Method for embedding astaxanthin in different carriers based on whey protein / pullulan

Abstract

The application discloses a method for embedding astaxanthin (AST) in different carriers based on a whey protein / polysaccharide compound, and adopts a WPC / PUL compound to embed AST in different carriers, namely, a nano-dispersion loaded with AST, an emulsion gel and a microcapsule powder, wherein the three carriers have good embedding efficiency and protection effect on AST, and significantly improve the simulated gastrointestinal digestion stability, bioavailability and storage stability of AST, and have important significance for expanding the preparation of the AST embedding delivery system and the application of AST in the food industry.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food processing, and particularly relates to a preparation method for effectively embedding astaxanthin based on whey protein / polysaccharide of Ulocladium. BACKGROUND

[0002] Astaxanthin (AST) is a fat-soluble carotenoid with the chemical name of 3, 3'-dihydroxy-4, 4'-diketo-β, β'-carotene, which is a deep red crystalline substance. Benefiting from the ketone group, hydroxyl group and long-chain conjugated unsaturated double bond in its molecular structure, AST is the strongest natural bioactive substance with antioxidant activity, which has the ability to quench singlet oxygen and scavenge free radicals, thereby having multiple benefits to human health, such as anti-tumor, prevention of cardiovascular diseases, enhancement of immune system function, protection of central nervous system, etc. However, this structure also makes AST sensitive to light, heat and oxygen, leading to its easy isomerization and degradation in the processes of processing, storage and digestion, etc. In addition, AST has the disadvantages of not easy to dissolve in water and not easy to disperse in the body, resulting in low bioavailability and many limitations in practical application. Therefore, how to effectively improve the water dispersibility, stability and bioavailability of AST has become an important problem to be solved for efficient development and utilization of AST.

[0003] In order to overcome the application limitations of AST, one strategy to expand its industrial application is to construct a carrier for embedding and delivering AST. Natural biological macromolecules have been widely used in embedding delivery systems. Whey protein concentrate (WPC) is a by-product in the process of cheese production, accounting for 20% of the total amount of milk protein, which contains various nutritional ingredients and has functions of anti-cancer, antioxidant, antibacterial, antiviral and immune regulation, etc., and is known as the "king of protein". As an amphiphilic protein, WPC contains both hydrophilic and hydrophobic groups, and has good gelation, film formation and small molecule compound binding ability. Pullulan (PUL), also known as polysaccharide, is a neutral water-soluble exopolysaccharide derived from microorganisms, which has good heat resistance, solubility, non-hygroscopicity, film formation and strong oxygen barrier ability to reduce the oxidation degree of products. At the same time, PUL also has good biological safety and can be degraded by microorganisms, thereby not causing environmental harm, and is a multifunctional new biological material with great development value, so it is widely used in the field of food and medicine.

[0004] 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 the interaction between the two, and thus are widely used as carriers for embedding and delivering bioactive substances. At present, various embedding systems such as nanoparticles, gels and microcapsules have been developed. However, due to different preparation techniques, the embedding efficiency and protection effect of the carrier on bioactive substances are different. Therefore, it is of great significance to study the embedding efficiency and protection effect of different types of embedding carriers on AST for expanding the preparation of AST embedding and delivery system and its application in food industry. SUMMARY

[0005] The purpose of the present application is to overcome the application limitations of AST, and to provide a method for embedding astaxanthin based on whey protein / pullulan to construct different carriers. Based on the WPC / PUL complex, three embedding systems of nanodispersion, emulsion gel and microcapsule powder loaded with AST are constructed to improve the water dispersibility, stability and bioavailability of AST.

[0006] The method for embedding astaxanthin based on whey protein / pullulan comprises:

[0007] 1) Preparation of WPC / PUL complex

[0008] (1) Dissolve whey protein WPC powder in distilled water, stir thoroughly at room temperature for 2 h, and store at 4℃ overnight to make it fully hydrated, and obtain WPC solution;

[0009] (2) Add pullulan PUL to the WPC solution to obtain WPC / PUL solution;

[0010] (3) Heat the WPC / PUL solution in a water bath at 80℃ for 20 min to form WPC / PUL complex, and cool to room temperature for standby;

[0011] 2) Embed astaxanthin AST using WPC / PUL complex;

[0012] The WPC / PUL complex of step 1) has a WPC concentration of 10% and a PUL concentration of 1-2.5%;

[0013] In step 2), the AST is added to vegetable oil (soybean oil, peanut oil, rapeseed oil or sunflower seed oil, etc.), magnetically stirred to prepare an AST soybean oil solution with a concentration of 0.1-0.3 mg / mL;

[0014] Mix the AST soybean oil solution and WPC / PUL complex, and use a high-speed shearing emulsifier to prepare a WPC / PUL / AST emulsion;

[0015] The WPC / PUL / AST emulsion is added with gluconic acid-delta-lactone GDL, magnetically stirred, and left to form a WPC / PUL / AST emulsion gel;

[0016] The WPC / PUL compound in step 1 has a PUL concentration of 2% mg / mL;

[0017] The AST soybean oil solution has a concentration of 0.2 mg / mL;

[0018] The AST soybean oil solution and the WPC / PUL compound are mixed in a volume ratio of 1:4;

[0019] The GDL has a content of 1% in the WPC / PUL / AST emulsion; and the leaving is at 25°C;

[0020] The WPC / PUL compound in step 1 has a WPC concentration of 1.0 mg / mL and a PUL concentration of 0.4-1.0 mg / mL;

[0021] The astaxanthin in step 2 is prepared by magnetically stirring an AST ethanol solution with a concentration of 30 μg / mL at room temperature in the dark;

[0022] The WPC / PUL compound and the AST ethanol solution are mixed and magnetically stirred at room temperature in the dark until the solution color does not change;

[0023] The ethanol is removed by vacuum rotary evaporation to obtain a WPC / PUL / AST nanodispersion;

[0024] The WPC / PUL compound in step 1 has a PUL concentration of 0.6-1.0 mg / mL;

[0025] The WPC / PUL compound and the AST ethanol solution are mixed in a volume ratio of 2:1;

[0026] The vacuum rotary evaporation is performed at 25°C and 100 rpm;

[0027] The obtained WPC / PUL / AST nanodispersion is centrifuged at 3000 rpm for 10 min to remove unembedded free AST.

[0028] The WPC / PUL compound in step 1 has a WPC concentration of 10% and a PUL concentration of 7.0-9.0%;

[0029] The astaxanthin AST in step 2 is dissolved in anhydrous ethanol and magnetically stirred at room temperature in the dark for 1 h to obtain an AST ethanol solution with a mass concentration of 1 mg / mL;

[0030] AST ethanol solution and WPC / PUL complex, mixed in a ratio of 1:1-6 by volume, freeze-dried in a vacuum freeze dryer, and microcapsule powder was obtained;

[0031] The AST ethanol solution and WPC / PUL complex are mixed in a ratio of 1:1-4 by volume, and the PUL concentration is 8.0%.

[0032] The present application provides a method for embedding astaxanthin based on whey protein / zymolytic polysaccharide, and a method for embedding astaxanthin based on WPC / PUL complex, which are nanodispersion, emulsion gel and microcapsule powder loaded with AST, respectively. The three carriers have good embedding efficiency and protection effect on AST, significantly improving the simulated gastrointestinal digestion stability, bioavailability and storage stability of AST, and have important significance for expanding the preparation of AST embedding delivery system and the application of AST in the food industry. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 . Appearance of free AST in water and WPC / PUL / AST nanodispersion with different PUL concentrations;

[0034] Figure 2 . Effect of PUL concentration on the embedding rate of WPC / PUL / AST nanodispersion;

[0035] Figure 3 . Effect of PUL concentration on the gastrointestinal digestion stability (A) and bioavailability (B) of AST in WPC / PUL / AST nanodispersion;

[0036] Figure 4 . Effect of PUL concentration on the storage stability of WPC / PUL / AST nanodispersion at different temperatures. A, 4℃; B, 25℃;

[0037] Figure 5 . Appearance of WPC / PUL / AST emulsion gel with different PUL concentrations;

[0038] Figure 6 . Effect of PUL concentration on the embedding rate of WPC / PUL / AST emulsion gel;

[0039] Figure 7 . Effect of PUL concentration on the gastrointestinal digestion stability (A) and bioavailability (B) of AST in WPC / PUL / AST emulsion gel;

[0040] Figure 8. Effect of PUL concentration on the storage stability of WPC / PUL / AST emulsion gels at different temperatures. A, 4℃; B, 25℃;

[0041] Figure 9 . Entrapment efficiency of WPC / PUL / AST microcapsule powders at different core-to-wall ratios;

[0042] Figure 10 . Micro-morphology of WPC / PUL / AST microcapsule powders;

[0043] Figure 11 . Effect of WPC / PUL gels as wall materials on the simulated gastrointestinal digestion stability (A) and bioaccessibility (B) of AST;

[0044] Figure 12 . Effect of WPC / PUL gels as wall materials on the storage stability (A) and degradation profile (B) of AST at different temperatures;

[0045] Figure 13 . In vivo absorption test of three AST-loaded nanodispersions, emulsion gels and microcapsule powders. DETAILED DESCRIPTION

[0046] Example 1 Preparation and characterization of WPC / PUL / AST nanodispersions

[0047] 1. Preparation of WPC / PUL / AST nanodispersions

[0048] WPC powder was dissolved in distilled water, stirred thoroughly at room temperature for 2 h and stored at 4℃ overnight for complete hydration to obtain 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, and after complete dissolution, the WPC / PUL complex was formed by heating at 80℃ for 20 min in a water bath and then cooled to room temperature for use.

[0049] An appropriate amount of AST was accurately weighed and dissolved in absolute ethanol under room temperature and light-protected conditions with magnetic stirring for 1 h to obtain an AST ethanol solution with a concentration of 30 μg / mL. The AST solution was mixed with the WPC / PUL complex obtained in the previous step at a mixing ratio of 1:2 (v:v) under room temperature and light-protected conditions with magnetic stirring for 5 h until the solution color remained unchanged. Then, the ethanol was removed by vacuum rotary evaporation at 25℃ and 100 rpm to obtain WPC / PUL / AST nanodispersions, which were centrifuged at 3000 rpm for 10 min to remove unentrapped free AST. During the preparation process, tin paper was used to avoid light to reduce the loss of AST.

[0050] Results: FromFigure 1 It can be found that the aggregation precipitation of AST alone in water can be observed obviously. When the WPC / PUL / AST nanodispersion is formed, the solution shows a clear transparent powder purple appearance under different PUL concentrations, which indicates that the WPC / PUL composite significantly improves the water solubility of AST.

[0051] 2. Measurement of nanodispersion embedding rate

[0052] Accurately take 1 mL of nanodispersion into a centrifuge tube, add 2 mL of ethyl acetate, and ultrasonic for 5 min, so that the particles are fully broken, then centrifuge at 8000 rpm for 5 min, collect the upper layer colored ethyl acetate solution. Repeat the above operation until the upper organic phase is colorless, then combine the organic phase, measure its absorbance at 475 nm with a UV spectrophotometer to calculate the embedded AST content, and calculate the embedding rate according to the formula.

[0053]

[0054]

[0055] In the formula: A is the absorbance; D (mL) is the total volume of supernatant; G (g) is the mass of the sample; d (cm) is the width of the cuvette; E is the extinction coefficient.

[0056] Results: From Figure 2 It can be seen from the above table that when the PUL concentration is 0, the embedding rate of the nanodispersion is only 63.33%. With the increase of PUL concentration, the embedding rate of WPC / PUL / AST nanodispersion gradually increases, and reaches the highest value of 73.93% when the PUL concentration is 0.8 mg / mL. Then, when the PUL concentration continues to increase to 1.0 mg / mL, the embedding rate of the nanodispersion slightly decreases to 71.01%.

[0057] 3. Simulated gastrointestinal digestion of nanodispersion

[0058] Firstly, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared. 10 mL of nanodispersion 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 at 37°C for 2 h, and 2 mL of the digest was taken out 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 shaking bed (37°C, 100 rpm) for 4 h, and 2 mL of the digest was taken out every 1 h. AST in the digest was extracted with ethyl acetate, and the mixture was ultrasonicated for 5 min, and after centrifugation at 8000 rpm for 5 min, the upper solution containing AST was collected, and the process was repeated until the organic phase was colorless. The organic phase was combined, and its absorbance at 475 nm was measured, and the retention rate of AST during simulated gastrointestinal digestion was calculated according to the formula.

[0059]

[0060] In the formula: C1 and C0 represent the content of AST in the digest and the initial sample, respectively.

[0061] After the end of the simulated gastrointestinal digestion process, 3 mL of the digest was taken out, centrifuged at 12,000 rpm for 30 min, and the micellar phase containing dissolved AST was collected, and its bioaccessibility was calculated using the formula.

[0062]

[0063] In the formula: C2 and C0 represent the content of AST in the micellar phase and the initial sample, respectively.

[0064] Results: From Figure 3 As can be seen from Table A, during the simulated gastrointestinal digestion process, the retention rate of AST in all nanodispersions decreased. After 2 h of simulated gastric digestion, the retention rate of AST in the nanodispersion without PUL was only 73.4%, and as the PUL concentration increased, the retention rate of AST in the WPC / PUL / AST nanodispersion also increased, reaching 76.0%, 79.1%, 82.7% and 80.7%, respectively. After further digestion in simulated intestinal fluid for 4 h, only 60.4% of AST was retained in the nanodispersion without PUL, while the retention rate of AST in the WPC / PUL / AST nanodispersion with PUL increased, and the AST retention rate of the WPC / PUL / AST nanodispersion with a PUL concentration of 0.8 mg / mL was the highest, reaching 70.9%. Figure 3B shows the bioaccessibility of AST in nanodispersion with different PUL concentrations. When the PUL concentration is 0, the bioaccessibility of AST in the nanodispersion is 47.67%. With the increase of PUL concentration, the bioaccessibility of AST in WPC / PUL / AST nanodispersion is increased to 51.73%, 53.77%, 57.29% and 57.27% respectively. The addition of PUL significantly improves the bioaccessibility of AST, but there is no significant difference in the bioaccessibility of AST in the nanodispersion formed at PUL concentrations of 0.8 mg / mL and 1.0 mg / mL.

[0065] 4. Storage stability of nanodispersion

[0066] The WPC / PUL / AST nanodispersion was sealed in a sample bottle and stored in a dark environment at 4°C and 25°C respectively for 2 months. Every time a sample of 1 mL was taken, AST was extracted using ethyl acetate, and the change in AST retention rate was calculated according to the formula.

[0067]

[0068] In the formula: C3 and C0 represent the AST content after the sample is stored for a period of time and at the initial time respectively.

[0069] Results: From Figure 4 As can be seen from A, after 2 months of storage at 4°C, the AST retention rate in the nanodispersion without PUL is 72.41%. With the addition of PUL, the AST retention rate in the nanodispersion is significantly improved. Among them, when the PUL concentration is 0.8 mg / mL, the AST retention rate reaches the highest of 85.06%. From Figure 4 As can be seen from B, during the 2 months of storage at 25°C, the AST retention rate of WPC / PUL / AST nanodispersion with different PUL concentrations has a similar trend to that when stored at 4°C, but the AST retention rate is lower than that when stored at 4°C. Among them, the AST retention rate in the nanodispersion without PUL is 61.97%, and with the increase of PUL concentration, the AST retention rate in WPC / PUL / AST nanodispersion is 66.08%, 68.99%, 75.42% and 72.01% respectively.

[0070] Example 2 Preparation and characterization of WPC / PUL / AST emulsion gel

[0071] 1. Preparation of WPC / PUL / AST emulsion gel

[0072] Preparation of 10% WPC solution, 4℃ overnight storage, respectively, add 0%, 1%, 1.5%, 2% and 2.5% PUL, fully dissolved, heated in water bath at 80℃ for 20 min to form WPC / PUL composite solution as the aqueous phase, cooled to room temperature for standby.

[0073] Accurately weigh the appropriate amount of AST and add it to the soybean oil. Stir magnetically at room temperature in the dark for 2 h to obtain an AST solution with a concentration of 0.2 mg / mL as the oil phase. Add the oil phase to the WPC / PUL aqueous phase solution obtained in the previous step, which has been cooled to room temperature, to achieve a volume fraction of 20% oil phase. Use a high-speed shear emulsifier to emulsify at 10000 rpm for 3 min to prepare a WPC / PUL / AST emulsion. Add 1% GDL to the WPC / PUL / AST emulsion and stir magnetically for 2 min. Let it stand at 25℃ for 3 h to form a WPC / PUL / AST emulsion gel, which is stored at 4℃ overnight for further analysis. A portion of the emulsion gel is pre-frozen at -80℃ for 12 h, then freeze-dried in a vacuum freeze dryer for 48 h to collect a powder sample for testing. During preparation, use tin paper to avoid light to reduce the loss of AST.

[0074] Results: From Figure 5 It can be seen that under different PUL concentrations, the WPC / PUL / AST emulsion gel all presents a powder yellow appearance, and has no downward flow trend during inversion, proving the formation of the gel. This is because the close accumulation and mutual cross-linking between the droplets trap the water as the continuous phase, greatly reducing the flowability of the emulsion gel, thus presenting a good solid-like state.

[0075] 2. Determination of emulsion gel entrapment rate

[0076] Mix 0.2 g of freeze-dried sample with 5 mL of ethyl acetate, vortex for 1 min, and then ultrasonic for 5 min to extract AST thoroughly. Then, centrifuge the sample at 8000 rpm for 5 min, and collect the supernatant. Repeat the above operation until the organic phase is colorless, and combine the organic phases. Measure the absorbance of the organic phase at 475 nm using a UV spectrophotometer to calculate the content of entrapped AST, and calculate the entrapment rate according to the formula.

[0077]

[0078]

[0079] In the formula, A is the 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.

[0080] Results: From Figure 6It can be seen that the entrapment efficiency of emulsion gels was 82.63% when the PUL concentration was 0. With the increase of PUL concentration, the entrapment efficiency of WPC / PUL / AST nanodispersion gradually increased. When the PUL concentration was 2.0%, the entrapment efficiency of emulsion gels reached the highest value of 91.70%. When the PUL concentration continued to increase to 2.5%, the entrapment efficiency of emulsion gels decreased to 90.36%.

[0081] 3. Simulated in vitro gastrointestinal digestion of emulsion gels

[0082] Firstly, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared. 5 mL of prepared WPC / PUL / AST emulsion gels were 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 at 37°C for 2 h, and 2.0 mL of the digestion solution was taken out 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 obtained mixture was further digested in a shaking bed (37°C, 100 rpm) for 4 h, and 2 mL of the digestion solution was taken out every 1 h. AST in the digestion solution was extracted with ethyl acetate, and the mixture was ultrasonicated for 5 min, and centrifuged at 8000 rpm for 5 min, and the upper solution containing AST was collected, and the operation was repeated until the organic phase was colorless. The organic phase was combined, and the absorbance at 475 nm was measured, and the retention rate of AST during simulated in vitro gastrointestinal digestion was calculated. After the end of simulated in vitro gastrointestinal digestion, 3 mL of the digestion solution was centrifuged at 12,000 rpm for 30 min, and the micellar phase containing dissolved AST was collected, and the bioaccessibility of AST in the emulsion gels was calculated.

[0083]

[0084]

[0085] In the formula: C1, C2 and C0 represent the content of AST in the digestion solution, the micellar phase and the initial sample, respectively.

[0086] Results: From the results shown in Table 2, it can be seen that the bioaccessibility of AST in the emulsion gels was 0.63% when the PUL concentration was 0. With the increase of PUL concentration, the bioaccessibility of AST in the emulsion gels gradually increased. When the PUL concentration was 2.0%, the bioaccessibility of AST in the emulsion gels reached the highest value of 2.70%. When the PUL concentration continued to increase to 2.5%, the bioaccessibility of AST in the emulsion gels decreased to 2.36%. Figure 7As can be seen in Figure A, the retention rate of AST in all samples decreased during the simulated gastrointestinal digestion. After 2 h of simulated gastric juice digestion, the retention rate of AST in emulsion gels without PUL was 80.23%, and with the increase of PUL concentration, the retention rate of AST in WPC / PUL / AST emulsion gels also increased, which were 83.06%, 87.21%, 91.69% and 89.11%, respectively. After further digestion for 4 h in simulated intestinal juice, only 67.36% of AST was retained in emulsion gels without PUL, while the retention rate of AST in WPC / PUL / AST emulsion gels with PUL was significantly improved, and the AST retention rate of WPC / PUL / AST emulsion gels with PUL concentration of 2.0% was the highest, which was 80.96%. Figure 7 Figure B shows the bioaccessibility of AST in emulsion gels with different PUL concentrations. When the PUL concentration was 0, the bioaccessibility of AST in emulsion gels was 33.31%, and with the increase of PUL concentration, the bioaccessibility 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 bioaccessibility of AST, but there was no significant difference in the bioaccessibility of AST in emulsion gels formed at PUL concentrations of 2.0% and 2.5%.

[0087] 4. Storage stability of emulsion gels

[0088] WPC / PUL / AST emulsion gels were sealed in sample bottles and stored in the dark at 4°C and 25°C for 15 d, respectively. Every time, 0.5 g of sample was taken out, and AST was extracted using ethyl acetate. The change in AST retention rate was calculated according to the formula.

[0089]

[0090] In the formula, C3 and C0 represent the AST content after storage for a period of time and at the initial time, respectively.

[0091] Results: From Figure 8 As can be seen in Figure A, after storage at 4°C for 15 d, the retention rate of AST in emulsion gels without PUL was 70.22%, and with the addition of PUL, the retention rate of AST in WPC / PUL / AST emulsion gels was significantly improved, and when the PUL concentration was 2.0%, the AST retention rate reached the highest, which was 80.36%. Figure 8B shows that the change trend of AST retention rate of WPC / PUL / AST emulsion gel with different PUL concentrations during storage at 25°C for 15 d is similar to that during storage at 4°C, but the AST retention rate is lower than that during storage at 4°C. Among them, the AST retention rate of the emulsion gel without PUL is only 65.12%, and with the increase of PUL concentration, the AST retention rates of WPC / PUL / AST emulsion gel are 69.26%, 70.28%, 76.26% and 73.69%, respectively.

[0092] Example 3 Preparation and characterization of WPC / PUL / AST microcapsule powder

[0093] 1. Preparation of WPC / PUL / AST microcapsule powder

[0094] WPC was dispersed in distilled water and stirred at room temperature for 2 h to prepare a 10% (by weight) WPC solution, which was fully hydrated at 4°C overnight. Under magnetic stirring, PUL was fully dissolved in the WPC solution at a final concentration of 8.0% (by weight) to prepare a WPC / PUL solution, which was heated at 80°C for 20 min to form a WPC / PUL gel, and then cooled to room temperature for use as the wall material of the microcapsule.

[0095] AST was accurately weighed and dissolved in anhydrous ethanol, and then magnetically stirred at room temperature in the dark for 1 h to obtain an AST ethanol solution with a mass concentration of 1 mg / mL as the core material of the microcapsule. The AST solution and the WPC / PUL gel obtained in the previous step were uniformly mixed at a ratio of 1:6, 1:4, 1:2 and 1:1 (v:v), and then stirred at a speed of 2000 rpm for 30 min on a magnetic stirrer to form a WPC / PUL / AST composite system. Finally, the obtained solution was pre-frozen at -80°C for 12 h, and then freeze-dried in a vacuum freeze-drier for 48 h to obtain the microcapsule powder. During the preparation process, tin paper was used to avoid light to reduce the loss of AST.

[0096] 2. Determination of the embedding rate of the microcapsule powder

[0097] The embedding rate of WPC / PUL / AST microcapsule powder is determined by the total AST content and the surface AST content in the microcapsule, and the calculation formula is as follows:

[0098]

[0099] 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:

[0100]

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 3. Microscopic morphology of microcapsule powder

[0105] 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.

[0106] 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.

[0107] 4. Simulated Gastrointestinal Digestion of Microcapsule Powder

[0108] Simulated Gastric Digestion: 3.2 g / L of pepsin was added to 2.0 g / L of sodium chloride solution, and the pH was adjusted to 2.0 to make simulated gastric fluid (SGF). 0.2 g of microcapsule powder was dissolved in 10 mL of distilled water, and 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 at 37 °C for 2 h.

[0109] Simulated Intestinal Digestion: First, 6.8 g / L of KH2PO4 solution was prepared, and after adjusting the pH to 7.0, 10.0 g / L of porcine bile salt and 5.0 g / L of trypsin were added to make simulated intestinal fluid (pH 7.0). After the completion of simulated gastric digestion, an equal volume of SIF was added to the digest, and the pH of the system was adjusted to 7.0. The resulting mixture was further digested in a shaking bed (37 °C, 100 rpm) for 4 h.

[0110] During the entire digestion process, 1 mL of the digest was taken out every 0.5 h, and AST was extracted with ethyl acetate. After the mixture was centrifuged at 5000 rpm for 5 min, the upper solution containing AST was collected, and the process was repeated until the solution was colorless. The absorbance at 475 nm was measured, and the retention rate of AST during simulated gastrointestinal digestion was calculated. After the completion of simulated gastrointestinal digestion, 1 mL of the digest was centrifuged at 12,000 rpm for 30 min, and the micellar phase containing dissolved AST was collected, and the bioaccessibility of AST in the emulsion gel was calculated.

[0111]

[0112]

[0113] In the formula: C1, C2 and C0 represent the contents of AST in the digest, the micellar phase and the initial sample, respectively.

[0114] Results: From Figure 11 As can be seen from Figure A, during the simulated gastrointestinal digestion, the retention rate of AST in both samples decreased. After 2 h of simulated gastric digestion, 41.33% of free AST was degraded, while only 14.96% of AST embedded in the microcapsule powder was degraded. After further digestion in simulated intestinal fluid for 4 h, only 43.64% of free AST was retained, while the retention rate of AST embedded in the microcapsule powder reached 75.50%, which was 1.73 times that of free AST. From Figure 11As can be seen in B, the bioavailability of free AST is only 9.11%, while the bioavailability of AST encapsulated by microcapsule powder is 46.50%, which is 5.1 times that of free AST. Compared with free AST, AST encapsulated by microcapsule powder has higher gastrointestinal digestion stability and bioavailability.

[0115] 5. Storage stability of microcapsule powder

[0116] Free AST and WPC / PUL / AST microcapsule powder were sealed in sample bottles and stored in a dark environment at 4°C and 25°C, respectively, for 26 weeks. Samples were taken at regular intervals, and the change in AST retention was calculated according to the formula.

[0117]

[0118] In the formula, C3 and C0 represent the AST content in the sample after a period of storage and at the initial time, respectively.

[0119] The half-life (t 1 / 2 ) and degradation constant (k) of AST were calculated by the following formula.

[0120]

[0121]

[0122] Results: From Figure 12 As can be seen in A, the AST retention rate in both samples decreases with the extension of storage time, with a faster degradation rate in the first week of storage, but the retention rate of AST encapsulated by microcapsule powder is significantly higher than that of free AST. After 26 weeks of storage at 4°C, the retention rate of free AST is 42.25%, while the retention rate of AST encapsulated by microcapsule powder is 85.02%, an increase of 42.77%. After 26 weeks of storage at 25°C, the change trend of AST retention rate in both samples is similar to that when stored at 4°C, but the retention rate of AST is lower than that when stored at 4°C. Among them, the retention rate of free AST is only 30.99%, while the retention rate of AST encapsulated by microcapsule powder increases to 75.77%. At the same time, Figure 12B indicates that the degradation curves of all samples are well fitted. After 26 weeks of storage at 4°C, the degradation rate constant value of free AST is 0.0309, and the half-life is 157.02 d, while the degradation rate constant value of AST embedded in the microcapsule powder is 0.0059, and the half-life is 882.38 d, which is 5.62 times that of free AST. The change trend at 25°C is similar to that at 4°C, but the AST degradation rate constant value is larger and the half-life is shorter. Among them, the degradation rate constant value of free AST is 0.0408, and the half-life is 118.92 d, while the degradation rate constant value of AST in the microcapsule powder is 0.0094, and the half-life is 516.17 d, which is 4.34 times that of free AST.

[0123] Example 4 Mouse in vivo absorption test of three AST-loaded nanodispersions, emulsion gels and microcapsule powders

[0124] Animal test and grouping After 8-week-old C57BL / 6N male mice were adaptively fed for one week, they were randomly assigned to 5 groups (10 mice per group), and the test 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), (5) WPC / PUL / AST emulsion gel group (AST-EG). The environmental temperature was 20 ± 2°C, the relative humidity was 50 ± 5%, the 12 h / 12 h day-night light cycle, and the environment was kept clean during the test period. The normal control group was gavaged with tap water, the astaxanthin group was gavaged with AST tap water suspension (astaxanthin is insoluble in water), and the other three groups were gavaged with WPC / PUL / AST microcapsule, WPC / PUL / AST nanodispersion and WPC / PUL / AST emulsion gel pure water solution (the final concentration of ASTA in all systems was 0.1 mmol / L) respectively, continuous gavage for 10 days, gavage amount 0.2 mL per mouse. The mice were taken blood from the eyeball 4 h after gavage, and the blood of the same group of mice was mixed, centrifuged at 8000 g for 5 min, and the upper serum was taken and stored in a -80°C refrigerator.

[0125] Serum astaxanthin detection 0.4 mL of mouse serum was taken and divided into two 5 mL centrifuge tubes, 1.6 mL of dichloromethane and methanol (dichloromethane:methanol = 2:1, v / v) was added to the centrifuge tube, shaken for 1 min, then 0.8 mL of n-hexane was added to the mixture, shaken for 30 s, centrifuged at 8000 g for 5 min, and the supernatant was collected. Repeat 2 times, collect all the supernatant in a brown centrifuge tube, dry with nitrogen, dissolve in 0.1 mL of methanol solution, filter through a 0.22 µm organic membrane, and then perform liquid phase analysis. The results showed that there was no astaxanthin detected in the serum of the normal control group (Con) mice, the astaxanthin in the group of astaxanthin suspension with tap water (AST-w) was about 26 pmol / mL, and the astaxanthin in the serum of the mice in the WPC / PUL / AST microcapsule group (AST-MS), the WPC / PUL / AST nanodispersion group (AST-ND), and the WPC / PUL / AST emulsion gel group (AST-EG) was 7.8 times, 10.0 times and 9.3 times higher than that in the AST-w group, respectively, indicating that the astaxanthin treated by different methods had good in vivo absorption effect.

Claims

1. A method for constructing a carrier embedding astaxanthin based on whey protein / Pullulan, comprising: 1) preparing a whey protein / Pullulan complex (1) Dissolve whey protein powder in distilled water, fully stir at room temperature for 2 h, and store at 4℃ overnight to make it fully hydrated, to obtain a whey protein solution; (2) Add Pullulan to the whey protein solution to obtain a whey protein / Pullulan solution; (3) Heat the whey protein / Pullulan solution in a water bath at 80℃ for 20 min to form a whey protein / Pullulan complex, and cool to room temperature for standby; The whey protein / Pullulan complex has a whey protein concentration of 10% and a Pullulan concentration of 2%; 2) embedding astaxanthin with the whey protein / Pullulan complex Add astaxanthin to soybean oil and magnetically stir to prepare an astaxanthin soybean oil solution with a concentration of 0.2 mg / mL; Mix the astaxanthin soybean oil solution and the whey protein / Pullulan complex, and use a high-speed shearing emulsifier to prepare a whey protein / Pullulan / astaxanthin emulsion; Add 1% gluconic acid-δ-lactone to the whey protein / Pullulan / astaxanthin emulsion, magnetically stir, and stand to form a whey protein / Pullulan / astaxanthin emulsion gel; The astaxanthin soybean oil solution and the whey protein / Pullulan complex are mixed at a volume ratio of 1:

4.

2. The method for embedding astaxanthin with whey protein / pullulan based construct carrier according to claim 1, characterized in that: The standing is at 25℃.

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