Preparation method of whey protein / pullulan / sugar astaxanthin nano dispersion

Abstract

The invention discloses a preparation method of a whey protein / pullulan / sugar astaxanthin nano-dispersion, WPC / PUL compounds are adopted to construct different carriers to embed AST, the carriers are respectively a nano-dispersion loaded with AST, emulsion gel and microcapsule powder, the three carriers have good embedding efficiency and protection effect on AST, the simulated gastrointestinal digestion stability of AST is remarkably improved, and the stability of AST is improved. And the method has great significance in expanding the preparation of an AST embedding delivery system and the application of AST in industries such as food and the like.

Description

[0001] This application is application number 2023111852269, entitled "Method for constructing different carriers to encapsulate astaxanthin based on whey protein / Zygopyrrolidone". Technical Field

[0002] This invention belongs to the field of food processing technology, specifically relating to a method for preparing whey protein / astaxanthin / sugar nanodispersions. Background Technology

[0003] Astaxanthin (AST) is a fat-soluble carotenoid, chemically named 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene, and appears as deep red crystals. Benefiting from the presence of ketone groups, hydroxyl groups, and long-chain conjugated unsaturated double bonds in its molecular structure, AST is currently the most potent natural bioactive antioxidant, capable of quenching singlet oxygen and scavenging free radicals. This gives AST various health benefits, such as anti-tumor activity, prevention of cardiovascular disease, enhancement of the immune system, and protection of the central nervous system. However, this structure also makes AST sensitive to light, heat, and oxygen, leading to isomerization and degradation during processing, storage, and digestion. Furthermore, AST is poorly soluble in water and difficult to disperse in the body, resulting in low bioavailability and limitations in practical applications. Therefore, effectively improving the water dispersibility, stability, and bioavailability of AST has become a crucial issue that urgently needs to be addressed for its efficient development and utilization.

[0004] To overcome the limitations of AST (associated protein synthesizer) applications and expand its industrial use, one strategy is to construct carriers for its encapsulation and delivery. Natural biomacromolecules have been widely used in encapsulation and delivery systems. Whey protein concentrate (WPC), a byproduct of cheese production, accounts for 20% of total milk protein and contains various nutrients. It possesses anti-cancer, antioxidant, antibacterial, antiviral, and immunomodulatory functions, earning it the title of "King of Proteins." As an amphiphilic protein, WPC contains both hydrophilic and hydrophobic groups, exhibiting excellent gelling, film-forming, and small molecule binding abilities. Pullulan (PUL), also known as microbial polysaccharide, is a neutral, water-soluble extracellular polysaccharide derived from microorganisms. It possesses good heat resistance, solubility, non-hygroscopicity, film-forming properties, and a strong ability to block oxygen, thus reducing the oxidation of products. Furthermore, PUL exhibits good biocompatibility, being biodegradable without causing environmental harm. It is a multifunctional novel biomaterial with significant development potential and is therefore widely used in the food and pharmaceutical fields.

[0005] Due to the excellent nutritional and functional characteristics of proteins and polysaccharides, food scientists have developed complexes with different structures, sizes, and functional characteristics based on their interactions using various technologies. These complexes are widely used as carriers for the encapsulation and delivery of bioactive substances. Currently, various encapsulation systems have been developed, such as nanoparticles, gels, and microcapsules. However, due to differences in preparation techniques, the encapsulation efficiency and protective effect of carriers on bioactive substances vary. Therefore, studying the encapsulation efficiency and protective effect of different types of encapsulation carriers on AST is of great significance for expanding the preparation of AST encapsulation and delivery systems and their application in the food and other industries. Summary of the Invention

[0006] The purpose of this invention is to overcome the application limitations of AST and to provide a method for constructing different carriers to encapsulate astaxanthin based on whey protein / pyrolytic polysaccharide. Based on the WPC / PUL complex, three encapsulation systems for AST were constructed: nano-dispersions, emulsion gels, and microcapsule powders, in order to improve the water dispersibility, stability, and bioaccessibility of AST.

[0007] Methods for constructing different carriers to encapsulate astaxanthin based on whey protein / Zygopyrrolizidine include:

[0008] 1) Preparation of WPC / PUL composite

[0009] (1) Dissolve whey protein (WPC) powder in distilled water, stir thoroughly at room temperature for 2 h, and store at 4°C overnight to allow it to fully hydrate, thus obtaining a WPC solution;

[0010] (2) Add the polysaccharide PUL to the WPC solution to obtain a WPC / PUL solution;

[0011] (3) Heat the WPC / PUL solution in an 80°C water bath for 20 min to form a WPC / PUL complex, and then cool it to room temperature for later use;

[0012] 2) Astaxanthin AST was encapsulated using the WPC / PUL complex.

[0013] The preparation method of whey protein / astaxanthin / sugar emulsion gel includes:

[0014] The WPC / PUL composite described in step 1) has a WPC concentration of 10% and a PUL concentration of 1-2.5%.

[0015] The AST described in step 2) is added to vegetable oil (soybean oil, peanut oil, rapeseed oil or sunflower oil, etc.), and magnetically stirred to prepare an AST soybean oil solution with a concentration of 0.1-0.3 mg / mL.

[0016] An AST soybean oil solution and a WPC / PUL complex were mixed and a WPC / PUL / AST emulsion was prepared using a high-speed shear emulsifier.

[0017] Add gluconate-δ-lactone GDL to WPC / PUL / AST emulsion, stir magnetically, and let stand to form WPC / PUL / AST emulsion gel.

[0018] The WPC / PUL complex described in step 1) has a PUL concentration of 2% mg / mL;

[0019] The concentration of the AST soybean oil solution is 0.2 mg / mL;

[0020] The AST soybean oil solution and the WPC / PUL complex were mixed at a volume ratio of 1:4.

[0021] The GDL content in the WPC / PUL / AST emulsion is 1%; the standing is carried out at 25°C.

[0022] Another object of the present invention is to provide a method for preparing whey protein / astaxanthin / sugar nanodispersions.

[0023] The preparation method of whey protein / Zhuomeiduo / sugar astaxanthin nanodispersion includes:

[0024] The WPC / PUL complex described in step 1) has a WPC concentration of 1.0 mg / mL and a PUL concentration of 0.4-1.0 mg / mL;

[0025] Step 2) The astaxanthin was prepared as an AST ethanol solution with a concentration of 30 μg / mL by magnetic stirring under room temperature and light-protected conditions.

[0026] Mix the WPC / PUL complex and AST ethanol solution, and stir magnetically at room temperature in the dark until the solution color remains unchanged.

[0027] Ethanol was removed by vacuum rotary evaporation to obtain WPC / PUL / AST nanodispersion;

[0028] The WPC / PUL complex described in step 1) has a PUL concentration of 0.6-1.0 mg / mL;

[0029] The WPC / PUL complex and AST ethanol solution are mixed at a volume ratio of 2:1.

[0030] The vacuum rotary evaporation conditions are 25°C and 100 rpm.

[0031] The obtained WPC / PUL / AST nanodispersion was centrifuged at 3000 rpm for 10 min to remove unencapsulated free AST.

[0032] The preparation method of whey protein / Zhuomeiduo / sugar astaxanthin nanodispersion includes:

[0033] The WPC / PUL composite described in step 1) has a WPC concentration of 10% and a PUL concentration of 7.0-9.0%.

[0034] The astaxanthin AST described in step 2) was dissolved in anhydrous ethanol and magnetically stirred for 1 h at room temperature in the dark to obtain an AST ethanol solution with a mass concentration of 1 mg / mL.

[0035] AST ethanol solution and WPC / PUL complex were mixed in a volume ratio of 1:1-6 and freeze-dried in a vacuum freeze dryer to obtain microcapsule powder.

[0036] The AST ethanol solution and the WPC / PUL complex were mixed at a volume ratio of 1:1-4, and the PUL concentration was 8.0%.

[0037] This invention provides a method for encapsulating astaxanthin using different carriers based on whey protein / pyromellitic polysaccharide, and for encapsulating AST using different carriers based on the WPC / PUL complex, namely, AST-loaded nanodispersions, emulsion gels, and microcapsule powders. All three carriers exhibit good encapsulation efficiency and protective effect on AST, significantly improving the simulated gastrointestinal digestion stability, bioaccessibility, and storage stability of AST. This is of great significance for expanding the preparation of AST encapsulation and delivery systems and the application of AST in the food and other industries. Attached Figure Description

[0038] Figure 1 Appearance of free AST in water and WPC / PUL / AST nanodispersions with different PUL concentrations;

[0039] Figure 2 The effect of PUL concentration on the encapsulation efficiency of WPC / PUL / AST nanodispersions;

[0040] Figure 3 Effects of PUL concentration on the gastrointestinal digestibility (A) and bioavailability (B) of AST in WPC / PUL / AST nanodispersion;

[0041] Figure 4 Effect of PUL concentration on the storage stability of WPC / PUL / AST nanodispersions at different temperatures. A, 4℃; B, 25℃;

[0042] Figure 5The appearance morphology of WPC / PUL / AST emulsion gels with different PUL concentrations;

[0043] Figure 6 The effect of PUL concentration on the embedding efficiency of WPC / PUL / AST emulsion gel;

[0044] Figure 7 Effects of PUL concentration on the gastrointestinal digestive stability (A) and bioavailability (B) of AST in WPC / PUL / AST emulsion gel;

[0045] Figure 8 Effect of PUL concentration on the storage stability of WPC / PUL / AST emulsion gel at different temperatures. A, 4℃; B, 25℃;

[0046] Figure 9 Encapsulation efficiency of WPC / PUL / AST microcapsules with different core-to-wall ratios;

[0047] Figure 10 Microstructure of WPC / PUL / AST microcapsule powder;

[0048] Figure 11 The effects of WPC / PUL gel as a wall material on the stability (A) and bioaccessibility (B) of gastrointestinal digestion in AST simulations;

[0049] Figure 12 The effect of WPC / PUL gel as a wall material on the storage stability (A) and degradation curve (B) of AST at different temperatures;

[0050] Figure 13 In vivo absorption assays of three AST-loaded nanodispersions, emulsion gels, and microcapsules in mice. Detailed Implementation

[0051] Example 1: Preparation and Characterization of WPC / PUL / AST Nanodispersions

[0052] 1. Preparation of WPC / PUL / AST nanodispersions

[0053] WPC powder was dissolved in distilled water and stirred thoroughly at room temperature for 2 h. The solution was then stored overnight at 4 °C to ensure complete hydration, yielding a 1.0 mg / mL WPC solution. PUL was added to the WPC solution at concentrations of 0, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively. After complete dissolution, the solutions were heated in an 80 °C water bath for 20 min to form WPC / PUL complexes. The solutions were then cooled to room temperature for later use.

[0054] An appropriate amount of AST was accurately weighed and dissolved in anhydrous ethanol. The solution was magnetically stirred for 1 h at room temperature in the dark to obtain an AST ethanol solution with a concentration of 30 μg / mL. The AST solution was then mixed with WPC / PUL complexes of different PUL concentrations obtained in the previous step at a 1:2 (v:v) ratio and magnetically stirred for 5 h at room temperature in the dark until the solution color remained unchanged. Ethanol was then removed by vacuum rotary evaporation at 25 °C and 100 rpm to obtain WPC / PUL / AST nanodispersions. These nanodispersions were centrifuged at 3000 rpm for 10 min to remove unencapsulated free AST. During the preparation process, tin foil was used to protect the solution from light to minimize AST loss.

[0055] Result: From Figure 1 It can be observed that when AST is alone in water, obvious aggregated precipitation is observed. However, when the WPC / PUL / AST nanodispersion is formed, the solution exhibits a clear and transparent pinkish-purple appearance at different PUL concentrations, indicating that the WPC / PUL complex significantly improves the water solubility of AST.

[0056] 2. Determination of the encapsulation efficiency of nano-dispersions

[0057] Accurately transfer 1 mL of the nano-dispersion into a centrifuge tube, add 2 mL of ethyl acetate, sonicate for 5 min to fully break down the particles, then centrifuge at 8000 rpm for 5 min and collect the colored ethyl acetate solution in the upper layer. Repeat the above operation until the upper organic phase is colorless, then combine the organic phases and measure the absorbance at 475 nm using a UV spectrophotometer to calculate the encapsulated AST content. Calculate the encapsulation rate according to the formula.

[0058] ;

[0059] ;

[0060] In the formula: A is absorbance; D (mL) is the total volume of the supernatant; G (g) is the mass of the sample; d (cm) is the width of the cuvette; and E is the extinction coefficient.

[0061] Result: From Figure 2 As can be seen, when the PUL concentration is 0, the encapsulation efficiency of the nanodispersion is only 63.33%. With the increase of PUL concentration, the encapsulation efficiency of the WPC / PUL / AST nanodispersion gradually increases, reaching a maximum of 73.93% at a PUL concentration of 0.8 mg / mL. Subsequently, when the PUL concentration continues to increase to 1.0 mg / mL, the encapsulation efficiency of the nanodispersion decreases slightly to 71.01%.

[0062] 3. Simulated gastrointestinal digestion of nano-dispersions

[0063] First, simulated gastric juice (SGF) and simulated intestinal juice (SIF) were prepared. 10 mL of nano-dispersion was mixed with 10 mL of SGF. The pH of the mixture was adjusted to 2.0, and the mixture was continuously shaken at 100 rpm for 2 h at 37°C, with 2 mL of digestion solution collected every 0.5 h. After simulated gastric digestion, an equal volume of SIF was added, and the pH of the system was adjusted to 7.0. The resulting mixture was further digested in a shaker (37°C, 100 rpm) for 4 h, with 2 mL of digestion solution collected every 1 h. AST was extracted from the digestion solution with ethyl acetate. The mixture was sonicated for 5 min, centrifuged at 8000 rpm for 5 min, and the supernatant containing AST was collected. This process was repeated until the organic phase was colorless. The organic phases were combined, and their absorbance at 475 nm was measured. The retention rate of AST during simulated gastrointestinal digestion was calculated using the formula.

[0064] ;

[0065] In the formula: C1 and C0 represent the contents of AST in the digestion solution and the initial sample, respectively.

[0066] After the simulated gastrointestinal digestion process was completed, 3 mL of digestive fluid was taken out, centrifuged at 12,000 rpm for 30 min, and micelles containing dissolved AST were collected. The bioaccessibility was calculated using a formula.

[0067] ;

[0068] In the formula: C2 and C0 represent the contents of AST in the micelle phase and the initial sample, respectively.

[0069] Result: From Figure 3 As shown in Figure A, the AST retention rate decreased in all nanodispersions during simulated gastrointestinal digestion. After 2 hours of simulated gastric digestion, the AST retention rate in the PUL-free nanodispersion was only 73.4%. With increasing PUL concentration, the AST retention rate in the WPC / PUL / AST nanodispersion increased to 76.0%, 79.1%, 82.7%, and 80.7%, respectively. After further 4 hours of simulated intestinal digestion, only 60.4% of the AST was retained in the PUL-free nanodispersion, while the AST retention rate increased in the PUL-added WPC / PUL / AST nanodispersions. The highest AST retention rate (70.9%) was observed in the WPC / PUL / AST nanodispersion with a PUL concentration of 0.8 mg / mL. Figure 3B shows the bioavailability of AST in nanodispersions with different PUL concentrations. When the PUL concentration is 0, the bioavailability of AST in the nanodispersion is 47.67%. With the increase of PUL concentration, the bioavailability of AST in WPC / PUL / AST nanodispersion increases to 51.73%, 53.77%, 57.29%, and 57.27%, respectively. The addition of PUL significantly improves the bioavailability of AST, but there is no significant difference in the bioavailability of AST in nanodispersions formed at PUL concentrations of 0.8 mg / mL and 1.0 mg / mL.

[0070] 4. Storage stability of nano-dispersions

[0071] The WPC / PUL / AST nanodispersion was sealed in sample vials and stored in the dark at 4°C and 25°C for 2 months, respectively. At regular intervals, 1 mL samples were taken, and AST was extracted using ethyl acetate. The change in AST retention was calculated using a formula.

[0072] ;

[0073] In the formula: C3 and C0 represent the AST content after the sample has been stored for a period of time and at the beginning, respectively.

[0074] Result: From Figure 4 As shown in Figure A, after storage at 4°C for 2 months, the retention rate of AST in the PUL-free nanodispersion was 72.41%. With the addition of PUL, the retention rate of AST in the nanodispersion was significantly improved. The highest AST retention rate of 85.06% was achieved when the PUL concentration was 0.8 mg / mL. Figure 4 As shown in Figure B, during the two-month storage at 25°C, the trend of AST retention in WPC / PUL / AST nanodispersions with different PUL concentrations was similar to that during storage at 4°C, but the AST retention was lower compared to that during storage at 4°C. Specifically, the AST retention in the PUL-free nanodispersion was 61.97%. With increasing PUL concentration, the AST retention in the WPC / PUL / AST nanodispersions were 66.08%, 68.99%, 75.42%, and 72.01%, respectively.

[0075] Example 2: Preparation and Characterization of WPC / PUL / AST Emulsion Gel

[0076] 1. Preparation of WPC / PUL / AST emulsion gel

[0077] Prepare a 10% WPC solution, store it overnight at 4°C, and then add 0%, 1%, 1.5%, 2% and 2.5% PUL respectively. After complete dissolution, heat it in a water bath at 80°C for 20 min to form a WPC / PUL composite solution as the aqueous phase, and cool it to room temperature for later use.

[0078] An appropriate amount of AST was accurately weighed and added to soybean oil. The mixture was magnetically stirred for 2 h at room temperature in the dark to obtain an AST solution with a concentration of 0.2 mg / mL as the oil phase. This oil phase was added to the WPC / PUL aqueous solution obtained in the previous step and cooled to room temperature, bringing the oil phase volume fraction to 20%. The mixture was then emulsified using a high-speed shear emulsifier at 10,000 rpm for 3 min to prepare a WPC / PUL / AST emulsion. 1% GDL was added to the WPC / PUL / AST emulsion, and the mixture was magnetically stirred for 2 min. The mixture was then allowed to stand at 25°C for 3 h to form a WPC / PUL / AST emulsion gel, which was stored overnight at 4°C for further analysis. A portion of the emulsion gel was pre-frozen at -80°C for 12 h and then freeze-dried in a vacuum freeze dryer for 48 h before collecting the powder sample for analysis. During the preparation process, aluminum foil was used to protect the sample from light to minimize AST loss.

[0079] Result: From Figure 5 As can be seen, the WPC / PUL / AST emulsion gels all exhibit a pinkish-yellow appearance at different PUL concentrations and show no downward flow tendency during inversion, proving gel formation. This is because the tight packing and cross-linking between the droplets trap water, which is the continuous phase, greatly reducing the fluidity of the emulsion gel and thus presenting a good solid-like state.

[0080] 2. Determination of emulsion-gel embedding efficiency

[0081] 0.2 g of freeze-dried sample was mixed with 5 mL of ethyl acetate, vortexed for 1 min, and then sonicated for 5 min to fully extract AST. The sample was then centrifuged at 8000 rpm for 5 min, and the supernatant was collected. This process was repeated until the organic phase was colorless. The organic phases were combined, and the absorbance at 475 nm was measured using a UV spectrophotometer to calculate the encapsulated AST content. The encapsulation rate was then calculated using the formula.

[0082] ;

[0083] ;

[0084] In the formula: A is absorbance; D (mL) is the total volume of the supernatant; G (g) is the mass of the sample; d (cm) is the width of the cuvette; and E is the extinction coefficient.

[0085] Result: From Figure 6As can be seen, when the PUL concentration is 0%, the encapsulation efficiency of the emulsion gel is 82.63%. With increasing PUL concentration, the encapsulation efficiency of the WPC / PUL / AST nanodispersion gradually increases. The encapsulation efficiency reaches a maximum of 91.70% when the PUL concentration is 2.0%. Upon further addition of PUL to a concentration of 2.5%, the encapsulation efficiency of the emulsion gel decreases to 90.36%.

[0086] 3. The mimicry of gastrointestinal digestion by emulsion gels

[0087] First, simulated gastric juice (SGF) and simulated intestinal juice (SIF) were prepared. 5 mL of the prepared WPC / PUL / AST emulsion gel was crushed and mixed with 15 mL of SGF. The pH of the mixture was adjusted to 2.0, and the mixture was continuously shaken at 100 rpm for 2 h at 37°C. 2.0 mL of the digestion solution was collected every 0.5 h. After simulated gastric digestion, an equal volume of SIF was added, and the pH of the system was adjusted to 7.0. The resulting mixture was further digested in a shaker (37°C, 100 rpm) for 4 h, with 2 mL of the digestion solution collected every 1 h. AST was extracted from the digestion solution with ethyl acetate. The mixture was sonicated for 5 min, centrifuged at 8000 rpm for 5 min, and the supernatant containing AST was collected. This process was repeated until the organic phase was colorless. The organic phases were combined, and their absorbance at 475 nm was measured to calculate the retention rate of AST during simulated gastrointestinal digestion. After simulating the gastrointestinal digestion process, 3 mL of digestive fluid was centrifuged at 12,000 rpm for 30 min, and the micelle phase containing dissolved AST was collected to calculate the bioaccessibility of AST in the emulsion gel.

[0088] ;

[0089] ;

[0090] In the formula: C1, C2 and C0 represent the contents of AST in the digestion solution, micelle phase and initial sample, respectively.

[0091] Result: From Figure 7As shown in Figure A, the AST retention rate decreased in all samples during simulated gastrointestinal digestion. After 2 hours of simulated gastric digestion, the AST retention rate in the PUL-free emulsion gel was 80.23%. With increasing PUL concentration, the AST retention rate in the WPC / PUL / AST emulsion gel also increased, reaching 83.06%, 87.21%, 91.69%, and 89.11%, respectively. After further 4 hours of simulated intestinal digestion, only 67.36% of the AST was retained in the PUL-free emulsion gel, while the AST retention rate in the PUL-added WPC / PUL / AST emulsion gel was significantly improved. The highest AST retention rate (80.96%) was observed in the WPC / PUL / AST emulsion gel with a PUL concentration of 2.0%. Figure 7 B shows the bioavailability of AST in emulsion gels with different PUL concentrations. When the PUL concentration is 0, the bioavailability of AST in the emulsion gel is 33.31%. With the increase of PUL concentration, the bioavailability of AST in WPC / PUL / AST emulsion gels increased to 35.35%, 37.52%, 40.69%, and 39.22%, respectively. The addition of PUL significantly improved the bioavailability of AST, but there was no significant difference in the bioavailability of AST in emulsion gels formed at PUL concentrations of 2.0% and 2.5%.

[0092] 4. Storage stability of emulsion gels

[0093] The WPC / PUL / AST emulsion gel was sealed in sample vials and stored in the dark at 4°C and 25°C for 15 days, respectively. At regular intervals, 0.5 g samples were taken, and AST was extracted using ethyl acetate. The change in AST retention was calculated according to the formula.

[0094] ;

[0095] In the formula: C3 and C0 represent the AST content after the sample has been stored for a period of time and at the beginning, respectively.

[0096] Result: From Figure 8 As shown in Figure A, after storage at 4°C for 15 days, the retention rate of AST in the PUL-free emulsion gel was 70.22%. With the addition of PUL, the retention rate of AST in the WPC / PUL / AST emulsion gel was significantly improved. The highest AST retention rate of 80.36% was achieved when the PUL concentration was 2.0%. Figure 8B indicates that during storage at 25℃ for 15 days, the trend of AST retention in WPC / PUL / AST emulsion gels with different PUL concentrations was similar to that during storage at 4℃, but the AST retention was lower. Specifically, the AST retention in the PUL-free emulsion gel was only 65.12%. With increasing PUL concentration, the AST retention in the WPC / PUL / AST emulsion gels were 69.26%, 70.28%, 76.26%, and 73.69%, respectively.

[0097] Example 3 Preparation and characterization of WPC / PUL / AST microcapsule powder

[0098] 1. Preparation of WPC / PUL / AST microcapsule powder

[0099] WPC was dispersed in distilled water and stirred at room temperature for 2 h to obtain a 10% (by weight) WPC solution, which was then incubated overnight at 4°C to allow for full hydration. Under magnetic stirring, PUL was dissolved in the WPC solution to a final concentration of 8.0% (by weight) to prepare a WPC / PUL solution. The solution was heated in a water bath at 80°C for 20 min to form a WPC / PUL gel, which was then cooled to room temperature and used as the wall material for microcapsules.

[0100] A precise mass of AST was dissolved in anhydrous ethanol and magnetically stirred for 1 h at room temperature in the dark to obtain an AST ethanol solution with a mass concentration of 1 mg / mL, which was used as the core material for the microcapsules. The AST solution was then uniformly mixed with the WPC / PUL gel obtained in the previous step at ratios of 1:6, 1:4, 1:2, and 1:1 (v:v), and stirred at 2000 rpm for 30 min on a magnetic stirrer to form a WPC / PUL / AST composite system. Finally, the resulting solution was pre-frozen at -80℃ for 12 h and then freeze-dried in a vacuum freeze dryer for 48 h to obtain the microcapsule powder. During the preparation process, tin foil was used to shield the microcapsules from light to minimize AST loss.

[0101] 2. Determination of microcapsule encapsulation efficiency

[0102] The encapsulation efficiency of WPC / PUL / AST microcapsule powder is determined by the total AST content in the microcapsules and the surface AST content, calculated using the following formula:

[0103] ;

[0104] To determine the total AST content of the microcapsules, 50 mg of microcapsule powder was dissolved in 5 mL of distilled water, vortexed for 1 min, and centrifuged at 5000 g for 5 min. The supernatant was removed. Then, 5 mL of dimethyl sulfoxide was added, vortexed for 1 min, and centrifuged at 5000 g for 10 min at 4 °C. The supernatant was collected. After three extractions, the absorbance at 475 nm was measured using a UV / Vis spectrophotometer, and the AST content was calculated according to the following formula:

[0105] ;

[0106] In the formula: A is absorbance; D (mL) is the total volume of the supernatant; G (g) is the mass of the sample; d (cm) is the width of the cuvette; and E is the extinction coefficient.

[0107] Hexane was used to extract surface AST from the microcapsules. 50 mg of microcapsule powder was thoroughly mixed with 5 mL of hexane, and the mixture was centrifuged at 8000 g for 5 min at 4 °C to obtain the supernatant. This process was repeated twice. The absorbance at 475 nm was recorded, and the surface AST content was determined using the formula described above.

[0108] Result: As Figure 9 As shown, when the ratio of AST solution to WPC / PUL gel was 1:6, the encapsulation efficiency of WPC / PUL / AST microcapsules was 85.95%. With increasing ratio, the encapsulation efficiency gradually increased. The highest encapsulation efficiency (93.14%) was achieved when the ratio of AST solution to WPC / PUL gel was 1:2. However, when the mixing ratio was further increased to 1:1, the encapsulation efficiency significantly decreased to 86.58%. Therefore, in subsequent studies, WPC / PUL / AST microcapsules prepared with a mixing ratio of AST solution and WPC / PUL gel of 1:2 were selected as the optimal research material.

[0109] 3. Microscopic morphology of microcapsule powder

[0110] The microstructure of the microcapsule powder prepared by mixing AST solution and WPC / PUL gel at a volume ratio of 1:2 was observed using high-resolution field emission electron microscopy. Before observation, the sample was fixed to the sample stage with conductive tape and sputtered with gold.

[0111] Result: From Figure 10 As can be seen, the WPC / PUL / AST microcapsule powder exhibits the irregular structure and adhesion common during freeze-drying. According to the scale bar, the microcapsule size is within the micrometer range. Furthermore, the microcapsule surface remains intact without obvious cracks or gaps, indicating that microcapsules prepared using WPC / PUL gel as the wall material can provide good encapsulation and protection for AST.

[0112] 4. Microencapsulated powder mimics gastrointestinal digestion

[0113] Simulated gastric digestion process: 3.2 g / L pepsin was added to a 2.0 g / L sodium chloride solution, and the pH was adjusted to 2.0 to prepare simulated gastric juice (SGF). 0.2 g of microcapsule powder was dissolved in 10 mL of distilled water, then mixed with 10 mL of SGF. The pH of the mixture was adjusted to 2.0, and the mixture was continuously shaken at 100 rpm for 2 h at 37°C.

[0114] Simulated intestinal digestion process: First, a 6.8 g / L KH₂PO₄ solution was prepared, and the pH was adjusted to 7.0. Then, 10.0 g / L porcine bile salts and 5.0 g / L trypsin were added to prepare simulated intestinal fluid (pH 7.0). After simulated gastric digestion, an equal volume of SIF was added to the digestive fluid, and the pH of the system was adjusted to 7.0. The resulting mixture was further digested in a shaker (37℃, 100 rpm) for 4 h.

[0115] Throughout the digestion process, 1 mL of digestate was collected every 0.5 h, and AST was extracted with ethyl acetate. The mixture was centrifuged at 5000 rpm for 5 min, and the supernatant containing AST was collected. This process was repeated until colorless. The absorbance at 475 nm was measured, and the retention rate of AST during simulated gastrointestinal digestion was calculated. After the simulated gastrointestinal digestion process was completed, 1 mL of digestate was centrifuged at 12,000 rpm for 30 min, and the micelle phase containing dissolved AST was collected. The bioavailability of AST in the emulsion gel was calculated.

[0116] ;

[0117] ;

[0118] In the formula: C1, C2 and C0 represent the contents of AST in the digestion solution, micelle phase and initial sample, respectively.

[0119] Result: From Figure 11 As shown in Figure A, the AST retention rate decreased in both samples during simulated gastrointestinal digestion. After 2 hours of simulated gastric digestion, 41.33% of free AST was degraded, while only 14.96% of the AST encapsulated in microcapsules was degraded. After further digestion in simulated intestinal fluid for 4 hours, only 43.64% of free AST was retained, while the retention rate of AST encapsulated in microcapsules reached 75.50%, which was 1.73 times that of free AST. Figure 11As shown in Figure B, the bioavailability of free AST is only 9.11%, while the bioavailability of AST encapsulated in microcapsules reaches 46.50%, which is 5.1 times that of free AST. Compared with free AST, AST encapsulated in microcapsules has higher gastrointestinal digestibility and bioavailability.

[0120] 5. Storage stability of microencapsulated powder

[0121] Free AST and WPC / PUL / AST microcapsule powders were sealed in sample vials and stored for 26 weeks in the dark at 4°C and 25°C, respectively. Samples were taken at regular intervals, and the change in AST retention was calculated using a formula.

[0122] ;

[0123] In the formula: C3 and C0 represent the AST content in the sample after a period of storage and at the initial stage, respectively.

[0124] The half-life of AST (t 1 / 2 The degradation constant (k) and the degradation constant (k) are calculated by the following formula.

[0125] ;

[0126] ;

[0127] Result: From Figure 12 As shown in Figure A, the AST retention rate in both samples decreased with prolonged storage time, with a faster degradation rate during the first week of storage. However, the retention rate of AST encapsulated in microcapsules was significantly higher than that of free AST. After 26 weeks of storage at 4°C, the retention rate of free AST was 42.25%, while the retention rate of AST encapsulated in microcapsules was 85.02%, an increase of 42.77%. After 26 weeks of storage at 25°C, the trend of AST retention rate changes in both samples was similar to that at 4°C, but the AST retention rate was even lower. Specifically, the retention rate of free AST was only 30.99%, while the retention rate of AST encapsulated in microcapsules increased to 75.77%. Figure 12B indicates that the degradation curves of all samples showed a good fit. After 26 weeks of storage at 4°C, the degradation rate constant of free AST was 0.0309, with a half-life of 157.02 days, while the degradation rate constant of AST encapsulated in microcapsules was 0.0059, with a half-life of 882.38 days, which was 5.62 times that of free AST. The trend of degradation at 25°C was similar to that at 4°C, but the degradation rate constant of AST was larger and the half-life was shorter. Specifically, the degradation rate constant of free AST was 0.0408, with a half-life of 118.92 days, while the degradation rate constant of AST in microcapsules was 0.0094, with a half-life of 516.17 days, which was 4.34 times that of free AST.

[0128] Example 4: In vivo absorption test of three AST-loaded nanodispersions, emulsion gels, and microcapsules in mice:

[0129] Animal Experiments and Grouping: Eight-week-old male C57BL / 6N mice were acclimatized for one week and then randomly assigned to five groups (n=10 per group). The experimental groups were as follows: (1) Normal control group (Con), (2) Astaxanthin group (AST-w), (3) WPC / PUL / AST microcapsule group (AST-MS), (4) WPC / PUL / AST nanodispersion group (AST-ND), and (5) WPC / PUL / AST emulsion gel group (AST-EG). The ambient temperature was 20 ± 2℃, the relative humidity was 50 ± 5%, and the day-night light cycle was 12 h / 12 ​​h. The environment was kept clean during the experiment. The normal control group was administered tap water by gavage, the astaxanthin group was administered an AST tap water suspension (astaxanthin is insoluble in water), and the other three groups were administered pure aqueous solutions of WPC / PUL / AST microcapsules, WPC / PUL / AST nanodispersions, and WPC / PUL / AST emulsion gels, respectively (the final concentration of ASTA in all systems was 0.1 mmol / L). This gavage was performed continuously for 10 days, with a gavage volume of 0.2 mL per mouse. Four hours after gavage, blood was collected from the eyeballs of the mice, and the blood samples from the same group were mixed and centrifuged at 8000 g for 5 min. The supernatant serum was collected and stored at -80℃.

[0130] For the detection of astaxanthin in serum, 0.4 mL of mouse serum was aliquoted into two 5 mL centrifuge tubes. 1.6 mL of dichloromethane and methanol (dichloromethane:methanol = 2:1, v / v) were added to each tube, and the mixture was shaken for 1 min. Then, 0.8 mL of n-hexane was added, and the mixture was shaken for 30 s. The mixture was centrifuged at 8000 g for 5 min, and the supernatant was collected. This process was repeated twice. All supernatants were collected in brown centrifuge tubes, dried under nitrogen, dissolved in 0.1 mL of methanol, filtered through a 0.22 µm organic membrane, and then subjected to liquid chromatography analysis. The results showed that astaxanthin was not detected in the serum of mice in the normal control group (Con). The serum astaxanthin concentration in the astaxanthin-tap water suspension group (AST-w) was approximately 26 pmol / mL. The serum astaxanthin concentrations in the WPC / PUL / AST microcapsule group (AST-MS), WPC / PUL / AST nanodispersion group (AST-ND), and WPC / PUL / AST emulsion gel group (AST-EG) were 7.8-fold, 10.0-fold, and 9.3-fold higher than those in the AST-w group, respectively, indicating that astaxanthin after different treatments had good in vivo absorption.

Claims

1. A method for preparing whey protein / Pullulan / astaxanthin nanodispersion, comprising: 1) preparing WPC / PUL complex (1) Dissolving whey protein WPC powder in distilled water, stirring thoroughly at room temperature for 2 h and storing at 4℃ overnight to make it fully hydrated, obtaining WPC solution; (2) Adding Pullulan PUL to the WPC solution to obtain WPC / PUL solution; The WPC / PUL solution has a WPC concentration of 1.0 mg / mL and a PUL concentration of 0.4-1.0 mg / mL; (3) Heating the WPC / PUL solution in a water bath at 80℃ for 20 min to form WPC / PUL complex, and cooling to room temperature for standby; 2) The astaxanthin is prepared as an AST ethanol solution with a concentration of 30 μg / mL by magnetic stirring in the dark at room temperature; Mixing the WPC / PUL complex and the AST ethanol solution, and magnetic stirring in the dark at room temperature until the solution color is unchanged; removing ethanol by vacuum rotary evaporation to obtain WPC / PUL / AST nanodispersion.

2. The method for embedding astaxanthin in different carriers based on whey protein / Pullulan according to claim 1, characterized in that: Step 1) The WPC / PUL complex has a PUL concentration of 0.6-1.0 mg / mL; Mixing the WPC / PUL complex and the AST ethanol solution is mixing at a volume ratio of 2:1; The vacuum rotary evaporation is performed at 25℃ and 100 rpm; The obtained WPC / PUL / AST nanodispersion is centrifuged at 3000 rpm for 10 min to remove unembedded free AST.

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