Method for improving bioavailability of astaxanthin in phaffia rhodozyma powder and application thereof

Abstract

The present application relates to the technical field of chemical preparations, in particular to a method for improving the bioavailability of astaxanthin in phaffia rhodozyma powder and application. The present application uses composite enzymes to pretreat phaffia rhodozyma powder, so that astaxanthin is fully released from the cells, and then specially-made eutectic solvent is used to efficiently extract the pretreated phaffia rhodozyma powder, and the extraction rate can reach more than 90%. The astaxanthin extract prepared by the present application has high antioxidant activity and high bioavailability, and the DPPH free radical scavenging rate can be up to 89.14%, the ABTS free radical scavenging rate can be up to 90.09%, and the bioavailability can be up to 81.41%. In addition, the present application is simple to operate, does not need ultrasonic, microwave and other assisted methods, has low requirements on equipment, can be suitable for industrial large-scale production, provides a feasible scheme for the green and efficient extraction of astaxanthin, and has a wide application prospect in the food, cosmetic and feed industries.

Description

Technical Field

[0001] This application relates to the field of chemical preparation technology, specifically to a method and application for improving the bioavailability of astaxanthin in red Pharfoay yeast powder. Background Technology

[0002] Astaxanthin (3,3'-dihydroxy-4,4'-diketo-β-carotene) has the molecular formula C 40 H 52 O4, or carotenoids, possesses strong antioxidant, anti-inflammatory, and anti-aging bioactivities and has broad application prospects in food, pharmaceuticals, feed, and cosmetics. *Phaeocystis erythrorhizoma* has become the most promising microorganism for the bioproduction of astaxanthin due to its advantages such as rapid heterotrophic metabolism of various sugars, short culture cycle, simple culture conditions, and ability to achieve high-density culture in fermenters. However, astaxanthin is an intracellular pigment of *Phaeocystis erythrorhizoma*, and enzymes in the animal intestines are unable to break down the cell walls of *Phaeocystis erythrorhizoma*, preventing the release of astaxanthin and making it difficult to digest and absorb, resulting in extremely low bioavailability of astaxanthin in *Phaeocystis erythrorhizoma* powder.

[0003] Deep eutectic solvents (DES) are a novel type of green solvent composed of a mixture of hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) in a specific ratio. The formation of hydrogen bonds between HBAs and HBDs lowers the lattice energy of the components themselves, resulting in a relatively stable mixture with a lower melting point than the individual components. The raw materials for deep eutectic solvents are mostly derived from green and natural substances, reducing environmental impact and improving their acceptability in food production. Although deep eutectic solvents have shown great potential in astaxanthin extraction, current methods often require the addition of ultrasonic or microwave techniques to achieve efficient extraction.

[0004] Currently, several technologies exist for extracting astaxanthin from *Phaeff's red yeast*. Existing technology 1 involves using dimethyl sulfoxide (DMSO) to disrupt the cell walls of *Phaeff's red yeast*, combined with ultrasonic treatment, followed by acetone extraction to obtain the extract. Compared to existing technology 1, this invention uses a natural, green, and non-toxic eutectic solvent as the extraction medium. This eutectic solvent is composed of natural components such as choline chloride, amino acids, organic acids, and egg white peptides, offering significant advantages in terms of being green, safe, and biodegradable. More importantly, by selecting hydrogen bond donors and acceptors in the eutectic solvent and adjusting the molar ratio and water content, a solvent system with high solubility and stability for astaxanthin can be formed, laying the foundation for improving its bioavailability. Existing technology 2 discloses a method for extracting intracellular astaxanthin from *Phaeff's red yeast* using β-glucanase and chitinase. This technology relies on supercritical CO2 extraction, which suffers from high equipment investment, high operating pressure, high CO2 consumption, and low astaxanthin content in the extract. Compared with prior art 2, this invention uses eutectic solvent extraction, avoiding the use of complex equipment, simplifying operation, and making it easier to achieve large-scale production. Prior art 3 discloses a method for extracting astaxanthin using a hydrophobic eutectic solvent. This method uses Pharfovia rubescens powder or Haematococcus pluvialis as raw materials. After cell disruption, a hydrophobic eutectic solvent, represented by menthol-propionic acid, is added and ultrasonic extraction is assisted to obtain astaxanthin extract, achieving efficient and rapid extraction of astaxanthin. Prior art 4 discloses equipment and process for efficient continuous extraction of astaxanthin using a low-frequency microwave-assisted eutectic solvent method. This method pretreats the eutectic solvent with low-frequency microwaves, and then combines ultrasonic-assisted extraction with subsequent solvent extraction to achieve astaxanthin extraction. Although techniques 3 and 4 use eutectic solvent extraction to improve extraction efficiency to some extent, they rely on physical auxiliary means such as ultrasound and microwaves, resulting in complex equipment systems and high energy consumption. Compared with existing technologies 3 and 4, this invention pre-treats the red Pharbitis yeast powder with enzymatic hydrolysis, allowing astaxanthin to be fully released from the cells. Then, the pre-treated red Pharbitis yeast powder is efficiently extracted using a eutectic solvent, with an extraction rate of over 90%.

[0005] In summary, this invention provides a method and application for improving the bioavailability of astaxanthin in red phaf yeast powder. It has the advantages of simple operation, high extraction efficiency, and environmental friendliness. It does not require ultrasonic or microwave methods to assist in cell wall disruption and extraction, has low production costs, and is suitable for large-scale industrial production. It is of great significance for food, cosmetics, feed processing and other fields. Summary of the Invention

[0006] The purpose of this invention is to provide a method and application for improving the bioavailability of astaxanthin in red phaf yeast powder.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 5-40 mL, add the compound enzyme, and enzymatically hydrolyze for 2-24 h at 30-50℃ and pH 4.5-6.5. After enzymatic hydrolysis, centrifuge at 3500-8000 rpm for 5-30 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0009] (2) Mix hydrogen bond donors and hydrogen bond acceptors in a molar ratio of 1:1 to 1:4, adjust the water content to 10% to 40%, heat to 40 to 80°C, stir for 1 to 2 h until a clear and transparent eutectic solvent is formed.

[0010] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). First, the eutectic solvent is preheated to 30~80℃, and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 5~40 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 30~80℃ for 1~4 h, centrifuged at 3500~8000 rpm for 5~30 min, and the supernatant is collected to obtain astaxanthin extract.

[0011] The complex enzyme mentioned in step (1) is two of β-glucanase, β-mannanase, flavor protease and papain, with an enzyme activity of 20,000~100,000 U / g and an enzyme activity ratio of 1:1-1:5.

[0012] The hydrogen bond donor in step (2) includes at least one of lactic acid, glycerol, citric acid, trehalose, mannose, sorbitol, and egg white peptide; the hydrogen bond acceptor includes at least one of betaine, choline chloride, glycine, lysine, and L-proline.

[0013] The amino acid sequence of the egg white peptide (hydrogen bond donor) described in this invention is shown in the table below:

[0014] Table 1. Amino acid sequence listing of the egg white peptides used in this invention (SEQ ID No. 1)

[0015] No peptide sequence m / z [Da] Protein source 1 KLTEWTSSNVMEERKIKVYLPR 1354.23363 ovalbumin 2 KLTEWTSSNVME 712.83994 ovalbumin 3 ELPFASGDLSML 640.31557 ovalbumin 4 ELPYASGDLSM 591.77100 ovalbumin 5 KLTEWTSSNVMEE 777.36124 ovalbumin 6 KLTEWTSSNVMEERKI 976.00131 ovalbumin 7 KLTEWTSSNVM 648.31865 ovalbumin 8 AFKDEDTQAMP 626.77935 ovalbumin 9 AFKDEDTQAMPF 700.31356 ovalbumin 10 AFNAEDTREMPFH 782.84609 ovalbumin 11 AFKDEDTQAMPFR 778.36412 ovalbumin 12 AFNAEDTREMP 640.78243 ovalbumin

[0016] This invention provides an astaxanthin extract with high bioavailability, which is more easily absorbed and utilized by the animal intestines and can be applied in feed formulation or nutritional food development.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) The present invention uses a compound enzyme to pre-treat the red Paffo yeast powder with mild cell wall breaking conditions and complete cell wall breaking, so that astaxanthin is fully released from the cell. Then, a low eutectic solvent is used to efficiently extract the red Paffo yeast powder after cell wall breaking, which greatly improves the extraction rate of astaxanthin, and the extraction rate of astaxanthin can reach more than 90%.

[0019] (2) The present invention uses a eutectic solvent to replace traditional organic solvents such as dimethyl sulfoxide and acetone for the extraction of astaxanthin. Its advantages are as follows: ① The eutectic solvent is composed of food-grade or natural components, which are non-toxic and harmless and conform to the principles of green chemistry. ② The eutectic solvent is designable. By selecting hydrogen bond donors and acceptors and adjusting the molar ratio and water content, a solvent system with high solubility and stability for astaxanthin can be formed, laying the foundation for improving its bioavailability. ③ The raw materials that make up the eutectic solvent are inexpensive and easy to prepare. The solvent is easy to recycle, which reduces the production cost and has the potential for industrial application. ④ The eutectic solvent replaces traditional organic solvents as a new type of green extractant for astaxanthin, avoiding the toxicity risk and environmental pollution of organic solvent residues.

[0020] (3) The astaxanthin extract prepared by the present invention has high antioxidant activity and high bioavailability. Its DPPH free radical scavenging rate can reach up to 89.14%, its ABTS free radical scavenging rate can reach up to 90.09%, and its bioavailability can reach up to 81.41%. The bioavailability is 16 times higher than that of untreated Pharbitis fulvidraco powder and 5 times higher than that of Pharbitis fulvidraco powder after enzymatic hydrolysis.

[0021] (4) The present invention is easy to operate, does not require ultrasonic or microwave methods to assist in cell wall breaking and extraction, has low equipment requirements, does not require expensive production line modifications or special equipment investment to adapt to special processes such as ultrasonic and microwave, reduces production costs, and is suitable for large-scale industrial production. Attached Figure Description

[0022] Figure 1 This is a standard curve diagram for the quantitative determination of astaxanthin involved in the embodiments of this application.

[0023] Figure 2 This is a visual diagram of the astaxanthin eutectic solvent extract involved in the embodiments of this application.

[0024] Figure 3 The image shows the appearance of extracts from astaxanthin extracted with other solvents involved in the comparative examples of this application.

[0025] Figure 4 This is a gastrointestinal digestion appearance diagram of the extract involved in the embodiments of this application. Detailed Implementation

[0026] The following embodiments are intended to further illustrate the technical solutions, operating steps and technical effects of the present invention through specific implementation methods and experimental data, so as to enable those skilled in the art to more intuitively and thoroughly understand the principles and application scenarios of the present invention.

[0027] Example 1:

[0028] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 5 mL, add β-glucanase and β-mannanase with an enzyme activity ratio of 1:1, and enzymatically hydrolyze for 2 h at 30℃ and pH 4.5. After enzymatic hydrolysis, centrifuge at 3500 rpm for 5 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0029] (2) Mix choline chloride and lactic acid in a molar ratio of 1:4, adjust the water content to 10%, heat to 40°C, stir for 1 h until a clear and transparent eutectic solvent is formed.

[0030] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is preheated to 30°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 5 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 30°C for 1 h. The mixture is centrifuged at 3500 rpm for 5 min and the supernatant is collected to obtain astaxanthin extract.

[0031] Example 2:

[0032] (1) Red Paffo yeast powder was dispersed in deionized water at a solid-liquid ratio of 1 g: 40 mL, and β-glucanase and papain were added at an enzyme activity ratio of 1:5. Enzymatic hydrolysis was carried out at 50℃ and pH 6.5 for 24 h. After the enzymatic hydrolysis was completed, the mixture was centrifuged at 8000 rpm for 30 min, and the supernatant was removed to obtain the pretreated red Paffo yeast powder;

[0033] (2) Mix choline chloride and citric acid in a 1:1 molar ratio, adjust the water content to 40%, heat to 80°C, stir for 2 h until a clear and transparent eutectic solvent is formed.

[0034] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is preheated to 80°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 40 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 80°C for 4 h and centrifuged at 8000 rpm for 30 min. The supernatant is collected to obtain astaxanthin extract.

[0035] Example 3:

[0036] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 15 mL, add β-mannanase and flavor protease with an enzyme activity ratio of 1:4, and enzymatically hydrolyze for 2 h at 35℃ and pH 5.0. After enzymatic hydrolysis, centrifuge at 5000 rpm for 10 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0037] (2) Mix choline chloride and glycerol in a molar ratio of 1:3, adjust the water content to 20%, heat to 60°C, stir for 1 h, until a clear and transparent eutectic solvent is formed;

[0038] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is preheated to 35°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 15 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 35°C for 3 h. The mixture is centrifuged at 5000 rpm for 10 min and the supernatant is collected to obtain astaxanthin extract.

[0039] Example 4:

[0040] (1) Disperse red Paffo yeast powder in deionized water at a solid-liquid ratio of 1 g: 25 mL, add papain and flavor protease with an enzyme activity ratio of 1:3, and enzymatically hydrolyze for 12 h at 45℃ and pH 5.5. After enzymatic hydrolysis, centrifuge at 7000 rpm for 20 min, remove the supernatant, and obtain the pretreated red Paffo yeast powder;

[0041] (2) Mix choline chloride and trehalose in a molar ratio of 1:2, adjust the water content to 20%, heat to 50°C, stir for 1 h until a clear and transparent eutectic solvent is formed.

[0042] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is first preheated to 50°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 35 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 50°C for 3 h and centrifuged at 7000 rpm for 20 min. The supernatant is collected to obtain astaxanthin extract.

[0043] Example 5:

[0044] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 35 mL, add β-glucanase and flavor protease with an enzyme activity ratio of 1:2, and enzymatically hydrolyze for 10 h at 35℃ and pH 5.0. After enzymatic hydrolysis, centrifuge at 6000 rpm for 25 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0045] (2) Mix choline chloride and sorbitol in a molar ratio of 1:4, adjust the water content to 10%, heat to 70°C, stir for 1 h until a clear and transparent eutectic solvent is formed.

[0046] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is first preheated to 70°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 25 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 70°C for 2 h and centrifuged at 6000 rpm for 25 min. The supernatant is collected to obtain astaxanthin extract.

[0047] Example 6:

[0048] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 20 mL, add papain and flavor protease with an enzyme activity ratio of 1:4, and enzymatically hydrolyze for 8 h at 40℃ and pH 6.0. After enzymatic hydrolysis, centrifuge at 8000 rpm for 25 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0049] (2) Mix betaine and lactic acid in a molar ratio of 1:3, adjust the water content to 20%, heat to 65°C, stir for 1 hour until a clear and transparent eutectic solvent is formed.

[0050] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is first preheated to 45°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 20 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 45°C for 4 h and centrifuged at 8000 rpm for 25 min. The supernatant is collected to obtain astaxanthin extract.

[0051] Example 7:

[0052] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g:15 mL, add β-glucanase and flavor protease with an enzyme activity ratio of 1:1, and enzymatically hydrolyze for 16 h at 45℃ and pH 6.5. After enzymatic hydrolysis, centrifuge at 6500 rpm for 15 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0053] (2) Mix betaine and glycerol in a molar ratio of 1:2, adjust the water content to 30%, heat to 75°C, stir for 2 hours until a clear and transparent eutectic solvent is formed;

[0054] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is first preheated to 65°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 15 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 65°C for 1 h and centrifuged at 6500 rpm for 15 min. The supernatant is collected to obtain astaxanthin extract.

[0055] Example 8:

[0056] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 8 mL, add β-glucanase and flavor protease with an enzyme activity ratio of 1:2, and enzymatically hydrolyze for 6 h at 32℃ and pH 4.8. After enzymatic hydrolysis, centrifuge at 4000 rpm for 8 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0057] (2) Mix betaine and mannose in a 1:1 molar ratio, adjust the water content to 30%, heat to 45°C, stir for 1.5 h until a clear and transparent eutectic solvent is formed;

[0058] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is first preheated to 32°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 8 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 32°C for 2.5 h, centrifuged at 4000 rpm for 8 min, and the supernatant is collected to obtain astaxanthin extract.

[0059] Example 9:

[0060] (1) Red Paffo yeast powder was dispersed in deionized water at a solid-liquid ratio of 1 g: 18 mL, and β-mannanase and papain were added at an enzyme activity ratio of 1:5. Enzymatic hydrolysis was carried out at 48℃ and pH 6.2 for 20 h. After the enzymatic hydrolysis was completed, the mixture was centrifuged at 7500 rpm for 18 min, and the supernatant was removed to obtain the pretreated red Paffo yeast powder.

[0061] (2) Mix betaine and egg white peptide in a molar ratio of 1:2, adjust the water content to 20%, heat to 70°C, stir for 1.2 h until a clear and transparent eutectic solvent is formed;

[0062] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is preheated to 60°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 18 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 60°C for 2 h and centrifuged at 7500 rpm for 18 min. The supernatant is collected to obtain astaxanthin extract.

[0063] Example 10:

[0064] (1) Red Paffo yeast powder was dispersed in deionized water at a solid-liquid ratio of 1 g: 30 mL, and β-glucanase and papain were added at an enzyme activity ratio of 1:4. Enzymatic hydrolysis was carried out at 42℃ and pH 5.8 for 14 h. After the enzymatic hydrolysis was completed, the mixture was centrifuged at 5500 rpm for 22 min, and the supernatant was removed to obtain the pretreated red Paffo yeast powder.

[0065] (2) Mix betaine and citric acid in a molar ratio of 1:3, adjust the water content to 40%, heat to 55°C, stir for 1.8 h until a clear and transparent eutectic solvent is formed;

[0066] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is first preheated to 55°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 30 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 55°C for 3.5 h and centrifuged at 5500 rpm for 22 min. The supernatant is collected to obtain astaxanthin extract.

[0067] Example 11:

[0068] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 12 mL, add flavor protease and papain with an enzyme activity ratio of 1:3, and enzymatically hydrolyze for 4 h at 37℃ and pH 5.2. After enzymatic hydrolysis, centrifuge at 7200 rpm for 12 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0069] (2) Mix lysine and trehalose in a 1:1 molar ratio, adjust the water content to 10%, heat to 65°C, stir for 1 h until a clear and transparent eutectic solvent is formed.

[0070] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is first preheated to 38°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 12 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 38°C for 1.5 h and centrifuged at 7200 rpm for 12 min. The supernatant is collected to obtain astaxanthin extract.

[0071] Example 12:

[0072] (1) Red Paffo yeast powder was dispersed in deionized water at a solid-liquid ratio of 1 g: 22 mL, and β-mannanase and flavor protease were added at an enzyme activity ratio of 1:1. Enzymatic hydrolysis was carried out at 35℃ and pH 4.6 for 18 h. After the enzymatic hydrolysis was completed, the mixture was centrifuged at 4800 rpm for 28 min, and the supernatant was removed to obtain the pretreated red Paffo yeast powder.

[0073] (2) Mix lysine and egg white peptides in a molar ratio of 1:4, adjust the water content to 40%, heat to 50°C, stir for 2 h until a clear and transparent eutectic solvent is formed.

[0074] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is preheated to 48°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 22 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 48°C for 2 h and centrifuged at 4800 rpm for 28 min. The supernatant is collected to obtain astaxanthin extract.

[0075] Example 13:

[0076] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 6 mL, add β-glucanase and β-mannanase with an enzyme activity ratio of 1:5, and enzymatically hydrolyze for 10 h at 40℃ and pH 6.0. After enzymatic hydrolysis, centrifuge at 3800 rpm for 6 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0077] (2) Mix L-proline and glycerol in a 1:1 molar ratio, adjust the water content to 30%, heat to 75°C, stir for 1.5 h until a clear and transparent eutectic solvent is formed;

[0078] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharrellis powder described in step (1). The eutectic solvent is first preheated to 72°C and then added to the pretreated Pharrellis powder. The solid-liquid ratio is 1 g: 6 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 72°C for 3.8 h, centrifuged at 3800 rpm for 6 min, and the supernatant is collected to obtain astaxanthin extract.

[0079] Example 14:

[0080] (1) Red Paffo yeast powder was dispersed in deionized water at a solid-liquid ratio of 1 g: 28 mL, and β-glucanase and papain were added at an enzyme activity ratio of 1:3. Enzymatic hydrolysis was carried out at 34℃ and pH 5.5 for 22 h. After the enzymatic hydrolysis was completed, the mixture was centrifuged at 6800 rpm for 14 min, and the supernatant was removed to obtain the pretreated red Paffo yeast powder;

[0081] (2) Mix L-proline and egg white peptide in a molar ratio of 1:2, adjust the water content to 20%, heat to 60°C, stir for 1 h, until a clear and transparent eutectic solvent is formed.

[0082] (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharbitis erythrorhizon powder described in step (1). The eutectic solvent is first preheated to 58°C and then added to the pretreated Pharbitis erythrorhizon powder. The solid-liquid ratio is 1 g: 28 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 58°C for 1.2 h and centrifuged at 6800 rpm for 14 min. The supernatant is collected to obtain astaxanthin extract.

[0083] The formulation and test results of the eutectic solvent used in the embodiments of the present invention are shown in the table below:

[0084] Table 2. Eutectic solvent formulations and test results used in the embodiments of the present invention.

[0085] Hydrogen bond acceptor / HBA Hydrogen bond donor / HBD Mole ratio Moisture content Extraction rate / % Content / μg / mL <![CDATA[OD 600 ]]> Example 1 choline chloride lactic acid 1：4 10% 88.60±0.34 294.14±1.13 0.39±0.02 Example 2 choline chloride Citric acid 1：1 40% 76.89±0.28 255.27±0.94 0.23±0.01 Example 3 choline chloride glycerin 1：3 20% 72.40±0.84 240.38±2.80 0.28±0.02 Example 4 choline chloride Trehalose 1：2 20% 85.04±0.50 282.33±1.66 0.27±0.01 Example 5 choline chloride Sorbitol 1：4 10% 77.00±0.34 255.64±1.13 0.27±0.01 Example 6 betaine lactic acid 1：3 20% 11.91±0.03 39.54±0.11 0.09±0.00 Example 7 betaine glycerin 1：2 30% 96.04±0.34 318.84±1.13 0.64±0.03 Example 8 betaine Mannose 1：1 30% 18.23±0.09 60.52±0.30 0.10±0.00 Example 9 betaine Egg white peptides 1：2 20% 97.08±0.59 322.29±1.96 1.57±0.01 Example 10 betaine Citric acid 1：3 40% 27.71±0.39 91.98±1.28 0.18±0.00 Example 11 Lysine Trehalose 1：1 10% 45.51±0.04 151.08±0.12 0.13±0.00 Example 12 Lysine Egg white peptides 1：4 40% 42.15±0.06 139.94±0.21 0.14±0.00 Example 13 L-proline glycerin 1：1 30% 9.87±0.02 32.77±0.07 0.07±0.00 Example 14 L-proline Egg white peptides 1：2 20% 9.87±0.02 32.76±0.07 0.07±0.00

[0086] Example 15:

[0087] Several enzymatic hydrolysis methods were selected, with 1 g of Pharrellis redis yeast powder added to each. The hydrolysis volume was 25 mL, the pH was adjusted to 5.0, the enzyme reaction temperature was 40℃, and the reaction was carried out with gentle shaking for 24 h. The hydrolysate was then placed in boiling water for 10 min, centrifuged at 8000 rpm for 15 min at 4℃, the supernatant was discarded, and the solution was extracted with acetone. The extract was centrifuged at 3500 rpm for 5 min at 4℃, and the supernatant was collected. The astaxanthin extraction rate was determined. The enzymatic hydrolysis effects of different methods are shown in the table below:

[0088] Table 3 Enzymatic hydrolysis effects of different enzymatic hydrolysis methods

[0089] Types of enzymes Types of enzymes enzyme activity ratio Extraction rate / % blank / / / 2.81±0.04 Enzymatic hydrolysis method 1 β-glucanase β-Mannanase 1：1 23.88±0.60 Enzymatic hydrolysis method 2 β-glucanase Flavor proteases 1：3 42.84±0.26 Enzymatic hydrolysis method 3 β-glucanase Papain 1：3 22.2±0.18 Enzymatic hydrolysis method 4 β-Mannanase Flavor proteases 1：2 93.79±0.58 Enzymatic hydrolysis method 5 β-Mannanase Papain 1：5 32.21±0.36 Enzymatic hydrolysis method 6 Flavor proteases Papain 1：4 33.168±3.78

[0090] Comparative Example 1:

[0091] Astaxanthin was extracted from pretreated Pharfoays yeast powder using acetone, following these steps:

[0092] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 25 mL, add β-glucanase and β-mannanase with an enzyme activity ratio of 1:1, and enzymatically hydrolyze for 3 h at 40℃ and pH 5.0. After enzymatic hydrolysis, centrifuge at 8000 rpm for 15 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0093] (2) Add 25 mL of acetone to the pretreated red Pharbitis yeast powder described in step (1), vortex to dissolve it quickly, then centrifuge at 8000 rpm for 5 min, collect the supernatant, and obtain the acetone extract of astaxanthin.

[0094] Comparative Example 2:

[0095] Astaxanthin was extracted from pretreated Phaefoetida yeast powder using ethanol, following these steps:

[0096] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 25 mL, add β-glucanase and β-mannanase with an enzyme activity ratio of 1:1, and enzymatically hydrolyze for 3 h at 40℃ and pH 5.0. After enzymatic hydrolysis, centrifuge at 8000 rpm for 15 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0097] (2) Add 25 mL of ethanol to the pretreated red Pharbitis yeast powder described in step (1), vortex to dissolve it quickly, then centrifuge at 8000 rpm for 5 min, collect the supernatant, and obtain the ethanol extract of astaxanthin.

[0098] Comparative Example 3:

[0099] Astaxanthin was extracted from pretreated Phaefoetida yeast powder using water extraction, following these steps:

[0100] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 25 mL, add β-glucanase and β-mannanase with an enzyme activity ratio of 1:1, and enzymatically hydrolyze for 3 h at 40℃ and pH 5.0. After enzymatic hydrolysis, centrifuge at 8000 rpm for 15 min, remove the supernatant, and obtain the pretreated Pharbitis rubescens powder;

[0101] (2) Add 25 mL of water to the pretreated red phaf yeast powder described in step (1), vortex to dissolve it quickly, then centrifuge at 8000 rpm for 5 min, collect the supernatant, and obtain the water extract of astaxanthin.

[0102] Comparative Example 4:

[0103] Red Pavlov yeast powder is unprocessed.

[0104] Comparative Example 5:

[0105] The steps for pre-treated Pharvestia redis yeast powder without extraction using eutectic solvents are as follows:

[0106] (1) Disperse Pharbitis rubescens powder in deionized water at a solid-liquid ratio of 1 g: 25 mL, add β-glucanase and β-mannanase with an enzyme activity ratio of 1:1, and enzymatically hydrolyze for 3 h at 40 °C and pH 5.0. After enzymatic hydrolysis, centrifuge at 8000 rpm for 15 min, remove the supernatant, and obtain pretreated Pharbitis rubescens powder, which is then dried at 37 °C for 1 h.

[0107] Experimental Example 1:

[0108] Astaxanthin of known concentration was dissolved in acetone, and the UV absorbance at 474 nm was measured under different concentration conditions. A standard curve of astaxanthin content was plotted, with an astaxanthin concentration gradient of 0. g / mL, 2 g / mL, 2.5 g / mL, 3 g / mL, 3.5 g / mL, 4 g / mL, 4.5 g / mL, 5 g / mL, 5.5 g / mL. Standard curve as follows: Figure 1 As shown.

[0109] Experimental Example 2:

[0110] The optical density (OD) values ​​of extracts from different types of eutectic solvents were measured at 600 nm using a UV spectrophotometer, with each sample measured at least three times in parallel. The test results are shown in Table 2.

[0111] Experimental Example 3:

[0112] 1 mL of the extract was added to 3 mL of 0.1 mmol / L DPPH ethanol solution and mixed. The mixture was incubated in the dark for 30 min, and the absorbance was measured at 517 nm. The DPPH radical scavenging rate of each extract was calculated. The test results are shown in Table 4.

[0113]

[0114] Where: A1 is the absorbance of the sample group; A0 is the absorbance of the blank group; A2 is the absorbance of the control group.

[0115] Sample group: 1 mL extract + 3 mL DPPH solution

[0116] Blank group: 1 mL extract + 3 mL anhydrous ethanol

[0117] Control group: 1 mL anhydrous ethanol + 3 mL DPPH solution

[0118] Experiment Example 4:

[0119] An equal volume of 2.45 mM potassium persulfate and 7 mM ABTS reagent was mixed and incubated at room temperature in the dark for 16 h. Before testing, the absorbance was adjusted to 0.70 ± 0.20 by diluting the stock solution with anhydrous ethanol. 0.5 mL of the extract was mixed with 3 mL of the adjusted ABTS ethanol solution and incubated in the dark for 6 min. The absorbance was measured at 734 nm. The ABTS radical scavenging rate of each extract was calculated. The test results are shown in Table 4.

[0120]

[0121] Where: A1 is the absorbance of the sample group; A0 is the absorbance of the blank group; A2 is the absorbance of the control group.

[0122] Sample group: 1 mL extract + 3 mL ABTS solution

[0123] Blank group: 1 mL extract + 3 mL anhydrous ethanol

[0124] Control group: 1 mL anhydrous ethanol + 3 mL ABTS solution

[0125] Table 4 Antioxidant activity of extracts from different types of eutectic solvents

[0126] DPPH free radical scavenging rate / % ABTS free radical scavenging rate / % Comparative Example 4 17.31±2.83 16.08±4.06 Comparative Example 5 16.48±3.51 14.88±3.82 Example 1 30.56±4.08 21.74±2.84 Example 2 24.34±3.83 15.79±3.78 Example 3 25.36±1.82 19.08±0.22 Example 4 25.57±3.32 22.43±2.89 Example 5 24.39±3.01 21.18±3.63 Example 7 89.14±0.80 89.77±2.56 Example 9 82.93±1.63 90.09±1.96

[0127] Experimental Example 5:

[0128] Different types of extracts were mixed with an equal volume of simulated gastric fluid electrolyte stock solution (SGF). The pH of the mixture was adjusted to 3.0 using 1.0 M hydrochloric acid solution and incubated for 2 h in a constant temperature shaker (37℃, 120 rpm). After gastric digestion, the pH was adjusted to 7.0 to terminate pepsin hydrolysis. An equal volume of simulated intestinal fluid electrolyte stock solution (SIF) was added to the gastric digestive fluid, and the mixture was incubated for 2 h in a constant temperature shaker (37℃, 120 rpm). Immediately after the reaction, the mixture was placed in an ice bath to terminate enzyme activity, centrifuged at 8000 rpm / min for 15 min at 4℃, and the supernatant was collected to determine the bioavailability of different types of extracts. The gastrointestinal digestion appearance of the extracts is shown in the figure below. Figure 4 As shown in Table 5, the bioavailability test results are as follows.

[0129]

[0130] Where: C m C represents the amount of astaxanthin remaining after enzymatic hydrolysis. i The amount of raw astaxanthin contained in the extract

[0131] Table 5 Bioavailability of extracts from different types of eutectic solvents

[0132] Bioavailability / % Comparative Example 4 5.16±0.01 Comparative Example 5 16.45±0.65 Example 1 57.16±2.44 Example 2 81.41±2.16 Example 7 31.30±1.04 Example 9 26.58±0.82

[0133] It should also be understood that the foregoing embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention. Equivalent changes or reasonable substitutions made by those skilled in the art to the relevant technical solutions without departing from the technical concept and substance of the present invention should all be considered to fall within the scope of protection of the present invention.

Claims

1. A method for improving the bioavailability of astaxanthin in Phaefurostyle erythrorhizon powder, characterized in that, Includes the following steps: (1) Disperse red Paffia yeast powder in deionized water at a solid-liquid ratio of 1 g: 5~40 mL, add compound enzyme, and enzymatically hydrolyze for 2~24 h at 30~50℃ and pH 4.5~6.5; after enzymatic hydrolysis, centrifuge at 3500~8000 rpm for 5~30 min, remove supernatant, and obtain pretreated red Paffia yeast powder; (2) Mix hydrogen bond donors and hydrogen bond acceptors in a molar ratio of 1:1 to 1:4, adjust the water content to 10% to 40%, heat to 40 to 80°C, stir for 1 to 2 h until a clear and transparent eutectic solvent is formed. (3) The eutectic solvent described in step (2) is used to extract the pretreated Pharrellis powder described in step (1). First, the eutectic solvent is preheated to 30~80℃, and then added to the pretreated Pharrellis powder. The solid-liquid ratio is 1g: 5~40 mL. The mixture is quickly vortexed to dissolve the powder. The extraction is carried out at 30~80℃ for 1~4 h, centrifuged at 3500~8000 rpm for 5~30 min, and the supernatant is collected to obtain astaxanthin extract.

2. The method for improving the bioavailability of astaxanthin in Pharfoays yeast powder according to claim 1, characterized in that: The complex enzyme mentioned in step (1) is two of β-glucanase, β-mannanase, flavor protease and papain, with an enzyme activity of 20,000~100,000 U / g and an enzyme activity ratio of 1:1-1:

5.

3. The method for improving the bioavailability of astaxanthin in Pharfoays yeast powder according to claim 1, characterized in that: In step (2), the eutectic solvent contains at least one hydrogen bond donor, including lactic acid, glycerol, citric acid, trehalose, mannose, sorbitol, and egg white peptide; and at least one hydrogen bond acceptor, including betaine, choline chloride, glycine, lysine, and L-proline.

4. The method for improving the bioavailability of astaxanthin in Pharfoays yeast powder according to claim 3, characterized in that: The egg white peptide is any one of the active peptides with the amino acid sequence shown in SEQ ID No.

1.

5. A method for improving the bioavailability of astaxanthin in Phaefuer's yeast powder according to any one of claims 1 to 4, characterized in that, The astaxanthin extraction rate of the Rhodopseudomonas erythrosporum powder was 9.87%~97.08%, and the astaxanthin content was 32.76~322.29 μg / mL.

6. A highly bioavailable astaxanthin extract, characterized in that, The astaxanthin extract was prepared by the method for improving the bioavailability of astaxanthin in Pharfia redis yeast powder as described in claim 5. The astaxanthin extract had a DPPH free radical scavenging rate of 16.48%~89.14%, an ABTS free radical scavenging rate of 15.79%~90.09%, and a bioavailability of 26.58%~81.41%.

7. The application of the highly bioavailable astaxanthin extract according to claim 6, characterized in that, It can be used in feed formulation or in the preparation of nutritional foods.

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