A sol of astaxanthin-loaded chitosan-cellulose composite nanoparticles and a method for preparing the same

Abstract

The application discloses a preparation method of astaxanthin-loaded chitosan-cellulose composite nanoparticle sol, which comprises the following steps: 1) astaxanthin acetone solution is added dropwise into cellulose nanofiber solution, and stirring reaction is carried out for 0.5-2 hours, then water-soluble chitosan solution is added, and stirring reaction is carried out for 0.5-2 hours, so as to obtain chitosan-astaxanthin-cellulose solution; 2) the chitosan-astaxanthin-cellulose solution obtained in the step 1) is subjected to rotary evaporation to remove acetone, and centrifugal separation is carried out at 4-25 DEG C to remove insoluble substances, and the upper clear liquid is taken, so as to obtain astaxanthin-loaded chitosan-cellulose composite nanoparticle sol. The astaxanthin-loaded chitosan-cellulose composite nanoparticle sol has the characteristics of small particle size and high zeta potential, and can endow astaxanthin with high environmental stability; moreover, the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol is bright orange, can improve the water solubility of astaxanthin, and has good intestinal delivery capacity of astaxanthin.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of astaxanthin-loaded chitosan-cellulose composite nanoparticle sol and its preparation method. BACKGROUND

[0002] Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β,β'-carotene) is a liposoluble ketone carotenoid with 11 conjugated double bonds and polar / non-polar structure. Its special structure enables astaxanthin to scavenge free radicals inside and outside the cell membrane, endowing astaxanthin with a series of biological activities such as antioxidant and anti-inflammatory, and can be used for the prevention and treatment of oxidative stress-related diseases. It is a promising synthetic antioxidant substitute. However, poor water solubility and stability result in low bioavailability of astaxanthin, which limits its application in functional foods and pharmaceutical industries. At present, various micro / nano carriers such as emulsions, liposomes, solid lipid nanoparticles, nanostructured lipid carriers, polymer nanoparticles, and gels have been constructed for the protection and delivery of astaxanthin.

[0003] Among various delivery systems, complex coacervates have the advantages of simple and mild processing conditions, high physicochemical stability, high entrapment efficiency, and controlled release of bioactive substances. In contrast, lipid-based carriers such as emulsions, liposomes, solid lipid nanoparticles, and nanostructured lipid carriers require large amounts of surfactants and complex preparation processes to achieve the desired effects, which can greatly enhance gastrointestinal irritation and toxicity. Complex coacervation is mainly driven by electrostatic interactions between oppositely charged polyelectrolytes in solution, and complex coacervates loaded with food ingredients are usually prepared using proteins and anionic polysaccharides. Therefore, polysaccharide-based complex coacervates containing a large number of reactive functional groups have attracted attention in the field of biomaterials.

[0004] Cellulose and chitin are the first and second most abundant polysaccharides in nature, and their properties can be enhanced or enriched through chemical modification. Different cellulose derivatives such as carboxymethyl cellulose and hydroxypropyl cellulose have been widely used in the preparation of delivery systems, but the water solubility and physical degradation of these derivatives limit the gastrointestinal delivery of astaxanthin. Chitosan, derived from the N-deacetylation reaction of chitin, is a natural polycationic electrolyte that is stable in neutral and alkaline environments but lacks sufficient protection for astaxanthin under acidic conditions. In addition, due to the poor water solubility of chitosan, it needs to be dissolved in acidic solvents such as acetic acid, which can cause degradation of acid-sensitive astaxanthin. SUMMARY

[0005] The present application solves the technical problems existing in the prior art and provides a kind of astaxanthin-loaded chitosan-cellulose composite nanoparticle sol, which has the characteristics of small particle size, high water solubility and pH response performance, and endows astaxanthin with high environmental stability and good intestinal delivery capacity of astaxanthin.

[0006] The technical scheme adopted by the present application to solve the above-mentioned problems is:

[0007] A preparation method of a chitosan-cellulose composite nanoparticle sol loaded with astaxanthin, comprising the following steps:

[0008] 1) astaxanthin acetone solution is added dropwise to cellulose nanofiber solution, stirred and reacted for 0.5-2 h, then water-soluble chitosan solution is added, stirred and reacted for 0.5-2 h, to obtain a chitosan-astaxanthin-cellulose solution;

[0009] 2) the chitosan-astaxanthin-cellulose solution obtained in step 1) is subjected to rotary evaporation to remove acetone, and is centrifuged at 4-25 ℃ to remove insoluble substances, and the upper clear liquid is taken, to obtain a chitosan-cellulose composite nanoparticle sol loaded with astaxanthin.

[0010] According to the above scheme, the cellulose nanofiber in step 1) is a cellulose nanofiber with a surface rich in carboxyl groups, and the surface carboxyl content is 0.5-1.8 mmol / g. The cellulose is prepared by a TEMPO (2,2,6,6-tetramethylpiperidine-n-oxide) mediated oxidation system, and needs to be ultrasonically pretreated before use. The ultrasonic pretreatment conditions are: 300-1200 W ultrasonic for 5-30 min.

[0011] According to the above scheme, the water-soluble chitosan in step 1) is a chitosan hydrochloride prepared by treating chitosan with HCl, and the degree of deacetylation is 70-90%, the viscosity (10 g / L solution, 20 ℃) is 10-100 mPa·s, and the pH (10 g / L solution) is 2-6.

[0012] According to the above scheme, the volume ratio of the cellulose nanofiber solution and the water-soluble chitosan solution in step 1) is 1:(0-9), and the volume of the water-soluble chitosan solution is not 0; the addition amount of the astaxanthin acetone solution is 5-50% of the total volume of the cellulose nanofiber solution and the water-soluble chitosan solution. The cellulose nanofiber solution is obtained by dissolving cellulose nanofiber in water, and the cellulose nanofiber content is 0.1-1 mg / mL, and is stored at 0-25 ℃; the water-soluble chitosan solution is obtained by dissolving water-soluble chitosan in water, and the water-soluble chitosan content is 0.01-0.1 mg / mL, and is stored at 0-25 ℃; the astaxanthin acetone solution is obtained by dissolving astaxanthin in acetone, and the astaxanthin content is 0.01-0.5 mg / mL, and is stored at -20-5 ℃.

[0013] According to the above scheme, the stirring speed in step 1) is 400-1500 rpm, and the reaction temperature is 0-25 ℃.

[0014] According to the above scheme, the conditions for the rotary evaporation in step 2) are 30-50℃ rotary evaporation for 5-30min at a rotation speed of 80-200rpm; and the conditions for the centrifugation are 6000-12000rpm centrifugation for 5-30min.

[0015] According to the above scheme, all the reactions in step 1) and step 2) are carried out under light shielding conditions.

[0016] The astaxanthin-loaded chitosan-cellulose composite nanoparticle sol prepared by the above method, wherein the average particle size of the composite nanoparticles is 50-200nm, the zeta potential is -40--55mV, the astaxanthin loading amount in the chitosan-cellulose composite nanoparticles is 2.5-7.5%, and the embedding rate of astaxanthin reaches more than 85%.

[0017] Compared with the prior art, the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol has the following beneficial effects:

[0018] (1) The astaxanthin-loaded chitosan-cellulose composite nanoparticle sol has the characteristics of small particle size and high zeta potential, which endows astaxanthin with high environmental stability; and the sol is transparent orange-red, which improves the water solubility of astaxanthin and exhibits good intestinal delivery capacity of astaxanthin.

[0019] (2) The cellulose nanofiber prepared by the TEMPO-mediated oxidation system and the water-soluble chitosan prepared by HCl modification are used as raw materials, wherein the cellulose nanofiber has good water dispersibility and the anti-digestion property of natural cellulose, can establish a physical barrier with pH response characteristics, and the water solubility of chitosan can avoid the degradation of astaxanthin caused by the use of acidic solvents.

[0020] (3) The astaxanthin-loaded chitosan-cellulose composite nanoparticle sol is prepared by blending astaxanthin through composite coagulation technology, compared with direct blending with cellulose or chitosan, the composite coagulation between the cellulose nanofiber and the water-soluble chitosan can maintain the stability of astaxanthin in a wide pH range, by adjusting the content and ratio of the cellulose nanofiber solution and the water-soluble chitosan solution, the interaction type and binding degree between polyelectrolytes can be controlled, so as to control the particle size, pH response performance, solubility and stability in water of the composite nanoparticles.

[0021] (4) The cellulose nanoparticle provides a new solution for astaxanthin steady-state processing and delivery, and is an environmentally friendly material, the preparation process is environmentally friendly and pollution-free, the process is simple, the preparation conditions are mild, the raw materials are widely available, the price is low, it is biodegradable, non-toxic and pollution-free, and has good biocompatibility. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1Macroscopic morphology and TEM image of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1;

[0023] Figure 2 Astaxanthin retention rate of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 and the free astaxanthin solution obtained in Comparative Example 2 under UV light exposure;

[0024] Figure 3 In (a), the astaxanthin retention rate of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 and the free astaxanthin solution obtained in Comparative Example 2 at different temperatures, and in (b), the particle size and zeta potential of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 at different temperatures;

[0025] Figure 4 In (a) and (b), the macroscopic image and astaxanthin retention rate of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 and the free astaxanthin solution obtained in Comparative Example 2 at different pH, and in (c), the particle size and zeta potential of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 at different pH;

[0026] Figure 5 In (a), the astaxanthin retention rate of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 and the free astaxanthin solution obtained in Comparative Example 2 at different NaCl concentrations, and in (b), the particle size and zeta potential of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 at different NaCl concentrations;

[0027] Figure 6 Astaxanthin retention rate of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 and the free astaxanthin solution obtained in Comparative Example 2 at different storage times;

[0028] Figure 7 In (a), (b) and (c), the astaxanthin release rate, particle size and zeta potential of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 during the simulated digestion in the gastrointestinal tract. DETAILED DESCRIPTION

[0029] In order to better understand the present application, the following examples are further illustrated in conjunction with the present application, but the present application is not limited to the following examples.

[0030] In the following examples, cellulose nanofiber is prepared from eucalyptus cellulose by TEMPO-mediated oxidation system, and the content of carboxyl group on the surface of cellulose nanofiber is 1.60±0.02 mmol / g. The cellulose nanofiber needs to be ultrasonically pretreated before use. The ultrasonic pretreatment conditions are as follows: 900 W ultrasonic for 20 min.

[0031] In the following examples, the cellulose nanofiber solution is obtained by dissolving cellulose nanofiber in water, the concentration of cellulose nanofiber is 0.1-1 mg / mL, and the solution is stored at 0-25°C; the water-soluble chitosan solution is obtained by dissolving water-soluble chitosan in water, the concentration of water-soluble chitosan is 0.01-0.1 mg / mL, and the solution is stored at 0-25°C; and the astaxanthin acetone solution is obtained by dissolving astaxanthin in acetone, the concentration of astaxanthin is 0.01-0.5 mg / mL, and the solution is stored at-20-5°C.

[0032] Example 1

[0033] A preparation method of a chitosan-cellulose composite nanoparticle sol loaded with astaxanthin, and the specific steps are as follows:

[0034] 1) At 20°C, 2 mL of astaxanthin acetone solution with a concentration of 0.1 mg / mL is added dropwise into 10 mL of cellulose nanofiber solution with a concentration of 0.3 mg / mL, and the mixture is stirred at a rotation speed of 800 rpm in the dark for 0.5 h. Then, 10 mL of water-soluble chitosan solution with a concentration of 0.04 mg / mL is added, and the mixture is stirred at a rotation speed of 800 rpm in the dark for 0.5 h to obtain a chitosan-astaxanthin-cellulose solution.

[0035] 2) At 37°C, the chitosan-astaxanthin-cellulose solution obtained in step 1) is subjected to rotary evaporation in the dark at a rotation speed of 120 rpm for 15 min to remove acetone. Then, the mixture is centrifuged at 10,000 rpm at 4°C for 15 min to remove insoluble substances. The supernatant is collected to obtain a chitosan-cellulose composite nanoparticle sol loaded with astaxanthin.

[0036] Example 2

[0037] A preparation method of a chitosan-cellulose composite nanoparticle sol loaded with astaxanthin, and the specific steps are as follows:

[0038] 1) At 20°C, 2 mL of astaxanthin acetone solution with a concentration of 0.1 mg / mL is added dropwise into 10 mL of cellulose nanofiber solution with a concentration of 0.3 mg / mL, and the mixture is stirred at a rotation speed of 800 rpm in the dark for 0.5 h. Then, 10 mL of water-soluble chitosan solution with a concentration of 0.04 mg / mL is added, and the mixture is stirred at a rotation speed of 800 rpm in the dark for 0.5 h to obtain a chitosan-astaxanthin-cellulose solution.

[0039] 2) In the condition of 37℃, the solution of chitosan-astaxanthin-cellulose obtained in step 1) was rotary evaporated for 15 min at 120 rpm in the dark to remove acetone, and centrifuged at 10000 rpm for 15 min at 4℃ to remove insoluble substances, and the clear supernatant was obtained, which was the sol of chitosan-astaxanthin-cellulose composite nanoparticles loaded with astaxanthin.

[0040] Example 3

[0041] A method for preparing a sol of chitosan-cellulose composite nanoparticles loaded with astaxanthin, the specific steps are as follows:

[0042] 1) At 20℃, 2 mL of astaxanthin acetone solution with a concentration of 0.1 mg / mL was added dropwise to 10 mL of cellulose nanofiber solution with a concentration of 0.3 mg / mL, and stirred at 800 rpm in the dark for 0.5 h, then 10 mL of water-soluble chitosan solution with a concentration of 0.05 mg / mL was added, and stirred at 800 rpm in the dark for 0.5 h to obtain a chitosan-astaxanthin-cellulose solution;

[0043] 2) In the condition of 37℃, the solution of chitosan-astaxanthin-cellulose obtained in step 1) was rotary evaporated for 15 min at 120 rpm in the dark to remove acetone, and centrifuged at 10000 rpm for 15 min at 4℃ to remove insoluble substances, and the clear supernatant was obtained, which was the sol of chitosan-astaxanthin-cellulose composite nanoparticles loaded with astaxanthin.

[0044] Example 4

[0045] A method for preparing a sol of chitosan-cellulose composite nanoparticles loaded with astaxanthin, the specific steps are as follows:

[0046] 1) At 20℃, 2 mL of astaxanthin acetone solution with a concentration of 0.1 mg / mL was added dropwise to 10 mL of cellulose nanofiber solution with a concentration of 0.3 mg / mL, and stirred at 800 rpm in the dark for 0.5 h, then 10 mL of water-soluble chitosan solution with a concentration of 0.05 mg / mL was added, and stirred at 800 rpm in the dark for 0.5 h to obtain a chitosan-astaxanthin-cellulose solution;

[0047] 2) In the condition of 37℃, the solution of chitosan-astaxanthin-cellulose obtained in step 1) was rotary evaporated for 15 min at 120 rpm in the dark to remove acetone, and centrifuged at 10000 rpm for 15 min at 4℃ to remove insoluble substances, and the clear supernatant was obtained, which was the sol of chitosan-astaxanthin-cellulose composite nanoparticles loaded with astaxanthin.

[0048] Example 5

[0049] A preparation method of astaxanthin-loaded chitosan-cellulose composite nanoparticle sol, the specific steps are as follows:

[0050] 1) Under the condition of 20℃, 2mL astaxanthin acetone solution with a concentration of 0.1mg / mL was added dropwise into 10mL cellulose nanofiber solution with a concentration of 0.5mg / mL, and the reaction was stirred under the condition of 800rpm and in the dark for 0.5h, then 10mL water-soluble chitosan solution with a concentration of 0.05mg / mL was added, and the reaction was stirred under the condition of 800rpm and in the dark for 0.5h to obtain chitosan-astaxanthin-cellulose solution;

[0051] 2) Under the condition of 37℃, the chitosan-astaxanthin-cellulose solution obtained in step 1) was rotary evaporated under the condition of 120rpm and in the dark for 15min to remove acetone, and then centrifuged under the condition of 10000rpm and at 4℃ for 15min to remove insoluble substances, and the upper clear liquid was taken to obtain astaxanthin-loaded chitosan-cellulose composite nanoparticle sol.

[0052] Example 6

[0053] A preparation method of astaxanthin-loaded chitosan-cellulose composite nanoparticle sol, the specific steps are as follows:

[0054] 1) Under the condition of 20℃, 2mL astaxanthin acetone solution with a concentration of 0.1mg / mL was added dropwise into 10mL cellulose nanofiber solution with a concentration of 0.6mg / mL, and the reaction was stirred under the condition of 800rpm and in the dark for 0.5h, then 10mL water-soluble chitosan solution with a concentration of 0.05mg / mL was added, and the reaction was stirred under the condition of 800rpm and in the dark for 0.5h to obtain chitosan-astaxanthin-cellulose solution;

[0055] 2) Under the condition of 37℃, the chitosan-astaxanthin-cellulose solution obtained in step 1) was rotary evaporated under the condition of 120rpm and in the dark for 15min to remove acetone, and then centrifuged under the condition of 10000rpm and at 4℃ for 15min to remove insoluble substances, and the upper clear liquid was taken to obtain astaxanthin-loaded chitosan-cellulose composite nanoparticle sol.

[0056] Comparative Example 1

[0057] The difference from Example 4 is that the concentration of water-soluble chitosan solution is different, and the specific steps are as follows:

[0058] 1) At 20℃, 2mL of astaxanthin acetone solution with a concentration of 0.1mg / mL was added dropwise to 10mL of cellulose nanofiber solution with a concentration of 0.4mg / mL, and the reaction was stirred at a rotation speed of 800rpm in the dark for 0.5h. Then 10mL of water-soluble chitosan solution with a concentration of 0.25mg / mL was added, and the reaction was stirred at a rotation speed of 800rpm in the dark for 0.5h to obtain a chitosan-astaxanthin-cellulose solution;

[0059] 2) At 37℃, the chitosan-astaxanthin-cellulose solution obtained in step 1) was subjected to rotary evaporation in the dark at a rotation speed of 120rpm for 15min to remove acetone. Then the solution was centrifuged at 10000rpm at 4℃ for 15min to remove insoluble substances. The upper clear liquid was taken to obtain an astaxanthin-loaded chitosan-cellulose composite nanoparticle sol.

[0060] Comparative Example 2

[0061] The difference between Examples 1-6 and Comparative Example 1 is that chitosan-cellulose composite nanoparticles were not used as the astaxanthin loading carrier. The specific steps are as follows:

[0062] At 20℃, 1mg of astaxanthin was weighed and added to 10mL of DMSO, and the mixture was shaken in the dark for 10min to fully dissolve. Then 5mL of the astaxanthin DMSO solution was taken and added to 45mL of deionized water, and the mixture was shaken in the dark for 1min to fully mix, thereby obtaining a free astaxanthin solution for Comparative Example 2.

[0063] Performance Test

[0064] 1. Particle size, zeta potential, entrapment efficiency and drug loading of nanoparticles

[0065] The astaxanthin-loaded chitosan-cellulose composite nanoparticle sol prepared in Examples 1-6 and Comparative Example 1 was diluted with deionized water to a certain concentration, and then the particle size and zeta potential were measured using a Malvern nanoparticle size potential instrument. The results are shown in Table 1.

[0066] The entrapment efficiency of astaxanthin in the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol prepared in Examples 1-6 and Comparative Example 1 was calculated as follows: 1mL of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol was taken and added to 5mL of extraction solution (dichloromethane and methanol in a volume ratio of 2:1), and the mixture was shaken for 5min. Then the mixture was centrifuged at 7000rpm for 5min, and the absorbance of the lower dichloromethane solution at a wavelength of 478nm was measured. The concentration of astaxanthin in the sample was calculated according to the astaxanthin standard curve, and then the entrapment efficiency EE and the loading capacity LC were calculated according to the following formula. The results are shown in Table 1.

[0067] EE(%) = m2 / m1 x 100

[0068] LC (%) = m2 / m0 × 100

[0069] Wherein: m0 is the total mass of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol (mg, on a dry weight basis); m1 is the amount of astaxanthin added (mg, on a dry weight basis); m2 is the astaxanthin content in the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol (mg, on a dry weight basis).

[0070] Table 1

[0071]

[0072]

[0073] As can be seen from the results in Table 1, the average particle size of the nanoparticles in the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol prepared in the embodiment of the present invention is approximately 55-100 nm, with a relatively small particle size and a zeta potential absolute value significantly greater than 30 mV, indicating that the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol is stable. The astaxanthin encapsulation efficiency of the composite nanoparticles is above 80%, demonstrating a high encapsulation efficiency and loading capacity. Comparison of Example 1 with Comparative Example 1 shows that by adjusting the concentration of water-soluble chitosan, the zeta potential of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol can be controlled, thereby changing the particle size and pH response of the composite nanoparticles. In Comparative Example 1, the water-soluble chitosan concentration is higher, and the zeta potential of the nanoparticles increases from a negative value to 30.82 mV, resulting in flocculation in a neutral environment.

[0074] like Figure 1 As shown, pure astaxanthin is insoluble in water. The astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 is translucent orange-red and has a nearly spherical microstructure, indicating that the combination of water-soluble chitosan and cellulose nanofibers imparts good water solubility to astaxanthin. Furthermore, testing has shown that this astaxanthin-loaded chitosan-cellulose composite nanoparticle sol exhibits a Tyndall effect.

[0075] 2. Environmental stability test

[0076] UV light stability: 2 mL of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 with an astaxanthin concentration of 10 μg / mL and 2 mL of the free astaxanthin solution obtained in Comparative Example 2 with an astaxanthin concentration of 10 μg / mL were added to a 24-well plate, exposed to UV light for 150 min, and samples were taken at specific times to determine the astaxanthin retention rate.

[0077] Temperature stability: 2.5 mL of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 with an astaxanthin concentration of 10 μg / mL and 2.5 mL of the free astaxanthin solution obtained in Comparative Example 2 with an astaxanthin concentration of 10 μg / mL were added to 5 mL light-proof centrifuge tubes, respectively. The tubes were placed in a water bath at 4, 20, 40, 60, and 80°C for 1 h, and then immediately placed in an ice bath. The particle size, ζ potential, and astaxanthin retention rate of the samples were measured.

[0078] pH stability: 3 mL of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 with an astaxanthin concentration of 10 μg / mL and 3 mL of the free astaxanthin solution obtained in Comparative Example 2 with an astaxanthin concentration of 10 μg / mL were added to 5 mL sample bottles, respectively. The pH of the solution was adjusted to 2, 3, 4, 5, 6, 7, 8, or 9 with 1 mol / L HCl or 1 mol / L NaOH. After storage at 4°C overnight, the particle size, ζ potential, and astaxanthin retention rate of the samples were measured.

[0079] Ionic strength stability: 2.5 mL of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 with an astaxanthin concentration of 10 μg / mL and 2.5 mL of the free astaxanthin solution obtained in Comparative Example 2 with an astaxanthin concentration of 10 μg / mL were respectively mixed with 2.5 mL of NaCl solution to obtain dispersions containing NaCl concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L. After storage at 4°C overnight, the particle size, ζ potential, and astaxanthin retention rate of the samples were measured.

[0080] Storage stability: The astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 with an astaxanthin concentration of 10 μg / mL and the free astaxanthin solution obtained in Comparative Example 2 with an astaxanthin concentration of 10 μg / mL were stored in the dark at 4°C for 0, 1, 3, 5, 7, 14, and 28 days, respectively. The particle size, ζ potential, and astaxanthin retention rate of the samples were measured.

[0081] like Figure 2 As shown in the data, after 150 minutes of UV treatment, the retention rate of astaxanthin in the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 was as high as 88.04%, while the retention rate of astaxanthin in the free astaxanthin solution obtained in Comparative Example 2 was only 3.47%, indicating that the chitosan-cellulose composite nanoparticle sol can protect astaxanthin from degradation by UV light.

[0082] like Figure 3As shown, compared with the free astaxanthin solution obtained in Comparative Example 2, the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 has a particle size and a zeta potential of 65.62 nm and -46.53 mV, respectively, at a high temperature of 80°C, and a retention rate of astaxanthin as high as 93.74%, while the retention rate of astaxanthin in the free astaxanthin solution obtained in Comparative Example 2 is only 79.48%.

[0083] As shown in Table 2, the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 has a higher stability (astaxanthin retention rate > 98.24%) in an environment with a pH of 2-9, and aggregates when the pH is lower than 3, thereby protecting astaxanthin from degradation by an acidic solution, while the retention rate of astaxanthin in the free astaxanthin solution obtained in Comparative Example 2 is only 39.58% in an environment with a pH of 2. Figure 4 As shown in Table 3, the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 still has a higher retention rate of astaxanthin (astaxanthin retention rate > 93.61%) in a high ionic strength environment, and tends to protect astaxanthin from degradation by gradually aggregating; while the astaxanthin in the free astaxanthin solution obtained in Comparative Example 2 is obviously aggregated and largely degraded, and the retention rate of astaxanthin is only 78.82%.

[0084] Figure 5 As shown in Table 4, the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 can be stably stored for more than 28 days, which confirms that the chitosan-cellulose composite nanoparticle sol has a good protective effect on astaxanthin, significantly improves the stability of astaxanthin in an environment with ultraviolet light, high temperature, acidity, alkalinity and high ionic strength, and prolongs the storage period of astaxanthin.

[0085] As shown in Table 4, the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 can be stably stored for more than 28 days, which confirms that the chitosan-cellulose composite nanoparticle sol has a good protective effect on astaxanthin, significantly improves the stability of astaxanthin in an environment with ultraviolet light, high temperature, acidity, alkalinity and high ionic strength, and prolongs the storage period of astaxanthin. Figure 6

[0086] 3. Simulated gastrointestinal digestion test

[0087] The 35 mL of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 with a concentration of 10 μg / mL of astaxanthin was preheated at 37°C for 15 min, mixed with the same volume of simulated gastric juice, and shaken at 100 rpm and 37°C for 2 h. The pH was adjusted to 7.4 to terminate the gastric juice digestion, the same volume of preheated simulated intestinal juice was added, and shaken at 100 rpm and 37°C for 4 h. Sampling was performed at a specific time, the supernatant was taken after centrifugation at 10,000 rpm for 10 min, mixed with 5 mL of extraction liquid (dichloromethane and methanol in a volume ratio of 2:1) to extract the released astaxanthin, and the release rate of astaxanthin was determined.

[0088] As shown in Table 5, the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol obtained in Example 1 has a higher stability (astaxanthin retention rate > 98.24%) in an environment with a pH of 2-9, and aggregates when the pH is lower than 3, thereby protecting astaxanthin from degradation by an acidic solution, while the retention rate of astaxanthin in the free astaxanthin solution obtained in Comparative Example 2 is only 39.58% in an environment with a pH of 2. Figure 7 ​​As shown, the zeta potential of the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol of Example 1 increases under the simulated gastric fluid condition, obvious flocculation occurs, thereby protecting astaxanthin from being released and degraded; under the simulated intestinal fluid condition, the composite nanoparticles in the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol of Example 1 quickly dissolve and release astaxanthin, further confirming that the astaxanthin-loaded chitosan-cellulose composite nanoparticle sol has the pH response characteristic, and can effectively protect astaxanthin to reach the intestinal tract and be released.

[0089] The above merely describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and changes can be made without departing from the inventive concept, and these all belong to the protection scope of the present application.

Claims

1. A method for preparing astaxanthin-loaded chitosan-cellulose composite nanoparticle sol, characterized in that: The steps include: 1) adding the astaxanthin acetone solution dropwise to the cellulose nanofiber solution, stirring and reacting for 0.5 to 2 hours, and then adding the water-soluble chitosan solution, stirring and reacting for 0.5 to 2 hours to obtain a chitosan-astaxanthin-cellulose solution; wherein the cellulose nanofibers are cellulose nanofibers rich in surface carboxyl groups, with a surface carboxyl group content of 0.5 to 1.8 mmol / g; 2) removing acetone from the chitosan-astaxanthin-cellulose solution obtained in step 1) by rotary evaporation, removing insoluble matter by centrifugation at 4-25° C., and collecting the upper clear liquid to obtain a chitosan-cellulose composite nanoparticle sol loaded with astaxanthin; In step 1), the volume ratio of the cellulose nanofiber solution to the water-soluble chitosan solution is 1:(0-9), and the volume of the water-soluble chitosan solution is not 0; the amount of the astaxanthin acetone solution added is 5-50% of the total volume of the cellulose nanofiber solution and the water-soluble chitosan solution; wherein the concentration of the cellulose nanofiber solution is 0.1-1 mg / mL, with water as the solvent; the concentration of the water-soluble chitosan solution is 0.01-0.1 mg / mL, with water as the solvent; the concentration of the astaxanthin acetone solution is 0.01-0.5 mg / mL, with acetone as the solvent.

2. The method for preparing an astaxanthin-loaded chitosan-cellulose composite nanoparticle sol according to claim 1, characterized in that: The water-soluble chitosan in step 1) is chitosan hydrochloride prepared by treating chitosan with HCl, with a deacetylation degree of 70-90%, a viscosity of 10-100 mPa·s, and a pH of 2-6 for a 10 g / L solution.

3. The method for preparing an astaxanthin-loaded chitosan-cellulose composite nanoparticle sol according to claim 1, characterized in that: The cellulose nanofibers in step 1) need to be ultrasonically pretreated before use, with ultrasound being applied at a power of 300 to 1200 W for 5 to 30 minutes.

4. The method for preparing an astaxanthin-loaded chitosan-cellulose composite nanoparticle sol according to claim 1, characterized in that: The stirring speed in step 1) is 400-1500 rpm, and the reaction temperature is 0-25° C.; the rotary evaporation conditions in step 2) are: rotary evaporation at 30-50° C. for 5-30 min, a rotation speed of 80-200 rpm, and the centrifugation conditions are: centrifugation at 6000-12000 rpm for 5-30 min; both steps 1) and 2) are carried out in the dark.

5. The astaxanthin-loaded chitosan-cellulose composite nanoparticle sol prepared by the method according to any one of claims 1 to 4.

6. The astaxanthin-loaded chitosan-cellulose composite nanoparticle sol according to claim 5, characterized in that: The zeta potential of the sol is -40 to -55 mV; the average particle size of the astaxanthin-loaded chitosan-cellulose composite nanoparticles is 50 to 200 nm, the astaxanthin loading amount is 2.5 to 7.5%, and the astaxanthin embedding rate is more than 85%.

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