Astaxanthin protein nano suspension and preparation method thereof

Abstract

The invention discloses an astaxanthin protein nano suspension and a preparation method thereof, and belongs to the technical field of preparation processing. According to the invention, astaxanthin is wrapped by silk fibroin, edible amaranth protein or bovine serum albumin, the astaxanthin-protein nano suspension with color removed or light can be obtained, and the DPPH clearance rate of the suspension is tested, so that the oxidation resistance of the suspension is not lost but also improved; particularly, the DPPH removal rate of the astaxanthin-amaranth protein nano suspension prepared by wrapping the astaxanthin with the amaranth protein is increased by 1.48 times. The astaxanthin-protein nano suspension prepared by the method can obviously lighten the color of the astaxanthin without loss of oxidation resistance, and the application range of the astaxanthin is widened.

Description

Technical Field

[0001] The invention relates to an astaxanthin protein nano suspension and a preparation method thereof, belonging to the technical field of preparation processing. Background Art

[0002] Astaxanthin is a carotenoid that is mainly found in aquatic animals such as shrimp, crab, and fish. It has a strong antioxidant effect and multiple biological activities. It not only gives aquatic animals bright colors, but also has antioxidant, anti-inflammatory and immune-enhancing effects. There are some limitations in the traditional method of preparing astaxanthin, such as poor water solubility, which limits its application in the fields of food, health products, and medicine. In addition, the stability and bioavailability of astaxanthin are also problems that need to be solved in the prior art. In some applications, such as cosmetics or food additives, lighter astaxanthin may be required to meet specific product requirements. However, due to its dark color, its application in the fields of cosmetics, food, and other fields is limited in terms of color diversity. Therefore, it is of practical application value to develop a method for preparing astaxanthin with a lighter color.

[0003] In recent years, researchers have been committed to improving the stability and water solubility of astaxanthin to increase its application range in different products. For example, the stability and water solubility of astaxanthin can be improved through emulsification technology, microemulsification technology and compounding with other ingredients. In addition, there are also studies that adjust the preparation conditions, such as stirring shear force, surfactant dosage, oil-water phase ratio, and use high-pressure homogenizers to prepare more uniform fine particle droplets to improve the stability of astaxanthin. With the improvement of environmental protection and health awareness, it is particularly important to develop safe and environmentally friendly astaxanthin preparation methods. Some new preparation methods emphasize mild preparation conditions, avoid damage to the astaxanthin structure, and ensure the safety and environmental protection of the product. Although there are some methods to improve the stability and water solubility of astaxanthin, the color will gradually deepen during the process of increasing the concentration of astaxanthin, affecting its subsequent application. How to further reduce the color intensity of astaxanthin while maintaining its biological activity and stability is still a technical challenge. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a light-colored astaxanthin-protein nano suspension in view of the problem that the solution color of the current astaxanthin micro / nano products is dark, resulting in a single product color.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a method for preparing an astaxanthin protein nanosuspension, wherein the preparation method is to prepare an astaxanthin protein nanosuspension by encapsulating astaxanthin with protein, wherein the protein is one or more of silk fibroin, amaranth protein, and bovine serum albumin.

[0007] In one embodiment of the present invention, the preparation method specifically comprises the following steps:

[0008] S1. prepare astaxanthin solution and protein solution respectively;

[0009] S2, adding the astaxanthin solution dropwise to the protein solution at a volume ratio of 1:2-5, stirring or ultrasonicating to obtain a mixed emulsion;

[0010] S3, removing the solvent in the mixed emulsion and hydrating it to obtain the astaxanthin protein nanosuspension.

[0011] In one embodiment of the present invention, the concentration of the astaxanthin solution in step S1 is 0.08-20 g / L.

[0012] In one embodiment of the present invention, the concentration of the astaxanthin solution is preferably 0.08-0.16 g / L.

[0013] In one embodiment of the present invention, the concentration of the astaxanthin solution is preferably 1 to 20 g / L.

[0014] In one embodiment of the present invention, the concentration of the protein solution in step S1 is 0.01-5 g / L.

[0015] In one embodiment of the present invention, the concentration of the protein solution is preferably 0.01-0.04 g / L.

[0016] In one embodiment of the present invention, the concentration of the protein solution is preferably 1-5 g / L.

[0017] In one embodiment of the present invention, the solvent is ethanol, acetone or dichloromethane, which is used to dissolve astaxanthin in the present invention to prepare an astaxanthin solution.

[0018] In one embodiment of the present invention, in step S2, the dropping of the astaxanthin solution and the stirring are both performed at 20-30° C., in the dark, and under the protection of an inert gas.

[0019] In one embodiment of the present invention, in step S2, the stirring is performed at 200 to 500 rpm for 5 to 10 minutes.

[0020] In one embodiment of the present invention, in step S2, the ultrasonic treatment is performed intermittently at 400-500W.

[0021] In one embodiment of the present invention, in step S3, the solvent in the mixed emulsion is removed by evaporation under reduced pressure.

[0022] In one embodiment of the present invention, in step S3, hydration is performed by intermittent ultrasonic treatment at 45-55°C and 250-350W.

[0023] The second object of the present invention is to provide an astaxanthin nanosuspension prepared by the preparation method.

[0024] The third object of the present invention is to provide the use of the astaxanthin nanosuspension in cosmetics, health products or feed.

[0025] Beneficial effects of the present invention:

[0026] The present invention uses silk fibroin, amaranth protein or bovine serum albumin to encapsulate astaxanthin, and can obtain a decolored or lightened astaxanthin-protein nanosuspension, and by testing its DPPH clearance rate, its antioxidant activity is not only not lost, but also improved, especially the DPPH clearance rate of the astaxanthin-amaranth protein nanosuspension prepared by encapsulating astaxanthin with amaranth protein is increased by up to 1.48 times. The astaxanthin-protein nanosuspension prepared by the method of the present invention can significantly lighten the color of astaxanthin without losing antioxidant activity, thereby broadening the application range of astaxanthin. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 is a flow chart for the preparation of astaxanthin-protein nanocomplexes in Examples 1 to 3;

[0029] Figure 2 The astaxanthin-protein nanocomplex sample pictures in Examples 1 to 3;

[0030] Figure 3 is a flow chart for the preparation of astaxanthin-protein nanocomplexes in Examples 4 to 6;

[0031] Figure 4 The astaxanthin-protein nanocomplex sample pictures in Examples 4 to 6;

[0032] Figure 5 This is a sample picture of the astaxanthin-protein nanocomplex in Comparative Examples 1-2. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific examples. These implementation cases are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the claims attached to the application.

[0034] Source of raw materials

[0035] Astaxanthin is a standard product with a purity of ≥98%, Haematococcus aureus oil (5% astaxanthin, provided by Kelani)

[0036] Detection method:

[0037] Determination of encapsulation efficiency of astaxanthin-silk fibroin nanocomplex:

[0038] (1) Determination of astaxanthin-acetone standard curve

[0039] Weigh 6 mg of astaxanthin powder and dissolve it in 10 mL of acetone to obtain 600 mg / L astaxanthin mother solution; dilute to different concentrations, and draw an astaxanthin standard curve based on the liquid phase results. The regression equation of the standard curve is y=14.996x+24.933, R 2 =0.994, where x is the astaxanthin concentration and y is the HPLC peak area.

[0040] (2) The encapsulation efficiency of astaxanthin-silk fibroin nanocomposite was determined by petroleum ether extraction method:

[0041] Take 0.5mL of the prepared astaxanthin-protein nanocomplex, add 5mL of petroleum ether, shake vigorously at 30°C for 5min, let stand for 30min for extraction, transfer the upper layer of liquid to a rotary evaporator, repeat twice until the upper layer has no color. Then vacuum evaporation of the upper layer of liquid at 50°C to remove petroleum ether and precipitate free astaxanthin. Add an appropriate amount of acetone to dissolve it again, and determine the astaxanthin content by high performance liquid chromatography.

[0042] Encapsulation efficiency calculation formula

[0043]

[0044] Antioxidant capacity assay

[0045] Weigh 25 mg of DPPH accurately in a 50 mL brown volumetric flask, dissolve in methanol and dilute to the mark as the DPPH reaction solution. Mix the treated astaxanthin-protein nanocomplex with the above DPPH reaction solution in a ratio of 1:1, react in a 37°C water bath in the dark for 30 minutes, and then perform high performance liquid chromatography to analyze the change in the peak area of ​​DPPH before and after the reaction, and calculate the corresponding DPPH concentration. The control group was treated as follows: the same concentration of astaxanthin ethanol solution was mixed with DPPH in a ratio of 1:1, and the reaction conditions were the same as above.

[0046] The scavenging rate of DPPH free radicals was calculated according to the following formula:

[0047] C o represents the DPPH concentration before the reaction, C i Indicates the DPPH concentration after the reaction.

[0048] Color difference measurement

[0049] The color difference of the sample is measured using a high-precision spectrophotometer and calculated according to the following formula:

[0050] Color difference △E=[(△L) 2 +(△A) 2 +(△B) 2 ] 1 / 2

[0051] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased from commercial sources, or prepared by conventional methods, or are commonly used in the industry.

[0052] Embodiment 1:

[0053] 1) Weigh 0.012 g of astaxanthin into a beaker, add 100 mL of anhydrous ethanol to dissolve the astaxanthin, stir magnetically at 200 r / min for 1 h at room temperature in the dark to prepare a 0.12 mg / mL astaxanthin solution, which is ready for use.

[0054] 2) Prepare silk fibroin into a 0.4 mg / mL stock solution at room temperature, and then prepare the protein solution into gradient solutions of 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, and 0.04 mg / mL. All solutions must be prepared fresh before use.

[0055] 3) At 25° C., in a dark environment and under inert gas protection, the astaxanthin solution of step 1) was rapidly added dropwise to the protein solution prepared in step 2) at a volume ratio of 1:3, and stirred at 300 rpm for 8 minutes until the color of astaxanthin gradually faded.

[0056] 4) In the dark, 25° C., vacuum degree 6 mbar conditions, the ethanol solution was removed by rotary evaporation to allow the ethanol in the system to completely evaporate to form a film, and then the mixture was hydrated at 50° C. and ultrasonicated in an ice bath at 300 W (3 s, 3 s) for 3 min to obtain an astaxanthin-silk fibroin nanosuspension.

[0057] Embodiment 2:

[0058] 1) Weigh 0.012 g of astaxanthin into a beaker, add 100 mL of anhydrous ethanol to dissolve the astaxanthin, stir magnetically at 200 r / min for 1 h at room temperature in the dark to prepare a 0.12 mg / mL astaxanthin solution, which is ready for use.

[0059] 2) Prepare a 0.4 mg / mL stock solution of amaranth protein at room temperature, and then prepare the protein solution into gradient solutions of 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, and 0.04 mg / mL. All solutions must be prepared fresh before use.

[0060] 3) At 25° C., in a dark environment and under inert gas protection, the astaxanthin solution of step 1) was rapidly added dropwise to the protein solution prepared in step 2) at a volume ratio of 1:3, and stirred at 300 rpm for 8 minutes until the color of astaxanthin gradually faded.

[0061] 4) In the dark, 25° C., vacuum degree 6 mbar conditions, the ethanol solution was removed by rotary evaporation to completely evaporate the ethanol in the system to form a film, hydrated at 50° C., 300 W (3s, 3s), 3 min ice bath ultrasound, to obtain astaxanthin-amaranth protein nanosuspension.

[0062] Embodiment 3:

[0063] 1) Weigh 0.012 g of astaxanthin into a beaker, add 100 mL of anhydrous ethanol to dissolve the astaxanthin, stir magnetically at 200 r / min for 1 h at room temperature in the dark to prepare a 0.12 mg / mL astaxanthin solution, which is ready for use.

[0064] 2) Prepare the bovine serum albumin into a 0.4 mg / mL stock solution at room temperature, and then prepare the protein solution into 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, and 0.04 mg / mL gradient solutions. All solutions must be prepared fresh before use.

[0065] 3) At 25° C., in a dark environment and under inert gas protection, the astaxanthin solution of step 1) was rapidly added dropwise to the protein solution prepared in step 2) at a volume ratio of 1:3, and stirred at 300 rpm for 8 minutes until the color of astaxanthin gradually faded.

[0066] 4) In the dark, 25° C., vacuum degree 6 mbar conditions, the ethanol solution was removed by rotary evaporation to completely evaporate the ethanol in the system to form a film, hydrated at 50° C., 300 W (3 s, 3 s), 3 min ice bath sonication, to obtain astaxanthin-bovine serum albumin nanosuspension.

[0067] Example 4

[0068] 1) Weigh a certain amount of Haematococcus pluvialis oil and add it to a beaker containing anhydrous ethanol solution to prepare an ethanol algae oil solution containing about 16 g / L astaxanthin, which is ready for use.

[0069] 2) Prepare silk fibroin into a 5 mg / mL stock solution at room temperature, and then prepare the protein solution into 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL gradient solutions. All solutions must be prepared fresh before use.

[0070] 3) At 25°C, in a dark environment and under inert gas protection, the ethanol algae oil solution of step 1) was quickly added dropwise to the protein solution prepared in step 2) at a volume ratio of 1:3, and emulsified using an ultrasonic crusher with an ultrasonic power of 450W for 3 seconds and a rest period of 3 seconds. Finally, the color of astaxanthin gradually faded.

[0071] 4) In the dark, 25° C., vacuum degree 6 mbar conditions, the ethanol solution was removed by rotary evaporation to allow the ethanol in the system to completely evaporate to form a film, and the mixture was hydrated at 50° C. and ultrasonicated in an ice bath at 300 W (3 s, 3 s) for 3 min to obtain a Haematococcus pluvialis oil-protein nanosuspension.

[0072] Embodiment 5:

[0073] 1) Weigh a certain amount of Haematococcus pluvialis oil and add it to a beaker containing anhydrous ethanol solution to prepare an ethanol algae oil solution containing about 16 g / L astaxanthin, which is ready for use.

[0074] 2) Prepare a 5 mg / mL stock solution of amaranth protein at room temperature, and then prepare the protein solution into 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL gradient solutions. All solutions must be prepared fresh for use.

[0075] 3) At 25°C, in a dark environment and under inert gas protection, the ethanol algae oil solution of step 1) was quickly added dropwise to the protein solution prepared in step 2) at a volume ratio of 1:3, and emulsified using an ultrasonic crusher with an ultrasonic power of 450W for 3 seconds and a rest period of 3 seconds. Finally, the color of astaxanthin gradually faded.

[0076] 4) In the dark, 25° C., vacuum degree 6 mbar conditions, the ethanol solution was removed by rotary evaporation to allow the ethanol in the system to completely evaporate to form a film, and the mixture was hydrated at 50° C. and ultrasonicated in an ice bath at 300 W (3 s, 3 s) for 3 min to obtain a Haematococcus pluvialis oil-protein nanosuspension.

[0077] Embodiment 6:

[0078] 1) Weigh a certain amount of Haematococcus pluvialis oil and add it to a beaker containing anhydrous ethanol solution to prepare an ethanol algae oil solution containing about 16 g / L astaxanthin, which is ready for use.

[0079] 2) Prepare bovine serum albumin at 5g / mL at room temperature, and then prepare the protein solution into 2mg / mL, 3mg / mL, 4mg / mL, and 5mg / mL gradients. All of them need to be prepared and used immediately.

[0080] 3) At 25°C, in a dark environment and under inert gas protection, the ethanol algae oil solution of step 1) was quickly added dropwise to the protein solution prepared in step 2) at a volume ratio of 1:3, and emulsified using an ultrasonic crusher with an ultrasonic power of 450W for 3 seconds and a rest period of 3 seconds. Finally, the color of astaxanthin gradually faded.

[0081] 4) In the dark, 25° C., vacuum degree 6 mbar conditions, the ethanol solution was removed by rotary evaporation to allow the ethanol in the system to completely evaporate to form a film, and the mixture was hydrated at 50° C. and ultrasonicated in an ice bath at 300 W (3 s, 3 s) for 3 min to obtain a Haematococcus pluvialis oil-protein nanosuspension.

[0082] Comparative Example 1:

[0083] 1) Weigh a certain amount of Haematococcus pluvialis oil and add it to a beaker containing anhydrous ethanol solution to prepare an ethanol algae oil solution containing about 16 g / L astaxanthin, which is ready for use.

[0084] 2) Prepare zein into a 5 mg / mL stock solution at room temperature, and then prepare the protein solution into 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL gradient solutions. All solutions must be prepared fresh before use.

[0085] 3) At 25°C, in a dark environment and under inert gas protection, the ethanol algae oil solution of step 1) was quickly added dropwise to the protein solution prepared in step 2) at a volume ratio of 1:3, and an ultrasonic crusher was used for auxiliary embedding. The ultrasonic power was 450W, and the work time was 3s, and the rest time was 3s. Finally, the color of astaxanthin gradually faded.

[0086] Comparative Example 2:

[0087] 1) Weigh a certain amount of Haematococcus pluvialis oil and add it to a beaker containing anhydrous ethanol solution to prepare an ethanol algae oil solution containing about 16 g / L astaxanthin, which is ready for use.

[0088] 2) Prepare wheat protein into a 5 mg / mL stock solution at room temperature, and then prepare the protein solution into 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL gradient solutions. All solutions must be prepared fresh before use.

[0089] 3) At 25°C, in a dark environment and under inert gas protection, the ethanol algae oil solution of step 1) was quickly added dropwise to the protein solution prepared in step 2) at a volume ratio of 1:3, and an ultrasonic crusher was used for auxiliary embedding. The ultrasonic power was 450W, and the work time was 3s, and the rest time was 3s. Finally, the color of astaxanthin gradually faded.

[0090] Test example:

[0091] The astaxanthin-protein nanosuspensions prepared in Examples 1 to 6 and Comparative Examples 1 to 2 were tested for encapsulation efficiency, antioxidant capacity, and color.

[0092] First, we analyze the effect of reducing color intensity. The results are shown in Table 1, Table 2, Figure 2 , Figure 4 and Figure 5 As shown in Table 1 and Figure 2 The effect is specifically based on the effect of Examples 1 to 3, where the protein solution concentration is 0.01 mg / mL. The astaxanthin-protein nanocomplex is compared with the control group. From △E, it can be seen that there are obvious differences between each astaxanthin-protein nanocomplex and the control group. This phenomenon is also consistent with Figure 2 The results are consistent; when the astaxanthin concentration reaches 4g / L, the color of the astaxanthin-protein nanocomplex is significantly reduced after being wrapped with silk fibroin, amaranth protein and bovine serum albumin compared with the control group, while the color is almost not reduced after being wrapped with corn protein and wheat protein, which seriously limits its later application.

[0093] Table 1 Astaxanthin-protein nanosuspension color difference results

[0094] L(depth) A(red, green) B (yellow, blue) △E Comparison 32.94 12.46 14.54 0 Silk Fibroin 38.07 1.38 3.10 16.73 Amaranth protein 37.21 1.55 3.15 16.34 BSA 36.92 2.20 1.44 17.11

[0095] Table 2 Fading degree of astaxanthin-protein nanoemulsion

[0096]

[0097] Furthermore, the encapsulation efficiency of astaxanthin encapsulated by silk fibroin, amaranth protein and bovine serum albumin was tested. The results are shown in Tables 3 and 4. It was found that there was no significant difference in the encapsulation effect of the three proteins, whether it was low concentration astaxanthin or high concentration astaxanthin, and there was no significant difference in the encapsulation effect of proteins of different concentrations. Among them, bovine serum albumin had the best encapsulation effect on astaxanthin, followed by amaranth protein.

[0098] Table 3 Astaxanthin encapsulation efficiency results

[0099]

[0100] Table 4 High concentration astaxanthin encapsulation efficiency results

[0101]

[0102] Further, after astaxanthin was wrapped with silk fibroin, amaranth protein and bovine serum albumin, the antioxidant capacity of astaxanthin-protein nanocomplex was tested. The results are shown in Tables 5 and 6. The results show that the DPPH clearance rate of astaxanthin-protein nanocomplex obtained by wrapping astaxanthin with amaranth protein was greatly improved, up to 1.48 times. The DPPH clearance rate of astaxanthin-protein nanocomplex wrapped with silk fibroin and bovine serum albumin was less than that of the control group. It can be seen that the antioxidant property of astaxanthin is greatly improved after being wrapped with amaranth protein.

[0103] Table 5 DPPH clearance experimental results

[0104]

[0105] Table 6 DPPH clearance experimental results

[0106]

[0107] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing an astaxanthin protein nanosuspension, characterized in that: The preparation method is to prepare astaxanthin protein nano suspension by encapsulating astaxanthin with protein, wherein the protein is one or more of silk fibroin, amaranth protein and bovine serum albumin.

2. The preparation method according to claim 1, characterized in that: The preparation method specifically comprises the following steps: S1. prepare astaxanthin solution and protein solution respectively; S2, adding the astaxanthin solution dropwise to the protein solution at a volume ratio of 1:2-5, stirring or ultrasonicating to obtain a mixed emulsion; S3, removing the solvent in the mixed emulsion and hydrating it to obtain the astaxanthin protein nanosuspension.

3. The preparation method according to claim 2, characterized in that: The concentration of the astaxanthin solution in step S1 is 0.08-20 g / L; the concentration of the protein solution is 0.01-5 g / L.

4. The preparation method according to claim 2, characterized in that: In step S2, the addition of the astaxanthin solution and the stirring are both carried out at 20-30° C., in the dark, and under the protection of an inert gas.

5. The preparation method according to any one of claims 2 or 4, characterized in that: In step S2, stirring is performed at 200 to 500 rpm for 5 to 10 minutes.

6. The preparation method according to claim 2, characterized in that: In step S2, the ultrasonic treatment is performed at 400-500W intermittently.

7. The preparation method according to claim 2, characterized in that: In step S3, the solvent in the mixed emulsion is removed by evaporation under reduced pressure.

8. The preparation method according to claim 2, characterized in that: In step S3, hydration is performed by intermittent ultrasonic treatment at 45-55°C and 250-350W.

9. An astaxanthin nanosuspension prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the astaxanthin nanosuspension according to claim 9 in cosmetics, health products or feed.

Images