery system through the triple synergistic effect of a covalent backbone, π-π stacking, and electrostatic network, and significantly improves the stability and antioxidant activity of anthocyanins. The preparation process of this invention is green and safe, and it is suitable for the fields of functional beverages and health foods.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, specifically to a nanocomposite carrier, its preparation method, and its application. Background Technology

[0002] Blueberry anthocyanins, a class of natural polyphenolic compounds, are widely found in blueberries. Due to their various bioactivities, including antioxidant, anti-inflammatory, anti-tumor, and vision-improving properties, they are widely used in functional foods, pharmaceuticals, and cosmetics. However, the anthocyanin molecule contains multiple unsaturated bonds and phenolic hydroxyl groups, exhibiting high chemical reactivity. During processing and storage, they are susceptible to degradation by factors such as light, temperature, pH changes, and oxygen, leading to color fading and decreased bioactivity. Therefore, improving the stability of blueberry anthocyanins has become a current research hotspot.

[0003] To address the aforementioned issues, existing technologies have explored various methods for stabilizing anthocyanins. For instance, CN110123826A discloses a method for preparing a blueberry anthocyanin-chitosan oligosaccharide complex. By cross-linking blueberry anthocyanins and chitosan oligosaccharides with glutaraldehyde to form a complex, the thermal stability and antioxidant activity of anthocyanins are improved, and certain antitumor functions are imparted. This method utilizes the positive charge and biocompatibility of chitosan oligosaccharides to achieve preliminary protection of anthocyanins. However, due to the use of glutaraldehyde in the cross-linking reaction, there are potential biosafety risks, and the complex structure is relatively loose, requiring further improvement in stability within complex food systems.

[0004] In addition, CN105919127B discloses a stable high anthocyanin blueberry lozenge and its preparation method. By encapsulating crude blueberry anthocyanin extract with carboxymethyl chitosan and chitosan hydrochloride, and then mixing it with excipients such as blueberry polysaccharide extract for granulation and tableting, the stability of anthocyanins in the preparation and storage of lozenges is significantly improved. This technology effectively delays the oxidative degradation of anthocyanins through physical encapsulation and the synergistic effect of excipients. However, this method is mainly applicable to solid lozenge products and is difficult to apply directly to liquid food systems such as beverages and oral liquids. Furthermore, it lacks controllability over the release behavior of anthocyanins.

[0005] In summary, while existing technologies have improved the stability of blueberry anthocyanins to some extent, they still have shortcomings in terms of structural controllability, application adaptability, and delivery efficiency. Therefore, developing an anthocyanin delivery system that combines high encapsulation efficiency, good stability, intestinal-targeted release capability, and applicability to various food systems remains a pressing technical challenge. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a nanocomposite carrier, its preparation method and application, to solve the technical problem that "existing carriers are difficult to simultaneously achieve gastric acid stability protection and intestinal targeted release of blueberry anthocyanins in the gastrointestinal environment".

[0007] To achieve the above objectives, the present invention is implemented using the following technical solution: In a first aspect, the present invention provides a nanocomposite carrier, which is formed by self-assembly of chitosan oligosaccharide-phenolic acid copolymer, blueberry anthocyanins and tremella oligosaccharide; the chitosan oligosaccharide-phenolic acid copolymer is prepared by reacting chitosan oligosaccharide with phenolic acid compounds.

[0008] Specifically, the chitosan oligosaccharide has a molecular weight of 1000-3000 Da and a degree of deacetylation ≥90%.

[0009] Specifically, the phenolic acid compound is at least one of chlorogenic acid, caffeic acid, rosmarinic acid, and salvianolic acid.

[0010] Specifically, the oligosaccharide from Tremella has a molecular weight of 10,000-30,000 Da and a glucuronic acid content of ≥10%.

[0011] Secondly, the present invention provides a method for preparing a nanocomposite carrier, comprising the following steps: (1) Chitosan oligosaccharide and phenolic acid compounds are mixed at a mass ratio of (1.2-1.6):1 and reacted in the presence of a condensing agent. After post-treatment, chitosan oligosaccharide-phenolic acid copolymer is obtained. (2) Prepare a first solution by mixing the chitosan oligosaccharide-phenolic acid copolymer obtained in the above steps with deionized water, prepare a second solution by mixing blueberry anthocyanins with deionized water, mix the second solution with the first solution, adjust the pH, and obtain a complex dispersion. (3) Add the aqueous solution of tremella oligosaccharide to the dispersion of the complex obtained in step (2), stir, and then perform high pressure homogenization and drying to obtain the nanocomposite carrier.

[0012] Preferably, the condensing agent in step (1) is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, with a mass ratio of (1.2 to 1.5):1.

[0013] Preferably, in step (2), the pH of the first solution is 3.4 to 3.6, the pH of the second solution is 2.3 to 2.6, and the pH is adjusted to 4.3 to 4.4 after mixing.

[0014] Preferably, the mass ratio of chitosan oligosaccharide-phenolic acid copolymer to blueberry anthocyanins in step (2) is (3-10):1.

[0015] Preferably, the mass ratio of the chitosan oligosaccharide-phenolic acid copolymer to the tremella oligosaccharide in step (3) is (1.2~1.8):1.

[0016] Thirdly, this invention provides an application of nanocomposite carriers in functional beverages and health foods.

[0017] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) This invention constructs a multi-level nanostructure with covalent backbone, π-π stacking anchoring and electrostatic network barrier by ternary self-assembly of chitosan oligosaccharide-phenolic acid copolymer, blueberry anthocyanin and tremella oligosaccharide. Compared with the single cross-linking or embedding method in the prior art, this invention achieves the dual functions of gastric anchoring protection and intestinal response release of blueberry anthocyanin. The cumulative release rate in simulated gastric juice is less than 10% in 2 hours, and the release is significantly accelerated after entering simulated intestinal juice, with a cumulative release rate of about 80% in 10 hours. This solves the technical problem that existing carriers are difficult to balance gastric stability and intestinal targeting.

[0018] (2) The nanocomposite carrier of the present invention significantly improves the encapsulation rate and activity retention of blueberry anthocyanins; tests show that its encapsulation rate of blueberry anthocyanins can reach more than 88%, which is much higher than that of the comparative sample lacking any core component. Moreover, this nanocomposite carrier exhibits excellent thermal stability, light stability and storage stability, effectively extending the shelf life of anthocyanins.

[0019] (3) This invention abandons toxic crosslinking agents such as glutaraldehyde and uses naturally sourced chitosan oligosaccharide, phenolic acid and tremella oligosaccharide to construct nanocarriers through self-assembly. The preparation process is green and safe and is suitable for functional foods and beverages. Attached Figure Description

[0020] Figure 1 The graph shows the comparison results of the encapsulation efficiency of blueberry anthocyanins by the nanocomposite carriers of Examples 1-3 and Comparative Examples 1-3 of the present invention.

[0021] Figure 2 The graph shows the comparison results of the DPPH free radical scavenging rate of the nanocomposite carriers in Examples 1-3 and Comparative Examples 1-3 of the present invention. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention provides a nanocomposite carrier and its preparation method. By assembling chitosan oligosaccharide, chlorogenic acid, and tremella oligosaccharide in a multi-level structure, a polysaccharide-polyphenol composite nanocarrier with pH responsiveness and intestinal-targeted delivery function is constructed. This technical solution is based on the following mechanism: First, chitosan oligosaccharide, as a positively charged backbone, covalently binds with chlorogenic acid through an amide reaction to form a copolymer, endowing it with an aromatic ring structure to provide π-π stacking sites; second, under weakly acidic conditions, blueberry anthocyanins exist in a stable flavonoid cation form and are efficiently anchored to the copolymer through π-π stacking and hydrogen bonding; finally, tremella oligosaccharide forms a negatively charged network under pH regulation, constructing a dense nanostructure through electrostatic assembly and physical encapsulation, achieving multiple protections for anthocyanins. In this multi-level structure, covalent bonds provide the basic framework and maintain structural integrity; electrostatic interactions drive self-assembly and endow the system with pH responsiveness. Tremella oligosaccharides endow the carrier with typical pH responsive characteristics. In the highly acidic environment of gastric juice, the carboxyl groups of Tremella oligosaccharides undergo carboxyl protonation, and the network structure relatively shrinks, which can effectively delay gastric juice penetration and protect anthocyanins from degradation. After entering the near-neutral environment of the intestine, the deprotonation of carboxyl groups causes the network to swell, allowing the anthocyanins to be released slowly, ensuring that they can reach the intestine, the optimal absorption site, intact, and achieve targeted intestinal delivery.

[0024] A method for preparing a nanocomposite carrier includes the following steps: (1) Weigh chitosan oligosaccharide and chlorogenic acid, dissolve them in MES buffer, and stir to obtain a mixed solution; dissolve 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in ethanol to obtain a condensing agent, add the condensing agent to the above mixed solution, stir at 25~30℃ in the dark for 4~6 hours, after the reaction is completed, put the reaction solution into a dialysis bag (molecular weight cutoff 1500Da), dialyze in deionized water for 36~48 hours, change the water every 6 hours, freeze dry the dialysate to obtain chitosan oligosaccharide-chlorogenic acid copolymer powder for later use; in the presence of EDC / NHS, the carboxyl group of chlorogenic acid and the amino group of chitosan oligosaccharide undergo amidation reaction to form a stable amide bond, generating chitosan oligosaccharide-chlorogenic acid copolymer, the covalent connection ensures the connection stability of chlorogenic acid and chitosan oligosaccharide, and at the same time the ortho-dihydroxy group retained on the chlorogenic acid molecule provides an active site for subsequent interaction.

[0025] (2) Dissolve chitosan oligosaccharide-chlorogenic acid copolymer powder in deionized water, and adjust the pH to 3.4-3.6 with 1M citric acid solution to obtain the first solution; weigh blueberry anthocyanin powder, dissolve it in deionized water, and adjust the pH to 2.3-2.6 with 1M citric acid aqueous solution to obtain the second solution; under stirring at 600r / min, pour the second solution into the first solution at once, continue stirring in the dark for 20-40 minutes, and slowly add 10% sodium citrate aqueous solution at a dropping rate of 1mL. The pH of the system was adjusted to 4.3-4.4 at a constant speed of 1000 min to obtain a dispersion of the complex. Under pH 3-4 conditions, blueberry anthocyanins bind to the copolymer through the following mechanisms: π-π stacking is formed between the benzopyran ring of anthocyanins and the caffeoyl aromatic ring of chlorogenic acid; the phenolic hydroxyl groups of anthocyanins form an extensive hydrogen bond network with the hydroxyl groups of chitosan oligosaccharides and the phenolic hydroxyl groups of chlorogenic acid; the local hydrophobic microenvironment formed after encapsulation further stabilizes the anthocyanins. The triple non-covalent interaction enables the anthocyanins to achieve efficient loading without destroying the natural structure.

[0026] (3) Weigh the tremella oligosaccharide powder and dissolve it in deionized water. Adjust the pH to 4.3-4.4 with 1M citric acid aqueous solution to obtain the third solution. Add the third solution dropwise to the composite dispersion obtained in step (2) at a rate of 2 mL / min under stirring. After the addition is complete, continue stirring for 30-40 minutes to obtain the nanocarrier dispersion. Pass the obtained dispersion through a high-pressure homogenizer and circulate it 2-3 times under a pressure of 30-35 MPa. Add 1-2% mannitol by mass and freeze dry to obtain the nanocomposite carrier. After the negatively charged tremella oligosaccharide is added, it will be electrostatically attracted to the positively charged chitosan oligosaccharide-chlorogenic acid copolymer. Tremella oligosaccharide acts as an anionic crosslinking agent to connect multiple copolymer chains to form a nanoscale polyelectrolyte complex. With electrostatic interaction as the main driving force, combined with the synergistic regulation of hydrogen bonding and steric repulsion, the system self-assembles into spherical nanoparticles with uniform particle size under appropriate mass ratio to obtain a structurally stable nanocomposite carrier.

[0027] In this invention, all raw materials used are commercially available. The molecular weight of the tremella oligosaccharide is 10,000-30,000 Da, and the glucuronic acid content is ≥10%; the molecular weight of the chitosan oligosaccharide is 1,000-3,000 Da, and the degree of deacetylation is ≥90%.

[0028] In this invention, the MES buffer solution is a 0.1M, pH 5.5 aqueous solution of MES.

[0029] In this invention, the pH of the first solution in step (2) is in the range of 3.4 to 3.6, which allows the chitosan oligosaccharide-chlorogenic acid copolymer to maintain highly protonated amino groups to carry a positive charge, while simultaneously exposing the aromatic ring of chlorogenic acid. Adjusting the second solution to pH 2.3 to 2.6 allows blueberry anthocyanins to exist in the most stable flavonoid cation form, maintaining their planar conjugated structure to possess the strongest π-π stacking ability. When the second solution is rapidly added to the first solution, the pH of the mixed system quickly rises to approximately 3.1 to 3.3. When the anthocyanins exist in their most stable flavonoid cation form, they are immediately... By anchoring the pH to avoid structural transformation and activity loss due to pH elevation, the pH was slowly adjusted to 4.3-4.4. At this pH, the carboxyl groups of Tremella oligosaccharides are activated, making them negatively charged, which provides a key driving force for subsequent electrostatic self-assembly. The negatively charged Tremella oligosaccharides interact electrostatically with the positively charged chitosan oligosaccharide-phenolic acid copolymer, and self-assemble into a dense nanonetwork, which physically encapsulates the blueberry anthocyanins that have been anchored by π-π stacking and hydrogen bonding, thus completing the construction of the final carrier. In addition, this pH value is also within the suitable acidity range for functional beverages, which is conducive to the direct application of the product.

[0030] In this invention, the chlorogenic acid can be replaced with other phenolic acid compounds having an unsubstituted or less substituted aromatic ring structure, such as caffeic acid, rosmarinic acid, and salvianolic acid. These compounds all contain benzene ring structures that can undergo π-π stacking with the aromatic ring of blueberry anthocyanins, thus achieving a similar anchoring effect.

[0031] In this invention, the reagents used to adjust the pH value are a 1M aqueous solution of citric acid and a 10% sodium citrate aqueous solution.

[0032] In this invention, mannitol is added as a freeze-drying protectant to prevent nanoparticles from agglomerating during the drying process. Mannitol is not an essential structural component of the nanocarrier, and in subsequent applications, the undried nanocarrier dispersion can also be used directly.

[0033] Preferably, the mass ratio of chitosan oligosaccharide to chlorogenic acid in step (1) is (1.2~1.6):1, and more preferably 1.4:1.

[0034] Preferably, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide in the condensing agent in step (1) is (1.2~1.5):1.

[0035] Preferably, the mass ratio of chitosan oligosaccharide-chlorogenic acid copolymer powder to blueberry anthocyanin powder in step (2) is (3~10):1, more preferably 5:1. The inventors have found through experiments that the mass ratio of chitosan oligosaccharide-chlorogenic acid copolymer powder to blueberry anthocyanin powder has an important influence on the encapsulation efficiency and stability of the nanocomposite carrier. When the mass ratio is lower than 3:1, the π-π stacking sites provided by the copolymer tend to be saturated, and some anthocyanins are difficult to be effectively anchored, resulting in a decrease in encapsulation efficiency. When the mass ratio is higher than 10:1, although it can be encapsulated, the anthocyanin loading per unit mass of carrier decreases, and the economy decreases. The 5:1 mass ratio used in Example 2 achieves the optimal loading efficiency while maintaining a high encapsulation efficiency, which is the preferred ratio of the present invention.

[0036] Preferably, the mass ratio of the chitosan oligosaccharide-chlorogenic acid copolymer powder to the tremella oligosaccharide powder is (1.2~1.8):1, more preferably 1.33:1. The inventors have found through experiments that the mass ratio of the chitosan oligosaccharide-chlorogenic acid copolymer powder to the tremella oligosaccharide powder has a significant impact on the particle size, encapsulation efficiency, and stability of the nanocomposite carrier. When the mass ratio is higher than 1.8:1, the negatively charged network is not dense enough, the nanoparticle size is too large, and the encapsulation efficiency decreases slightly. When the mass ratio is lower than 1.2:1, although a complete network can be formed, the loading of effective components per unit mass of carrier is reduced, and the anthocyanin release performance is affected due to the excessively dense network. The 1.33:1 mass ratio used in Example 2 results in the smallest nanoparticle size and the highest encapsulation efficiency, which is the preferred ratio of the present invention.

[0037] Example 1; (1) Weigh 2.0g of chitosan oligosaccharide (molecular weight 2000Da) and 1.25g of chlorogenic acid, and dissolve them in 200mL of MES buffer. Stir magnetically to obtain a mixed solution. Weigh 1.2g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.9g of N-hydroxysuccinimide and dissolve them in 5mL of ethanol to obtain a condensing agent. Add the condensing agent to the above mixed solution and stir at 25°C in the dark for 4 hours. After the reaction is completed, put the reaction solution into a dialysis bag (molecular weight cutoff 1500Da) and dialyze it in deionized water for 36 hours. Change the water every 6 hours. Freeze-dry the dialysate to obtain chitosan oligosaccharide-chlorogenic acid copolymer powder for later use. (2) Dissolve 1g of chitosan oligosaccharide-chlorogenic acid copolymer powder in 200mL of deionized water, adjust the pH to 3.4, and prepare the first solution; weigh 0.1g of blueberry anthocyanin powder and dissolve it in 50mL of deionized water, adjust the pH to 2.4, and prepare the second solution; pour the second solution into the first solution at 600r / min under stirring, continue stirring in the dark for 20 minutes, adjust the pH of the system to 4.3, and obtain the complex dispersion; (3) Weigh 0.45g of Tremella oligosaccharide powder (molecular weight 15000Da) and dissolve it in 150mL of deionized water. Adjust the pH to 4.4 to obtain the third solution. Add the third solution dropwise to the composite dispersion obtained in step (2) at a rate of 2mL / min under stirring. After the addition is complete, continue stirring for 30 minutes to obtain the nanocarrier dispersion. Pass the obtained dispersion through a high-pressure homogenizer and circulate it twice under a pressure of 30MPa. Add 2% mannitol by mass and freeze dry to obtain the nanocomposite carrier.

[0038] Example 2; (1) Weigh 2.0g of chitosan oligosaccharide (molecular weight 2000Da) and 1.43g of chlorogenic acid, and dissolve them in 200mL of MES buffer. Stir magnetically to obtain a mixed solution. Weigh 1.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.1g of N-hydroxysuccinimide and dissolve them in 5mL of ethanol to obtain a condensing agent. Add the condensing agent to the above mixed solution and stir at 28°C in the dark for 5 hours. After the reaction is completed, put the reaction solution into a dialysis bag (molecular weight cutoff 1500Da) and dialyze it in deionized water for 40 hours, changing the water every 6 hours. The dialysate is freeze-dried to obtain chitosan oligosaccharide-chlorogenic acid copolymer powder for later use. (2) Dissolve 1g of chitosan oligosaccharide-chlorogenic acid copolymer powder in 200mL of deionized water and adjust the pH to 3.5 to obtain the first solution; weigh 0.2g of blueberry anthocyanin powder and dissolve it in 50mL of deionized water and adjust the pH to 2.5 to obtain the second solution; pour the second solution into the first solution at 600r / min and continue stirring in the dark for 30 minutes to adjust the pH of the system to 4.3 to obtain the complex dispersion; (3) Weigh 0.75g of Tremella oligosaccharide powder (molecular weight 15000Da) and dissolve it in 150mL of deionized water. Adjust the pH to 4.3 to obtain the third solution. Add the third solution dropwise to the composite dispersion obtained in step (2) at a rate of 2mL / min under stirring. After the addition is complete, continue stirring for 30 minutes to obtain the nanocarrier dispersion. Pass the obtained dispersion through a high-pressure homogenizer and circulate it 3 times under a pressure of 32MPa. Add 1.5% mannitol by mass and freeze dry to obtain the nanocomposite carrier.

[0039] Example 3; (1) Weigh 2.0g of chitosan oligosaccharide (molecular weight 2000Da) and 1.43g of chlorogenic acid, and dissolve them in 200mL of MES buffer. Stir magnetically to obtain a mixed solution. Weigh 1.5g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.1g of N-hydroxysuccinimide and dissolve them in 5mL of ethanol to obtain a condensing agent. Add the condensing agent to the above mixed solution and stir at 28°C in the dark for 6 hours. After the reaction is completed, put the reaction solution into a dialysis bag (molecular weight cutoff 1500Da) and dialyze it in deionized water for 48 hours, changing the water every 6 hours. The dialysate is freeze-dried to obtain chitosan oligosaccharide-chlorogenic acid copolymer powder for later use. (2) Dissolve 1g of chitosan oligosaccharide-chlorogenic acid copolymer powder in 200mL of deionized water, adjust the pH to 3.6, and prepare the first solution; weigh 0.4g of blueberry anthocyanin powder and dissolve it in 50mL of deionized water, adjust the pH to 2.6, and prepare the second solution; pour the second solution into the first solution at 600r / min, continue stirring in the dark for 40 minutes, adjust the pH of the system to 4.4, and obtain the complex dispersion; (3) Weigh 1.2g of Tremella oligosaccharide powder (molecular weight 15000Da) and dissolve it in 150mL of deionized water. Adjust the pH to 4.4 to obtain the third solution. Add the third solution dropwise to the composite dispersion obtained in step (2) at a rate of 2mL / min under stirring. After the addition is complete, continue stirring for 40 minutes to obtain the nanocarrier dispersion. Pass the obtained dispersion through a high-pressure homogenizer and circulate it 3 times under a pressure of 35MPa. Add 2% mannitol by mass and freeze dry to obtain the nanocomposite carrier.

[0040] Comparative Example 1; The difference between Comparative Example 1 and Example 2 is that step (1) is omitted, and step (2) is changed to: take 1g of chitosan oligosaccharide and dissolve it in 200mL of deionized water, adjust the pH to 3.5, and prepare the first solution; weigh 0.2g of blueberry anthocyanin powder and dissolve it in 50mL of deionized water, adjust the pH to 2.5, and prepare the second solution; under stirring at 600r / min, pour the second solution into the first solution at once, continue stirring in the dark for 30 minutes, adjust the pH of the system to 4.3, and obtain the complex dispersion; the remaining steps are the same as in Example 2.

[0041] Comparative Example 2; The difference between Comparative Example 2 and Example 2 is that step (3) is omitted, and step (2) is changed to: take 1g of chitosan oligosaccharide-chlorogenic acid copolymer powder and dissolve it in 200mL of deionized water, adjust the pH to 3.5, and prepare the first solution; weigh 0.2g of blueberry anthocyanin powder and dissolve it in 50mL of deionized water, adjust the pH to 2.5, and prepare the second solution; under stirring at 600r / min, pour the second solution into the first solution at once, continue stirring in the dark for 30 minutes, adjust the pH of the system to 4.3, and obtain the nanocarrier dispersion. The obtained dispersion is passed through a high-pressure homogenizer and circulated 3 times under a pressure of 32MPa. After adding 1.5% mannitol by mass, it is freeze-dried to obtain the nanocomposite carrier.

[0042] Comparative Example 3; (1) Weigh 0.2g of blueberry anthocyanin powder and dissolve it in 50mL of deionized water. Adjust the pH to 2.5 with 1M citric acid aqueous solution to obtain the first solution. (3) Weigh 0.75g of Tremella oligosaccharide powder (molecular weight 15000Da) and dissolve it in 150mL of deionized water. Adjust the pH to 4.3 to obtain a second solution. Add the second solution dropwise to the first solution at a rate of 2mL / min while stirring. After the addition is complete, continue stirring for 30 minutes to obtain a nano-carrier dispersion. Pass the obtained dispersion through a high-pressure homogenizer and circulate it 3 times under a pressure of 32MPa. Add 1.5% mannitol by mass and freeze dry to obtain a nano-composite carrier.

[0043] Test Results Particle size and Zeta potential determination: 10 mg of the nanocomposite carrier powder prepared in Example 2 was dissolved in 10 mL of deionized water, mixed well, and 1.5 mL was injected into the sample cell. The particle size and Zeta potential were measured using a dynamic light scattering particle size analyzer under the following conditions: 25 °C and equilibration time of 120 seconds. The results showed that the average particle size of the nanocomposite carrier of this invention was 150-180 nm, the polydispersity index (PDI) was <0.2, and the Zeta potential was -25 to -35 mV, indicating that the prepared nanoparticles had uniform particle size, narrow distribution, and stable system.

[0044] Test Example 1; Encapsulation Efficiency Determination Test method: Take 20 mg of the nanocomposite carrier powder prepared in each example and comparative example, add 20 mL of deionized water to redissolve it, and obtain a uniform dispersion; take 2 mL of the above dispersion, dilute it with deionized water to 5 mL, shake well, and then determine the concentration using the pH differential method. The concentration is recorded as C. 测总 The total anthocyanin concentration in the original solution is C. 总 =C 测总×2.5; Take 2 mL of the above dispersion and place it in an ultrafiltration centrifuge tube (molecular cutoff 10 kDa). Centrifuge at 8000 rpm for 20 minutes to separate free anthocyanins. Collect the filtrate and determine the concentration C of free anthocyanins using the pH differential method. 游离 Encapsulation ratio = (C 总 -C 游离 ) / C 总 ×100%, specific results are as follows Figure 1 As shown.

[0045] Depend on Figure 1 It can be seen that the encapsulation rates of Examples 1-3 are all between 87-89%, indicating that the preparation process of the present invention is stable and has good reproducibility. Among them, the encapsulation rate of Example 2 reaches 88.9%, which is significantly higher than that of the comparative examples, proving that the complete system of the present invention has a high loading capacity for blueberry anthocyanins.

[0046] The encapsulation efficiency of Comparative Example 1 (without chlorogenic acid) decreased to 62.4%, indicating that the π-π stacking effect provided by chlorogenic acid is the key to the efficient anchoring of anthocyanins. The absence of this component makes it difficult for some anthocyanins to be effectively bound, resulting in a significant decrease in the encapsulation efficiency.

[0047] The encapsulation efficiency of Comparative Example 2 (without Tremella oligosaccharides) was only 28.3%, indicating that the negatively charged network formed by Tremella oligosaccharides is crucial for maintaining the stability of the nanoparticle structure. Without this component, it would be difficult to form a dense encapsulation structure. The encapsulation efficiency of Comparative Example 3 (without chitosan oligosaccharide and chlorogenic acid) was only 15.7%, indicating that it is difficult to achieve effective encapsulation by relying solely on the weak hydrogen bonding between Tremella oligosaccharide and anthocyanins. Chitosan oligosaccharide, as a positively charged framework, is the basis for electrostatic assembly.

[0048] Test Example 2; Intestinal Targeted Delivery Test First, two release media were prepared: simulated gastric juice (SGF) and simulated intestinal juice (SIF).

[0049] Simulated gastric juice (SGF): Take 2.0g of sodium chloride and 3.2g of pepsin, add 7.0mL of dilute hydrochloric acid, dissolve in water and bring the volume to 1000mL, and adjust the pH to 1.2±0.1.

[0050] Simulated intestinal fluid (SIF): Dissolve 6.8g of potassium dihydrogen phosphate in 500mL of water and adjust the pH to 6.8 with 0.1mol / L sodium hydroxide solution; dissolve 10.0g of pancreatic enzyme in 100mL of water, mix the two solutions and add water to make up to 1000mL.

[0051] Test method: Take 100 mg of each of the nanocomposite carrier powder prepared in each example and comparative example, add 50 mL of deionized water to redissolve and obtain a uniform dispersion. Take 5 mL of this dispersion and put it into a dialysis bag (molecular cutoff 10 kDa), place it in 100 mL of simulated gastric juice, and shake at 37℃ and 100 r / min. Take samples at 0.5 h, 1 h and 2 h to determine the anthocyanin concentration. After sampling, add an equal volume of fresh preheated simulated gastric juice and calculate the cumulative release rate. Each experiment is repeated 3 times and the average value is taken.

[0052] Two hours later, the dialysis bag was removed and transferred to 100 mL of simulated intestinal fluid, and the mixture was kept at 37°C and 100 rpm with constant temperature shaking. Samples were taken at 1, 4, and 8 hours, and anthocyanin concentrations were measured after treatment using the same method. The cumulative release rate was calculated. Each experiment was repeated three times, and the average value was taken. The specific results are shown in Table 1.

[0053] The cumulative release rate is calculated as follows: ; Among them, C n V: Anthocyanin concentration in the release medium at the nth sampling time (μg / mL); V: Total volume of the release medium (100mL); s : Sample volume per time (5 mL); C i m0: Drug concentration in the release medium during the i-th sampling (μg / mL); m0: Initial total anthocyanin content in the dialysis bag (μg).

[0054] Table 1

[0055] As shown in Table 1, the cumulative release rate of Examples 1-3 in simulated gastric fluid was no more than 10% after 2 hours. After entering simulated intestinal fluid, the release rate was significantly accelerated, with a cumulative release rate of about 80% after 10 hours. This demonstrates excellent gastric protection effect and typical pH-responsive intestinal targeted release characteristics, indicating that the complete system of the present invention can effectively protect anthocyanins from passing through the stomach and release them slowly in the intestine, achieving intestinal targeted delivery.

[0056] Comparative Example 1 (without chlorogenic acid) showed a cumulative release rate of 42.5% in simulated gastric fluid over 2 hours, which was significantly higher than that of Example 2. This indicates that the π-π stacking effect provided by chlorogenic acid is the key to the effective anchoring of anthocyanins in the stomach and the prevention of premature leakage. The absence of this component leads to a large loss of anthocyanins in the stomach.

[0057] Comparative Example 2 (without Tremella oligosaccharide) showed a cumulative release rate of up to 63.8% in simulated gastric juice after 2 hours, further demonstrating that the negatively charged network formed by Tremella oligosaccharide can construct a dense nanostructure and protect anthocyanins from gastric acid degradation. The absence of this component leads to a loose nanoparticle structure, making it difficult to effectively block gastric juice penetration.

[0058] Comparative Example 3 (without chitosan oligosaccharide and chlorogenic acid) had a cumulative release rate of 71.5% in simulated gastric juice after 2 hours, which was close to the free state. This indicates that the protection provided by the weak hydrogen bonding between tremella oligosaccharide and anthocyanins is limited, and chitosan oligosaccharide, as a positively charged framework, is the basis for electrostatic assembly and nanoparticle formation.

[0059] The above results indicate that the positively charged backbone provided by chitosan oligosaccharide, the π-π stacking sites provided by chlorogenic acid, and the negatively charged network formed by Tremella oligosaccharide in the complete system of this invention work synergistically and are indispensable. Together, they achieve gastric protection and intestinal targeted delivery of blueberry anthocyanins, significantly improving their oral bioavailability.

[0060] Test Example 3; Free Radical Scavenging Test Test method: Take 100 mg of each of the nanocomposite carrier powder prepared in each example and comparative example, add 50 mL of deionized water to redissolve and obtain a uniform dispersion. Release the above dispersion in simulated intestinal fluid according to the intestinal targeted delivery test method, and take the 10-hour release liquid as the test sample.

[0061] Blank control: Mix 2 mL of DPPH ethanol solution (0.1 mM) with 1 mL of deionized water as a blank control, and measure the absorbance A. 空白 .

[0062] Background control: Mix 2 mL of DPPH ethanol solution (0.1 mM) with 1 mL of blank simulated intestinal fluid (without anthocyanins) as a background control, and measure the absorbance A. 背景 , used to subtract the background absorbance of the simulated intestinal fluid itself.

[0063] Sample determination: Take 2 mL of DPPH ethanol solution (0.1 mM), add 1 mL of the sample to be tested, mix well, and react in the dark for 30 minutes. Measure the absorbance A at 517 nm. 样品 Calculate the clearance rate, clearance rate = A 空白 -(A 样品 -A 背景 ) / A 空白 ×100%, see details in the results. Figure 2 .

[0064] Depend on Figure 2 It can be seen that the DPPH free radical scavenging rate of Example 2 is the highest, indicating that the anthocyanins released by the complete system of the present invention still retain excellent antioxidant activity after achieving intestinal targeted delivery.

[0065] Comparative Example 1 shows that the absence of chlorogenic acid affected the encapsulation efficiency and impaired anthocyanin activity during delivery.

[0066] Comparative Example 2 shows that the network structure formed by Tremella oligosaccharides is very important for protecting anthocyanin activity.

[0067] Comparative Example 3 shows that relying solely on the hydrogen bonding between Tremella oligosaccharides and anthocyanins is insufficient to form an effective protective structure, and its protective effect on anthocyanins is significantly weaker than that of the complete system of this invention.

[0068] The above results indicate that the synergistic protection mechanism formed by chitosan oligosaccharide, chlorogenic acid, and tremella oligosaccharide not only achieves efficient loading and targeted delivery of anthocyanins, but also effectively maintains their activity during the delivery process.

[0069] Test Example 4; Stability Test Thermal stability: 100 mg of each of the nanocomposite carrier powders prepared in Example 2 and Comparative Examples 1-3 were taken and reconstituted in 100 mL of deionized water to obtain a uniform dispersion. 10 mL of each dispersion was placed in a 15 mL centrifuge tube and heated in an 80 °C water bath for 30 minutes. 1 mL of the anthocyanin concentration was measured before and after heating. The anthocyanin retention rate L1 was calculated as follows: Retention rate (%) = Anthocyanin concentration after heating / Anthocyanin concentration before heating × 100%. The specific results are shown in Table 2.

[0070] Light stability: Take 50 mg of each of the nanocomposite carrier powder prepared in Example 2 and Comparative Examples 1-3, add 50 mL of deionized water to redissolve and obtain a uniform dispersion. Place them in transparent glass bottles and irradiate them under natural light for 7 days. Take 1 mL before and after irradiation to determine the anthocyanin concentration and calculate the anthocyanin retention rate L2. The specific results are shown in Table 2.

[0071] Storage stability: Take 50 mg of each of the nanocomposite carrier powder prepared in Example 2 and Comparative Examples 1-3, add 50 mL of deionized water to redissolve and obtain a uniform dispersion, put it into a brown glass bottle, seal it and store it at 4°C in the dark. Samples were taken at 3, 6 and 8 months to determine the anthocyanin concentration and calculate the anthocyanin retention rate L3. The specific results are shown in Table 2.

[0072] Table 2

[0073] As shown in Table 2, the anthocyanin retention rate of Example 2 after heating at 80°C for 30 minutes was 92.5%, which was significantly higher than that of the other comparative examples, indicating that the complete system of the present invention has the best thermal protection effect on anthocyanins.

[0074] Example 2 showed that the anthocyanin retention rate was better than that of the control groups after 7 days of natural light irradiation, further demonstrating the stabilizing effect of the complete system.

[0075] In Example 2, the anthocyanin retention rates after storage at 4°C in the dark for 3, 6, and 8 months were 91.2%, 88.5%, and 85.3%, respectively. In contrast, the retention rates of Comparative Examples 1-3 all decreased significantly with prolonged storage time, indicating that the complete system of the present invention can effectively extend the shelf life of anthocyanins.

[0076] Comparative Example 1 shows that the π-π stacking effect provided by chlorogenic acid not only helps with encapsulation, but also enhances the stability of anthocyanins under heat, light and storage conditions.

[0077] The stability of Comparative Example 2 further decreased, especially its stability under light and storage conditions, indicating that the negatively charged network formed by the oligosaccharides from Tremella fuciformis provides a physical barrier to protect the anthocyanins.

[0078] Comparative Example 3 showed the worst stability, approaching a free state, indicating that the weak hydrogen bonding between Tremella oligosaccharides and anthocyanins alone is insufficient to provide effective protection.

[0079] The above results demonstrate that the present invention improves the stability of anthocyanins under heat, light, and storage conditions through the synergistic effects of covalent bonds, π-π stacking, and electrostatic networks, from three levels: molecular anchoring, physical barriers, and structural framework.

[0080] Application Example 1: Functional Beverages The ingredients in the formula are added in the following order, based on a total weight of 15,000 parts: 50 parts of Tremella oligosaccharide-blueberry anthocyanin nanocomposite carrier powder, 400 parts of fructose, 8 parts of blueberry flavoring, 0.5 parts of vitamin B6, and the remainder of purified water, to obtain a functional beverage containing Tremella oligosaccharide-blueberry anthocyanin nanocomposite.

[0081] Application Example 2; Health Food The formula, by weight, consists of the following components: 200 parts of Tremella oligosaccharide-blueberry anthocyanin nanocomposite carrier powder, 5 parts of lutein, 1 part of zeaxanthin, 50 parts of taurine, and 50 parts of vitamin C.

[0082] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. A nanocomposite carrier, characterized in that, The nanocomposite carrier is formed by the self-assembly of chitosan oligosaccharide-phenolic acid copolymer, blueberry anthocyanins and tremella oligosaccharide; the chitosan oligosaccharide-phenolic acid copolymer is prepared by reacting chitosan oligosaccharide with phenolic acid compounds.

2. The nanocomposite carrier according to claim 1, characterized in that, The chitosan oligosaccharide has a molecular weight of 1000-3000 Da and a degree of deacetylation ≥90%.

3. The nanocomposite carrier according to claim 1, characterized in that, The phenolic acid compound is at least one of chlorogenic acid, caffeic acid, rosmarinic acid, and salvianolic acid.

4. The nanocomposite carrier according to claim 1, characterized in that, The oligosaccharide from Tremella has a molecular weight of 10,000-30,000 Da and a glucuronic acid content of ≥10%.

5. A method for preparing a nanocomposite carrier, characterized in that, Includes the following steps: (1) Chitosan oligosaccharide and phenolic acid compounds are mixed at a mass ratio of (1.2-1.6):1 and reacted in the presence of a condensing agent. After post-treatment, chitosan oligosaccharide-phenolic acid copolymer is obtained. (2) Prepare a first solution by mixing the chitosan oligosaccharide-phenolic acid copolymer obtained in the above steps with deionized water, prepare a second solution by mixing blueberry anthocyanins with deionized water, mix the second solution with the first solution, adjust the pH, and obtain a complex dispersion. (3) Add the aqueous solution of tremella oligosaccharide to the dispersion of the complex obtained in step (2), stir, and then perform high pressure homogenization and drying to obtain the nanocomposite carrier.

6. The preparation method according to claim 5, characterized in that, The condensing agent mentioned in step (1) is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, with a mass ratio of (1.2 to 1.5):

1.

7. The preparation method according to claim 5, characterized in that, In step (2), the pH of the first solution is 3.4 to 3.6, the pH of the second solution is 2.3 to 2.6, and the pH is adjusted to 4.3 to 4.4 after mixing.

8. The preparation method according to claim 5, characterized in that, The mass ratio of chitosan oligosaccharide-phenolic acid copolymer to blueberry anthocyanins in step (2) is (3-10):

1.

9. The preparation method according to claim 5, characterized in that, The mass ratio of chitosan oligosaccharide-phenolic acid copolymer to tremella oligosaccharide in step (3) is (1.2-1.8):

1.

10. The application of a nanocomposite carrier as described in any one of claims 1-4 in functional beverages and health foods.

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