A method for improving the stability and bioavailability of astaxanthin by combining piperine with whey protein fiber-algin complex.

Abstract

This invention relates to the field of food processing technology and discloses a method for improving the stability and bioavailability of astaxanthin by combining piperine with a whey protein fibrillary-algin complex. The method specifically includes the following steps: S1. Preparation of whey protein fibrillary; S2. Loading piperine onto whey protein fibrillary; S3. Complexation of piperine-loaded whey protein fibrillary with sodium alginate; S4. Preparation of an astaxanthin-piperine co-encapsulated emulsion. This invention constructs a three-dimensional network resistant to pepsin hydrolysis through electrostatic complexation; enhances the effective concentration of piperine in the intestine through co-encapsulation, efficiently regulating the expression of P-gp efflux transport proteins and the transmembrane absorption of astaxanthin; and utilizes the functional property of piperine to inhibit the activity of P-gp efflux transport proteins to achieve efficient absorption of astaxanthin.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a method for improving the stability and bioavailability of astaxanthin by combining piperine with whey protein fiber-alginate complex. Background Technology

[0002] Astaxanthin (AST), a potent natural ketone carotenoid, has attracted considerable attention in the functional food and pharmaceutical fields due to its superoxide radical scavenging ability and unique anti-inflammatory and anti-apoptotic properties. However, its highly unsaturated conjugated double bond structure makes it prone to cis-isomerization and inactivation during processing and storage. Furthermore, its strong hydrophobicity makes it difficult to dissolve in aqueous digestive environments, and coupled with gastrointestinal degradation and intestinal epithelial barrier effects, its oral bioavailability is less than 10%.

[0003] Existing delivery systems struggle to address the efficiency degradation during intestinal absorption, primarily due to poor mucus permeability limiting access to the absorption interface and low transmembrane transport efficiency. Furthermore, active efflux mediated by P-gp efflux transport proteins further restricts absorption efficacy. Bioenhancement strategies can overcome absorption barriers by inhibiting P-gp efflux or CYP3A4 metabolic enzyme activity. Piperine, as a natural bioenhancement agent, can significantly improve the bioavailability of hydrophobic components like curcumin by altering cell membrane fluidity, inhibiting metabolic enzyme activity, and reducing drug efflux. However, piperine itself suffers from poor water solubility and photosensitivity, and its promoting effect and synergistic mechanism on astaxanthin absorption remain unclear. Summary of the Invention

[0004] The purpose of this invention is to provide a method for improving the stability and bioavailability of astaxanthin by combining piperine with whey protein fibrillary-algin complex, so as to solve the problems mentioned in the background art.

[0005] To address the aforementioned technical problems, this invention provides the following technical solution: a method for improving the stability and bioavailability of astaxanthin by combining piperine with a whey protein fiber / sodium alginate complex, comprising the following steps:

[0006] S1. Preparation of whey protein fibrils: Whey protein powder was prepared into a 2% whey protein solution, the pH was adjusted to 2.0, sonicated for 1 hour, hydrated at 4℃ for 6 hours, stirred in a high-temperature water bath to induce fibrillation, and then immediately placed in an ice-water bath for 30 minutes to prepare a whey protein fibril solution.

[0007] S2. Preparation of piperine solution: Piperine was dissolved in ethanol, and a 10 mg / ml piperine alcohol solution was prepared by stirring in a sealed and light-protected environment.

[0008] S3. Loading of piperine on whey protein fibrils: The whey protein fibrils were ultrasonically treated and then continuously stirred. During the process, piperine alcohol solution was added at a volume ratio of 100:1, ensuring that the ethanol content did not exceed 1% of the total system, thus forming a whey protein fibrils solution encapsulating piperine.

[0009] S4. Preparation of the complex of piperine-loaded whey protein fibrils and sodium alginate: Sodium alginate powder was dissolved in water and stirred for 4 hours to prepare a 1.5% sodium alginate solution. Then, the pH was adjusted to allow the sodium alginate to electrostatically adsorb the piperine-encapsulated whey protein fibrils to form a complex whey protein fibrils (piperine) / sodium alginate.

[0010] S5. Preparation of astaxanthin emulsion: Astaxanthin was dissolved in medium-chain triglycerides (MCT) to prepare 0.1% astaxanthin oil. The astaxanthin oil was added dropwise to the stirred whey protein fibrils (piperine) / sodium alginate aqueous phase and homogenized at high speed to prepare astaxanthin emulsion.

[0011] S6. Application of the piperine co-encapsulation system: The O / W emulsion obtained in step S5 was subjected to thermal stability, ultraviolet stability, bile salt resistance, in vitro simulated digestion, and Caco-2 cell uptake tests to evaluate the system's enhancement of the stability and bioavailability of encapsulated astaxanthin.

[0012] As a further aspect of the present invention, in step S1, the ultrasonic power is 100W, the temperature is controlled at 25±2℃, and the ultrasonic time is 1h.

[0013] As a further aspect of the present invention, the fibrinization conditions of the whey protein isolate solution of the active substance in step S1 are heating at 80°C for 16 hours and stirring at 600 rpm.

[0014] As a further aspect of the present invention, in step S2, the preparation of the piperine alcohol solution involves an ethanol content not exceeding 1% of the total system. The preparation process requires sealing and protection from light, and the stirring speed is 600 rpm.

[0015] As a further embodiment of the present invention, in step S3, whey protein fibrils are first sonicated at 100W for 1 hour, and under continuous stirring conditions (speed 600 r / min), piperine alcohol solution is slowly added; after the solution is added, it is immediately protected from light and sealed, and stirring is continued for 2 hours under the same conditions.

[0016] As a further embodiment of the present invention, in step S4, sodium alginate needs to be stirred at 50°C with a stirring speed of 800 rpm; the pH adjustment process requires first adjusting the pH of whey protein fiber (piperine) to about 4.0, then adding sodium alginate and stirring while adjusting the pH to 5.0, followed by high-speed homogenization at 6000 rpm, and then retesting the pH at 5.0.

[0017] As a further embodiment of the present invention, in step S5, the 0.1% astaxanthin oil is diluted with MCT oil by 10% astaxanthin oil, and the mixture is subjected to 3 cycles of stirring for 2 minutes and ultrasonication for 2 minutes, with a stirring speed of 800 rpm and an ultrasonic power of 100 W; the homogenization speed in the emulsion preparation process is 13000 rpm and the homogenization time is 2 minutes (with a 1-minute pause after each 1-minute homogenization).

[0018] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0019] First, by inducing whey protein fibrils (piperine) to form a stable complex with sodium alginate through electrostatic interaction, the encapsulation efficiency and bioavailability of astaxanthin were significantly improved. Simultaneously, sodium alginate formed a dense three-dimensional network structure through electrostatic complexation, endowing the emulsion with strong steric hindrance and high viscosity, effectively inhibiting droplet aggregation and enhancing resistance to phase separation.

[0020] Secondly, the whey protein fibrils (piperine) / sodium alginate electrostatic complex can resist pepsin hydrolysis, ensuring the complete delivery of astaxanthin to the intestine for efficient release and promoting micellar encapsulation.

[0021] Third, by enhancing the interfacial resistance to bile salt replacement and accelerating the release of free fatty acids through piperine, the bioavailability of astaxanthin is further improved.

[0022] Fourth, by precisely controlling the mass ratio of whey protein fibrils and sodium alginate, the network cross-linking degree and interfacial charge density were optimized, resulting in the best stability (particle size increase of only 7.5% during storage), bioavailability (72.17% ± 1.03%), and cellular uptake rate (37.47% ± 1.54%).

[0023] Fifth, by co-encapsulating piperine and astaxanthin, the effective concentration of piperine in the intestine is increased, which can effectively regulate the expression of P-gp efflux protein and the transmembrane absorption of astaxanthin. Attached Figure Description

[0024] Figure 1 This is a flowchart of an embodiment of the present invention.

[0025] Figure 2 The diagram shows the successful encapsulation of piperine in whey protein fibers and the successful formation of a complex between sodium alginate and whey protein fibers in this invention.

[0026] Figure 3 This is a comparison chart showing the encapsulation efficiency of astaxanthin in the emulsion systems of Examples 1 to 3 and Comparative Examples 1 to 3 of the present invention.

[0027] Figure 4This is a comparison chart of the astaxanthin retention rates in temperature stability of Examples 1 to 3 and Comparative Examples 1 to 3 of the present invention.

[0028] Figure 5 This is a comparison chart showing the astaxanthin retention rates in UV stability of Examples 1 to 3 and Comparative Examples 1 to 3 of the present invention.

[0029] Figure 6 This is a comparative graph showing the fluctuation results of emulsion potential at different bile salt concentrations in the anti-cholesterol replacement ability experiment of Examples 1 to 3 and Control Examples 1 to 3 of the present invention.

[0030] Figure 7 This is a comparison diagram of the emulsion state at the end of in vitro simulated gastric digestion of Examples 1 to 3 and Comparative Examples 1 to 3 of the present invention.

[0031] Figure 8 This is a comparison chart of the bioavailability of astaxanthin after in vitro simulated digestion of Examples 1 to 3 and Control Examples 1 to 3 of the present invention.

[0032] Figure 9 This is a graph comparing the release rates of free fatty acids from the emulsion at different time points during the in vitro simulated digestion process of Examples 1 to 3 and Comparative Examples 1 to 3 of the present invention.

[0033] Figure 10 This is a graph comparing the uptake rate of astaxanthin in Caco-2 after simulated digestion in vitro with or without piperine loading in Example 1, Example 4, and Example 5 of the present invention, respectively. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1

[0036] A method for improving the stability and bioavailability of astaxanthin by combining piperine with a whey protein fiber / sodium alginate complex includes the following steps:

[0037] S1. Preparation of whey protein fibrils: Dissolve whey protein powder in water and stir at 25℃±2℃ (600rpm) for 2h to prepare a 2% whey protein solution. Adjust the pH to 2.0, sonicate at 100W for 1h, hydrate at 4℃ for 6h, and then stir in an 80℃ water bath (600rpm) for 16h to induce fibrilation. After removing the solution, immediately place it in an ice water bath for 30min to prepare a whey protein fibril solution.

[0038] S2. Preparation of piperine solution: Dissolve piperine in ethanol, seal and stir (600 rpm) for 2 h in the dark to prepare a 10 mg / ml piperine alcohol solution;

[0039] S3. Loading of piperine on whey protein fibrils: The whey protein fibrils were ultrasonically treated at 100W for 1 hour, followed by stirring at 600rpm. At the beginning of stirring, piperine alcohol solution was added at a volume ratio of 100:1, ensuring that the ethanol content did not exceed 1% of the total system. Then, the system was immediately sealed and protected from light, and stirred continuously for 2 hours to form a whey protein fibrils (piperine) solution.

[0040] S4. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and subjected to 3 cycles of stirring for 2 minutes followed by ultrasonication for 2 minutes, with a stirring speed of 800 rpm and an ultrasonic power of 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the aqueous phase of whey protein fibrils (piperine) at pH 5.0 with water at a ratio of 1:1 (v / v). The mixture was homogenized at 13000 rpm for 2 minutes (with a 1-minute pause after each homogenization). The astaxanthin emulsion, consisting of 50% oil phase encapsulated by whey protein fibrils (piperine), was named W(P).

[0041] Compare with Example 1

[0042] Referring to the basic process framework of Example 1, the following control experimental system was set up:

[0043] Control group: whey protein fibrillary emulsion system without piperine loading;

[0044] S1. Preparation of whey protein fibrils (same as Example 1);

[0045] S2. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and subjected to 3 cycles of stirring for 2 minutes followed by ultrasonication for 2 minutes. The stirring speed was 800 rpm and the ultrasonic power was 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the aqueous phase of whey protein fibrils at pH 5.0 with water at a ratio of 1:1 (v / v). The mixture was homogenized at 13000 rpm for 2 minutes (with a 1-minute pause after each homogenization) to prepare an astaxanthin emulsion with 50% oil phase encapsulated by whey protein fibrils, named W.

[0046] Example 2

[0047] A method for improving the stability and bioavailability of astaxanthin by combining piperine with a whey protein fiber / sodium alginate complex includes the following steps:

[0048] S1. Preparation of whey protein fibrils: Dissolve whey protein powder in water and stir at 25℃±2℃ (600rpm) for 2h to prepare a 2% whey protein solution. Adjust the pH to 2.0, sonicate at 100W for 1h, hydrate at 4℃ for 6h, and then stir in an 80℃ water bath (600rpm) for 16h to induce fibrilation. After removing the solution, immediately place it in an ice water bath for 30min to prepare a whey protein fibril solution.

[0049] S2. Preparation of piperine solution: Dissolve piperine in ethanol, seal and stir (600 rpm) for 2 h in the dark to prepare a 10 mg / ml piperine alcohol solution;

[0050] S3. Loading of piperine on whey protein fibrils: The whey protein fibrils were ultrasonically treated at 100W for 1 hour, followed by stirring at 600rpm. At the beginning of stirring, piperine alcohol solution was added at a volume ratio of 100:1, ensuring that the ethanol content did not exceed 1% of the total system. Then, the system was immediately sealed and protected from light, and stirred continuously for 2 hours to form a whey protein fibrils (piperine) solution.

[0051] S4. Preparation of whey protein fiber (piperine) and sodium alginate complex: Sodium alginate powder was dissolved in water and stirred at 50℃ (800 rpm) for 4 hours to prepare a 1.5% sodium alginate solution. Then, the pH of the whey protein fiber (piperine) solution was adjusted to about 4.0, and sodium alginate was added at a ratio of 1:1 (v / v). While stirring, the pH was adjusted to 5.0. Then, the mixture was homogenized at 6000 rpm for 30 seconds. The pH was then measured and adjusted to 5.0 to produce a whey protein fiber (piperine) / sodium alginate complex (1:1).

[0052] S5. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and circulated by stirring for 2 min followed by ultrasonication for 2 min three times. The stirring speed was 800 rpm and the ultrasonic power was 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the stirred pH 5.0 whey protein fibrils (piperine) / sodium alginate (1:1) aqueous phase at a ratio of 1:1 (v / v) to water. The mixture was homogenized at 13000 rpm for 2 min (with a 1 min pause after each homogenization). The astaxanthin emulsion, consisting of 50% oil phase encapsulated by whey protein fibrils (piperine) / sodium alginate (1:1), was named W(P) / A(1:1).

[0053] Compare with Example 2

[0054] Referring to the basic process framework of Example 2, the following control experimental system was set up:

[0055] Control group: whey protein fiber / sodium alginate emulsion system without piperine loading but with sodium alginate complexation;

[0056] S1. Preparation of whey protein fibrils (same as Example 2);

[0057] S2. Preparation of whey protein fiber and sodium alginate complex: Sodium alginate powder was dissolved in water and stirred at 50℃ (800 rpm) for 4 h to prepare a 1.5% sodium alginate solution. Then, the pH of the whey protein fiber solution was adjusted to about 4.0, and sodium alginate was added at a ratio of 1:1 (v / v). While stirring, the pH was adjusted to 5.0. Then, the mixture was homogenized at 6000 rpm for 30 s. The pH was then measured and adjusted to 5.0 to produce a whey protein fiber / sodium alginate complex (1:1).

[0058] S3. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and subjected to 3 cycles of stirring for 2 minutes followed by ultrasonication for 2 minutes, with a stirring speed of 800 rpm and an ultrasonic power of 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the stirred pH 5.0 whey protein fibrils / sodium alginate (1:1) aqueous phase at a ratio of 1:1 (v / v) to water. The mixture was homogenized at 13000 rpm for 2 minutes (with a 1-minute pause after each homogenization) to prepare an astaxanthin emulsion with 50% oil phase encapsulated in whey protein fibrils / sodium alginate (1:1), named W / A (1:1).

[0059] Example 3

[0060] A method for improving the stability and bioavailability of astaxanthin by combining piperine with a whey protein fiber / sodium alginate complex includes the following steps:

[0061] S1. Preparation of whey protein fibrils: Dissolve whey protein powder in water and stir at 25℃±2℃ (600rpm) for 2h to prepare a 2% whey protein solution. Adjust the pH to 2.0, sonicate at 100W for 1h, hydrate at 4℃ for 6h, and then stir in an 80℃ water bath (600rpm) for 16h to induce fibrilation. After removing the solution, immediately place it in an ice water bath for 30min to prepare a whey protein fibril solution.

[0062] S2. Preparation of piperine solution: Dissolve piperine in ethanol, seal and stir (600 rpm) for 2 h in the dark to prepare a 10 mg / ml piperine alcohol solution;

[0063] S3. Loading of piperine on whey protein fibrils: The whey protein fibrils were ultrasonically treated at 100W for 1 hour, followed by stirring at 600rpm. At the beginning of stirring, piperine alcohol solution was added at a volume ratio of 100:1, ensuring that the ethanol content did not exceed 1% of the total system. Then, the system was immediately sealed and protected from light, and stirred continuously for 2 hours to form a whey protein fibrils (piperine) solution.

[0064] S4. Preparation of whey protein fiber (piperine) and sodium alginate complex: Sodium alginate powder was dissolved in water and stirred at 50°C (800 rpm) for 4 hours to prepare a 1.5% sodium alginate solution. Then, the pH of the whey protein fiber (piperine) solution was adjusted to about 4.0, and sodium alginate was added at a ratio of 1:2 (v / v). The pH was adjusted to 5.0 while stirring. Then, the mixture was homogenized at 6000 rpm for 30 seconds. The pH was then measured and adjusted to 5.0 to produce a whey protein fiber (piperine) / sodium alginate complex (1:2).

[0065] S5. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and circulated by stirring for 2 min followed by ultrasonication for 2 min three times. The stirring speed was 800 rpm and the ultrasonic power was 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the aqueous phase of whey protein fibrils (piperine) / sodium alginate (1:2) at pH 5.0 at a ratio of 1:1 (v / v) to water. The mixture was homogenized at 13000 rpm for 2 min (with a 1 min pause after each homogenization). The astaxanthin emulsion, consisting of 50% oil phase encapsulated by whey protein fibrils (piperine) / sodium alginate (1:2), was named W(P) / A(1:2).

[0066] Compare with Example 3

[0067] Referring to the basic process framework of Example 3, the following control experimental system was set up:

[0068] Control group: whey protein fiber (piperine) and sodium alginate 2:1 (v / v) emulsion system;

[0069] S1. Preparation of whey protein fibrils (same as Example 3);

[0070] S2. Preparation of piperine solution (same as in Example 3);

[0071] S3. Loading of piperine on whey protein fibrils (same as Example 3);

[0072] S4. Preparation of whey protein fiber (piperine) and sodium alginate complex: Sodium alginate powder was dissolved in water and stirred at 50°C (800 rpm) for 4 hours to prepare a 1.5% sodium alginate solution. Then, the pH of the whey protein fiber (piperine) solution was adjusted to about 4.0, and sodium alginate was added at a ratio of 2:1 (v / v). The pH was adjusted to 5.0 while stirring. Then, the mixture was homogenized at 6000 rpm for 30 seconds. The pH was then measured and adjusted to 5.0 to produce a whey protein fiber (piperine) / sodium alginate complex (2:1).

[0073] S5. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and circulated by stirring for 2 min followed by ultrasonication for 2 min three times. The stirring speed was 800 rpm and the ultrasonic power was 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the stirred pH 5.0 whey protein fibrils (piperine) / sodium alginate (2:1) aqueous phase at a ratio of 1:1 (v / v) to water. The mixture was homogenized at 13000 rpm for 2 min (with a 1 min pause after each homogenization). The astaxanthin emulsion, consisting of 50% oil phase encapsulated by whey protein fibrils (piperine) / sodium alginate (2:1), was named W(P) / A(2:1).

[0074] Example 4

[0075] A method for improving the stability and bioavailability of astaxanthin by combining piperine with a whey protein fiber / sodium alginate complex includes the following steps:

[0076] S1. Preparation of whey protein fibrils: Dissolve whey protein powder in water and stir at 25℃±2℃ (600rpm) for 2h to prepare a 2% whey protein solution. Adjust the pH to 2.0, sonicate at 100W for 1h, hydrate at 4℃ for 6h, and then stir in an 80℃ water bath (600rpm) for 16h to induce fibrilation. After removing the solution, immediately place it in an ice water bath for 30min to prepare a whey protein fibril solution.

[0077] S2. Preparation of piperine solution: Dissolve piperine in ethanol, seal and stir (600 rpm) for 2 h in the dark to prepare a 10 mg / ml piperine alcohol solution;

[0078] S3. Loading of piperine on whey protein fibrils: The whey protein fibrils were ultrasonically treated at 100W for 1 hour, followed by stirring at 600rpm. At the beginning of stirring, piperine alcohol solution was added at a volume ratio of 100:1, ensuring that the ethanol content did not exceed 1% of the total system. Then, the system was immediately sealed and protected from light, and stirred continuously for 2 hours to form a whey protein fibrils (piperine) solution.

[0079] S4. Preparation of whey protein fiber (piperine) and sodium alginate complex: Sodium alginate powder was dissolved in water and stirred at 50°C (800 rpm) for 4 hours to prepare a 1.5% sodium alginate solution. Then, the pH of the whey protein fiber (piperine) solution was adjusted to about 4.0, and sodium alginate was added at a ratio of 1:2 (v / v). The pH was adjusted to 5.0 while stirring. Then, the mixture was homogenized at 6000 rpm for 30 seconds. The pH was then measured and adjusted to 5.0 to produce a whey protein fiber (piperine) / sodium alginate complex (1:2).

[0080] S5. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and circulated by stirring for 2 min followed by ultrasonication for 2 min three times. The stirring speed was 800 rpm and the ultrasonic power was 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the aqueous phase of whey protein fibrils (piperine) / sodium alginate (1:2) at pH 5.0 at a ratio of 1:1 (v / v) to water. The mixture was homogenized at 13000 rpm for 2 min (with a 1 min pause after each homogenization). The astaxanthin emulsion, consisting of 50% oil phase encapsulated by whey protein fibrils (piperine) / sodium alginate (1:2), was named W(P) / A(1:2).

[0081] Compare with Example 4

[0082] Referring to the basic process framework of Example 2, the following control experimental system was set up:

[0083] Control group: whey protein fiber / sodium alginate emulsion system without piperine loading but with sodium alginate complexation;

[0084] S1. Preparation of whey protein fibrils (same as Example 2);

[0085] S2. Preparation of whey protein fiber and sodium alginate complex: Sodium alginate powder was dissolved in water and stirred at 50°C (800 rpm) for 4 hours to prepare a 1.5% sodium alginate solution. Then, the pH of the whey protein fiber solution was adjusted to about 4.0, and sodium alginate was added at a ratio of 1:2 (v / v). While stirring, the pH was adjusted to 5.0. Then, the mixture was homogenized at 6000 rpm for 30 seconds. The pH was then measured and adjusted to 5.0 to produce a whey protein fiber / sodium alginate complex (1:2).

[0086] S3. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and subjected to 3 cycles of stirring for 2 minutes followed by ultrasonication for 2 minutes. The stirring speed was 800 rpm and the ultrasonic power was 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the stirred pH 5.0 whey protein fibrils / sodium alginate (1:2) aqueous phase at a ratio of 1:1 (v / v) to water. The mixture was homogenized at 13000 rpm for 2 minutes (with a 1-minute pause after each homogenization) to prepare an astaxanthin emulsion with 50% oil phase encapsulated in whey protein fibrils / sodium alginate (1:2), named W / A (1:2).

[0087] Example 5

[0088] A method for improving the stability and bioavailability of astaxanthin by combining piperine with a whey protein fiber / sodium alginate complex includes the following steps:

[0089] S1. Preparation of whey protein fibrils: Dissolve whey protein powder in water and stir at 25℃±2℃ (600rpm) for 2h to prepare a 2% whey protein solution. Adjust the pH to 2.0, sonicate at 100W for 1h, hydrate at 4℃ for 6h, and then stir in an 80℃ water bath (600rpm) for 16h to induce fibrilation. After removing the solution, immediately place it in an ice water bath for 30min to prepare a whey protein fibril solution.

[0090] S2. Preparation of piperine solution: Dissolve piperine in ethanol, seal and stir (600 rpm) for 2 h in the dark to prepare a 10 mg / ml piperine alcohol solution;

[0091] S3. Loading of piperine on whey protein fibrils: The whey protein fibrils were ultrasonically treated at 100W for 1 hour, followed by stirring at 600rpm. At the beginning of stirring, piperine alcohol solution was added at a volume ratio of 100:1, ensuring that the ethanol content did not exceed 1% of the total system. Then, the system was immediately sealed and protected from light, and stirred continuously for 2 hours to form a whey protein fibrils (piperine) solution.

[0092] S4. Preparation of whey protein fiber (piperine) and sodium alginate complex: Sodium alginate powder was dissolved in water and stirred at 50°C (800 rpm) for 4 hours to prepare a 1.5% sodium alginate solution. Then, the pH of the whey protein fiber (piperine) solution was adjusted to about 4.0, and sodium alginate was added at a ratio of 2:1 (v / v). The pH was adjusted to 5.0 while stirring. Then, the mixture was homogenized at 6000 rpm for 30 seconds. The pH was then measured and adjusted to 5.0 to produce a whey protein fiber (piperine) / sodium alginate complex (2:1).

[0093] S5. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and circulated by stirring for 2 min followed by ultrasonication for 2 min three times. The stirring speed was 800 rpm and the ultrasonic power was 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the stirred pH 5.0 whey protein fibrils (piperine) / sodium alginate (2:1) aqueous phase at a ratio of 1:1 (v / v) to water. The mixture was homogenized at 13000 rpm for 2 min (with a 1 min pause after each homogenization). The astaxanthin emulsion, consisting of 50% oil phase encapsulated by whey protein fibrils (piperine) / sodium alginate (2:1), was named W(P) / A(2:1).

[0094] Compare with Example 5

[0095] Referring to the basic process framework of Example 2, the following control experimental system was set up:

[0096] Control group: whey protein fiber / sodium alginate emulsion system without piperine loading but with sodium alginate complexation;

[0097] S1. Preparation of whey protein fibrils (same as Example 2);

[0098] S2. Preparation of whey protein fiber and sodium alginate complex: Sodium alginate powder was dissolved in water and stirred at 50°C (800 rpm) for 4 hours to prepare a 1.5% sodium alginate solution. Then, the pH of the whey protein fiber solution was adjusted to about 4.0, and sodium alginate was added at a ratio of 2:1 (v / v). While stirring, the pH was adjusted to 5.0. Then, the solution was homogenized at 6000 rpm for 30 seconds. The pH was then measured and adjusted to 5.0 to produce a whey protein fiber / sodium alginate complex (2:1).

[0099] S3. Preparation of astaxanthin emulsion: 10% astaxanthin oil was diluted with MCT oil and subjected to 3 cycles of stirring for 2 minutes followed by ultrasonication for 2 minutes, with a stirring speed of 800 rpm and an ultrasonic power of 100 W to prepare 0.1% astaxanthin oil. The 0.1% astaxanthin oil was added dropwise to the stirred pH 5.0 whey protein fibrils / sodium alginate (2:1) aqueous phase at a ratio of 1:1 (v / v) to water. The mixture was homogenized at 13000 rpm for 2 minutes (with a 1-minute pause after each homogenization) to prepare an astaxanthin emulsion with 50% oil phase encapsulated in whey protein fibrils / sodium alginate (1:2), named W / A (2:1).

[0100] I. Experimental Methods

[0101] 1. Thermal stability of the emulsion

[0102] Take 3g of emulsion and incubate it in a water bath at different temperatures (room temperature, 60℃ and 90℃) for 30min. Then, vortex the sample for 1min and take 0.1g of the sample to determine the astaxanthin content.

[0103] 2. UV stability

[0104] Take 0.2g of emulsion sample, dilute it 30 times with deionized water, and then irradiate it under ultraviolet light for 0, 2, 4 and 6 hours respectively. Rotate the sample bottle by 90° every half hour, and then take 0.1g of sample for astaxanthin content determination.

[0105] 3. Bile salt resistance

[0106] Bile salts were dissolved in 10 mmol / L phosphate buffer (PBS, pH 7.0) to prepare bile salt solutions with concentration gradients of 0, 2.5, 5, 10, 20, and 50 mg / mL. These solutions were inactivated by boiling in a water bath for 5 min and then cooled. Freshly prepared emulsions were mixed with bile salt solutions of different concentrations at a 1:1 volume ratio and incubated in a 37°C constant-temperature shaking water bath (150 rpm) for 2 h to simulate a competitive environment at the intestinal interface. After terminating the reaction with an ice bath, the samples were diluted with 10 mM PBS (pH 7.2), and the zeta potential change was measured. The absolute value of the potential fluctuation was used to assess the emulsion's resistance to bile salt displacement.

[0107] 3. Determination of astaxanthin content

[0108] The emulsion sample was mixed with dichloromethane / methanol (2:1, v / v), vortexed for 2 min, and then centrifuged at 6000 rpm and 4℃ for 5 min. The absorbance of the organic phase was measured at a wavelength of 475 nm, and quantitative analysis was performed according to the standard curve.

[0109] 5. Biological accessibility

[0110] Mix 2.5g of emulsion with oral simulation solution, bring the volume to 5mL, adjust the pH to approximately 6.8, and stir for 30s to simulate oral chewing. Then, place the mixture in a 37℃ constant-temperature shaker (120r / min) for 30min. Add 5mL of simulated gastric juice (the mixture contains 2000U / ml pepsin), adjust the pH to 3.0 with 1M HCl, and react in a 37℃ constant-temperature shaker (120r / min) for 2h to simulate the gastric peristaltic digestive environment. After the reaction, adjust the pH to 7.0 with 2M NaOH to terminate the gastric digestion stage. Add an equal volume of simulated intestinal juice (total system contains 5mg / ml bile salts, 500U / ml pancreatic enzyme, and 2000U / ml pancreatic lipase) to the digested gastric chyme, and adjust the pH to 7.5 with 2M NaOH. Subsequently, transfer the system to a 37℃ constant-temperature shaker (120r / min) and incubate for 2h. The mixture was then immediately placed in a boiling water bath for 5 minutes to terminate the digestion.

[0111] The digestion solution was stirred at a constant temperature of 37℃ (100 r / min), and titrated with 0.2 M NaOH to maintain a constant pH of 7.0. The volume of NaOH consumed was recorded every 10 min, and the released FFA was calculated from the cumulative amount of NaOH consumed.

[0112]

[0113] Where V represents the cumulative volume of NaOH consumed in the titration (mL), c represents the concentration of the NaOH solution (0.20M), M lipid represents the molar amount of MCT (492g / mol), and W is the total mass of MCT initially present in the digestive emulsion (g).

[0114] The simulated digestion mixture was centrifuged at 4°C and 10000g for 30 min to separate the intermediate layer containing the micelle phase. 1 mL of the intermediate micelle phase was accurately measured and added to 4 mL of a dichloromethane / methanol mixed solvent (2:1 v / v). After vortex extraction for 2 min, the mixture was centrifuged at 6000 rpm for 10 min, and the organic phase was collected. The absorbance at 475 nm was measured using a UV-Vis spectrophotometer, and the astaxanthin content in the micelle phase was calculated based on the astaxanthin standard curve. The bioavailability of astaxanthin was calculated using the following formula:

[0115]

[0116] In the formula, m1 is the mass of astaxanthin in the micelle phase, and m0 is the initial mass of astaxanthin added to the digestion system.

[0117] 6. Cell uptake rate

[0118] Caco-2 cells cryopreserved in liquid nitrogen were rapidly thawed in a 37°C water bath until only trace amounts of ice crystals remained in the cryovials. They were then transferred to DMEM high-glucose medium (containing 10% (v / v) fetal bovine serum, 1% (v / v) non-essential amino acids, and 1% (v / v) penicillin-streptomycin-amphoteric B triple antibody) and centrifuged at 800 rpm for 3 min. The supernatant was discarded, and DMEM was added to the precipitate. The mixture was thoroughly mixed and placed in a T25 culture flask. The cells were incubated at 37°C with 5% relative CO2 and 90% relative humidity. The culture medium was changed every other day. Cells were passaged when they reached 80%-90% confluence.

[0119] Caco-2 cells in logarithmic growth phase were seeded into 96-well plates at approximately 5000-10000 cells per well and cultured at 37°C until cell adhesion (24 h). The culture medium was aspirated, and the cells were washed three times with PBS buffer. 20 μL of digested sample at different dilutions (20, 40, 60, and 80 times) was added to each well, with six replicates for each concentration. A blank control well (containing only culture medium) was also included. After culturing for another 24 h, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for 1-4 h. The absorbance was measured at 450 nm using a microplate reader to calculate cell viability and determine the non-toxic concentrations of each treatment solution for subsequent experiments.

[0120] Caco-2 cells in the logarithmic growth phase were divided into groups of 1 × 10⁻⁶ cells per well. 5 Six-well plates were inoculated and cultured at 37°C for 5 days, with DMEM medium changed every 24 hours to induce differentiation of Caco-2 cells. Cells were then washed three times with PBS buffer to remove surface impurities. A digested emulsion solution diluted 80-fold with DMEM and thoroughly mixed was added, with three replicates per group. Cells were then incubated at 37°C for 6 hours. Pre-chilled DMEM medium was added to stop cell uptake, followed by three washes with PBS. Cells were lysed on ice for 30 minutes using cell lysis buffer (PBS containing 10% ethanol), then sonicated. The cell lysate was centrifuged (10000 rpm, 2 minutes) and the supernatant was collected. The astaxanthin content was determined by HPLC. The cell uptake rate of astaxanthin was calculated using the following formula:

[0121] Cell uptake rate % = Astaxanthin content in cells / Astaxanthin content in the sample before loading × 100%

[0122] Astaxanthin content was determined using a high-performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II) equipped with a DAD detector. The chromatographic column was an Agilent ZORBAX SB-C18 (4.6 × 250 mm, 5 μm). Mobile phase A was 0.1% phosphoric acid water (v / v), and mobile phase B was HPLC-grade acetonitrile. The gradient program was: 0–15 min (B 90% → 95%), 15–35 min (B 95%), 35.1–45 min (B 90%). The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 470 nm. The sample was filtered through a 0.22 μm filter (dissolved in chromatographically pure dichloromethane), and the injection volume was 10 μL.

[0123] II. Experimental Results

[0124] 1. At pH 5.0±0.2, whey protein fibrils and sodium alginate can form a complex through electrostatic adsorption, which significantly enhances the fluorescence of whey protein fibrils. Simultaneously, the addition of piperine significantly reduces the fluorescence intensity of whey protein fibrils, because piperine loading causes fluorescence quenching in the whey protein fibrils. Figure 2 );

[0125] Wherein: WP is whey protein, WPF is whey protein fibrils, WPF(Pip) is whey protein fibrils (piperine), WPF / Alg(1:1) is whey protein fibrils / sodium alginate (1:1), and WPF(Pip) / Alg(1:1) is a complex of whey protein fibrils (piperine) / sodium alginate (1:1).

[0126] 2. In Examples 1 and 2, the loading of piperine did not significantly affect the encapsulation efficiency, heat retention rate, or UV irradiation retention rate of astaxanthin in the W / W / A (1:1) emulsion. However, the addition of piperine improved the emulsion's resistance to bile salt replacement. Figure 5 At a bile salt concentration of 5 mg / ml, the ζ-potential fluctuation of the unloaded piperine W emulsion sample was 6.72 mV. After piperine addition, the ζ-potential fluctuation of W(P) decreased to 3.65 mV. This phenomenon also existed for W(P) / A(1:1) and W / A(1:1). The potential fluctuation of W / A(1:1) without piperine loading was 3.43 mV, while the potential fluctuation of W(P) / A(1:1) with piperine loading was 0.6 mV. The comparison shows that piperine loading can improve the emulsion's resistance to bile salt replacement. This ability is presumably because piperine stabilizes the interfacial membrane through conformational modification of whey protein proficiency fibers.

[0127] The addition of piperine in Examples 1 and 2 (Comparative Examples 1 and 2) improved the bioavailability of astaxanthin. Compared to emulsions without piperine (W and W / A (1:1)), the added piperine emulsions (W(P) and W(P) / A (1:1)) showed significantly improved bioavailability of astaxanthin (21.30% ± 0.63% and 44.22% ± 0.58%, respectively); the addition of piperine emulsions (W and W / A (1:1)) showed significantly improved bioavailability of astaxanthin (17.23% ± 0.65% and 38.66% ± 0.71%, respectively). This is attributed to piperine enhancing resistance to bile salt replacement. Figure 5 This prevents aggregation and improves bioavailability. Furthermore, the potential presence of piperine was found to enhance the activity of digestive enzymes and promote the release of FFA. Figure 9 This promotes the formation of micelles.

[0128] 3. The emulsions prepared from whey protein fibrils / sodium alginate complexes in Examples 1 and 2, and Control Examples 1 and 2, can effectively improve the encapsulation efficiency, heat retention rate, UV irradiation retention rate, and resistance to bile salt replacement of astaxanthin in the emulsion system. Simultaneously, in in vitro simulated digestion, the emulsions prepared from whey protein fibrils / sodium alginate complexes can effectively improve the bioavailability of astaxanthin in the emulsion system. Figure 8 The addition of sodium alginate significantly improved the encapsulation efficiency of astaxanthin in the emulsion (from 95.75% to 99.36%). Figure 3 After the emulsions were incubated in a 90°C water bath for 30 minutes, the emulsion system with sodium alginate complexation showed a significantly higher retention rate of astaxanthin compared to the W and W(P) emulsions without sodium alginate complexation. The highest increase was observed in the W(P) / A (1:2) group (36.46%). Figure 4 During UV irradiation, the retention rate of astaxanthin in the sodium alginate complex emulsion was slightly improved in the first 4 hours after irradiation, but this improvement was not significant. However, after 6 hours of irradiation, the UV resistance advantage of the sodium alginate complex group began to appear. Figure 5 It can be seen that the astaxanthin retention rate of the sodium alginate complex group was significantly improved at this time; the sodium alginate complex group also had an advantage in the ability to resist bile salt replacement, and its potential change was very small regardless of the whey protein fibrin / sodium alginate ratio (Δζ=0.57-0.64mV). Figure 6 In in vitro simulated digestion processes, the sodium alginate complex group exhibited better resistance to gastric digestion, especially the W(P) / A(1:2) group. Figure 7 The improvement in emulsion stability due to sodium alginate complexation is attributed to three aspects: 1. The formation of the complex allows it to form a thicker and stronger adsorption shell on the surface of the oil droplets; 2. Sodium alginate fills the gaps between emulsion droplets, enhancing steric hindrance; 3. Sodium alginate imparts high viscosity to the emulsion, further enhancing steric hindrance.

[0129] 4. In Examples 2, 3, and Control Example 3, the amount of sodium alginate complexed had no significant effect on the temperature stability, UV stability, and resistance to bile salt replacement of astaxanthin in the emulsion, but showed significant differences in its resistance to gastric digestion. Figure 7 Among them, the W(P) / A(1:2) group has good resistance to gastric digestion, effectively resists hydrolysis by gastric enzymes, and ensures the integrity of the droplets until intestinal digestion. During intestinal digestion, bile salts and intestinal enzymes destroy the interfacial protection, while the large surface area of ​​small droplets accelerates digestion and the release of astaxanthin. Figure 9 This promoted micellar solubilization, and the bioavailability of the W(P) / A(1:2) group (72.17% ± 1.03%) was significantly higher than that of other sodium alginate complex groups. Figure 8 ).

[0130] 5. In Examples 2 and 4, and Examples 5 and 5, the loading of piperine significantly increased the uptake of astaxanthin in Caco-2 cells. Compared with the control group without piperine loading, the addition of piperine to the W(P) / A emulsion significantly enhanced cellular astaxanthin uptake. Figure 10 Following digestion of the piperine-free emulsion, the cellular uptake of astaxanthin in the W / A (1:1), W / A (1:2), and W / A (2:1) formulations was 8.35% ± 0.58%, 9.92% ± 0.01%, and 6.75% ± 0.38%, respectively. In contrast, the cellular uptake of astaxanthin after digestion of the peptide-loaded emulsion was significantly increased, reaching 24.82% ± 1.63%, 37.47% ± 1.54%, and 23.97% ± 4.21%, representing increases of 1.97-fold, 2.78-fold, and 2.55-fold, respectively. This improvement can be attributed to two key mechanisms. First, piperine inhibits P-gp efflux activity, thereby reducing cellular elimination of astaxanthin. Furthermore, piperine helps stabilize the micelle structures formed during digestion, thus promoting the solubilization and transport of astaxanthin. Additionally, because piperine is loaded in the aqueous whey protein fibrils, it is released earlier during digestion than in structures containing astaxanthin. This sequential release may promote enhanced absorption by intestinal cells, contributing to improved overall bioavailability.

[0131] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for improving the stability and bioavailability of astaxanthin by combining piperine with a whey protein fiber / sodium alginate complex, characterized in that, Includes the following steps: S1. Preparation of whey protein fibrils: whey protein powder was prepared into a 1.5%-2.5% whey protein solution, the pH was adjusted to 2.0±0.1, sonicated for 1 h, hydrated at 4℃ for 6 h, and then stirred in a high-temperature water bath to induce fibrillation. After taking it out, it was immediately placed in an ice water bath for 30 min to prepare a whey protein fibril solution. S2. Preparation of piperine solution: Dissolve piperine in ethanol, and prepare an alcoholic solution of 10 mg / ml piperine by stirring in a sealed and light-protected environment; S3. Loading of piperine on whey protein fibrils: whey protein fibrils were ultrasonically treated and then continuously stirred. During the process, piperine solution was added at a volume ratio of 100:1 to form a whey protein fibrils solution encapsulated with piperine. S4. Preparation of piperine-loaded whey protein fibrils and sodium alginate complex: Dissolve sodium alginate powder in water and stir for 4 hours to prepare a 1%-2% sodium alginate solution. Subsequently, by adjusting the pH, the whey protein fibers encapsulated with piperine were electrostatically adsorbed with sodium alginate, resulting in a complexation of piperine with whey protein fibers / sodium alginate. S5. Preparation of astaxanthin emulsion: Dissolve astaxanthin in medium-chain triglycerides (MCT) to prepare 0.1%-0.15% astaxanthin oil. Add the astaxanthin oil dropwise to a stirred solution of piperine and whey protein cellulose / sodium alginate complex, homogenize at high speed, and prepare astaxanthin emulsion.

2. The method for improving the stability and bioavailability of astaxanthin by combining piperine with whey protein fibrin / sodium alginate complex according to claim 1, characterized in that, In step S1, the ultrasonic power is 100W, the temperature is controlled at 25±2°C, and the ultrasonic time is 1h.

3. The method for improving the stability and bioavailability of astaxanthin by combining piperine with whey protein fibrin / sodium alginate complex according to claim 1, characterized in that, In step S1, the fibrinization conditions of the whey protein solution are heating at 80°C for 16 hours and stirring at 600 rpm.

4. The method for improving the stability and bioavailability of astaxanthin by combining piperine with whey protein fibrin / sodium alginate complex according to claim 1, characterized in that, In step S2, the piperine alcohol solution must ensure that the ethanol content does not exceed 1% of the total system. The preparation process requires sealing and protection from light, and the stirring speed is 600 rpm.

5. The method for improving the stability and bioavailability of astaxanthin by combining piperine with whey protein fibrin / sodium alginate complex according to claim 1, characterized in that, First, sonicate whey protein fibers at 100W for 1 hour. Then, under continuous stirring conditions, slowly add piperine alcohol solution at a speed of 600 r / min. After adding the solution, immediately protect it from light and seal it, and continue stirring under these conditions for 2 hours.

6. The method for improving the stability and bioavailability of astaxanthin by combining piperine with whey protein fibrin / sodium alginate complex according to claim 1, characterized in that, In step S4, sodium alginate needs to be stirred at 50°C with a stirring speed of 800 rpm. The pH adjustment process requires first adjusting the pH of piperine and whey protein fiber to about 4.0, then adding sodium alginate and stirring while adjusting the pH to 5.0±0.1, followed by high-speed homogenization at 6000 rpm, maintaining the pH at 5.0±0.1 throughout the process.

7. The method for improving the stability and bioavailability of astaxanthin by combining piperine with whey protein fibrin / sodium alginate complex according to claim 1, characterized in that, In step S5, the 0.1% astaxanthin oil is obtained by diluting 10% astaxanthin oil with MCT oil, followed by 2 minutes of stirring and 2 minutes of ultrasonic cycling three times. The stirring speed is 800 rpm and the ultrasonic power is 100W. The emulsion preparation homogenization speed is 13000 rpm and the homogenization time is 2 minutes, with a 1 minute pause after each 1 minute of homogenization.

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