Method for extracting astaxanthin by deep eutectic solvent in cooperation with natural catalyst

Abstract

The application discloses a method for extracting astaxanthin by using deep eutectic solvent and natural catalysts, and relates to the technical field of astaxanthin extraction. The method uses endogenous protease and esterase in shrimp and crab shells as natural catalysts, and comprises the following steps: S1, mixing crushed shrimp and crab shells with an activation solution containing choline chloride, glycerol, water and ascorbic acid, and activating endogenous enzymes by low-temperature infiltration of carbon dioxide; S2, adding lemon acid powder, reacting calcium carbonate in the shells with the lemon acid to generate carbon dioxide bubbles and calcium citrate precipitates, and performing semi-solid self-hydrolysis; S3, adding betaine and then dehydrating to a critical water content; S4, adding a hydrophobic deep eutectic solvent of menthol-n-octanoic acid-lauric acid, and then separating into layers after mixing and standing, wherein endogenous esterase at the interface continues to catalyze the hydrolysis of astaxanthin ester; and S5, precipitating free astaxanthin by low-temperature standing of the deep eutectic solvent phase, and obtaining the product through solid-liquid separation. The method does not need to add enzymes and perform saponification by using acid and alkali, has mild conditions, has high free astaxanthin yield, and can recycle the solvent.

Description

Technical Field

[0001] This invention relates to the field of astaxanthin extraction technology, and more specifically, to a method for extracting astaxanthin using a deep eutectic solvent in conjunction with a natural catalyst. Background Technology

[0002] Astaxanthin is a natural carotenoid that exists primarily in shrimp and crab shells as astaxanthin esters bound to proteins. Conventional methods for extracting astaxanthin from shrimp and crab shells require first disrupting the astaxanthin-protein complex with strong acids or alkalis, followed by extraction with organic solvents, and then alkaline saponification to convert the astaxanthin esters into free astaxanthin. These methods consume large amounts of acids and alkalis, generate significant amounts of wastewater, and the high-temperature, strongly alkaline conditions easily lead to the oxidative degradation of astaxanthin.

[0003] Deep eutectic solvents, as a green solvent, have been reported for astaxanthin extraction. However, existing deep eutectic solvent extraction methods mainly focus on obtaining astaxanthin esters, resulting in low yields of free astaxanthin and requiring subsequent saponification steps. Some studies have attempted to add commercially available enzymes to deep eutectic solvents for hydrolysis, but these enzymes are easily inactivated in non-aqueous deep eutectic solvents and are costly.

[0004] Shrimp and crab shells contain abundant endogenous proteases and esterases. The first step in traditional processing is often high-temperature steaming or drying, which directly deactivates these endogenous enzymes, preventing their utilization. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting astaxanthin using a deep eutectic solvent in conjunction with a natural catalyst. This method uses endogenous proteases and esterases from shrimp and crab shells as natural catalysts, eliminating the need for external commercial enzymes, and employing mild process conditions to achieve a high yield of free astaxanthin.

[0006] This invention is achieved through the following technical solution: A method for extracting astaxanthin using a deep eutectic solvent in conjunction with a natural catalyst includes the following steps: S1: After crushing the shrimp and crab shells, mix them with the activation solution at a weight ratio of 1:0.8 to 1:1.2. Introduce carbon dioxide into the mixture to maintain the pH at 5.0 to 5.5. Soak the mixture at 2 to 6°C for 6 to 10 hours to activate the endogenous proteases and esterases in the shrimp and crab shells.

[0007] The activating solution consists of choline chloride, glycerol, water, and ascorbic acid. The molar ratio of choline chloride to glycerol is 1:0.8 to 1:1.5, the molar ratio of choline chloride to water is 1:4 to 1:6, and the concentration of ascorbic acid is 5 to 15 mmol / L.

[0008] The choline chloride and glycerol in the activation solution can penetrate into the chitin-protein matrix of shrimp and crab shells, weakening the hydrogen bond network, making the matrix loose, and exposing the embedded calcium carbonate and astaxanthin-protein complex. Ascorbic acid acts as an antioxidant to protect astaxanthin while maintaining a reducing environment, which is beneficial for maintaining the activity of endogenous enzymes. The weakly acidic environment maintained by carbon dioxide can inhibit the growth of miscellaneous bacteria and keep the endogenous enzymes in a stable state of activity. Low-temperature wetting avoids thermal inactivation of enzymes. The material obtained in S1 contains water introduced by the activation solution, which serves as the medium for the reaction between citric acid and calcium carbonate in S2.

[0009] S2: The citric acid powder is evenly mixed into the material obtained in S1. The mixture is kept at 10-15℃ for 3-5 hours, and then heated to 32-38℃ for 1-3 hours. This allows the calcium carbonate contained in the shrimp and crab shells to react with the citric acid to generate carbon dioxide bubbles and calcium citrate precipitate. At the same time, endogenous proteases and esterases catalyze the degradation of the astaxanthin-protein complex and the hydrolysis of astaxanthin esters. Throughout the process, the material is in a semi-solid state without the precipitation of flowing water and is not stirred.

[0010] Shrimp and crab shells naturally contain a large amount of calcium carbonate, which reacts with added citric acid in the reaction medium provided by the moisture in the S1 material to produce calcium citrate, carbon dioxide, and water. The carbon dioxide bubbles generated in the reaction create micro-regional disturbances between the materials, replacing mechanical stirring and promoting mass transfer. Calcium citrate is an amorphous precipitate that deposits on the chitinous matrix and can adsorb some endogenous enzymes to form immobilized micro-regions.

[0011] Carbon dioxide partially dissolves in the water in the material, forming a buffer system with pH 5.0–5.5 together with the multi-stage ionization of citric acid. This is the optimal pH range for endogenous proteases and esterases. During the low-temperature stage, esterases are preferentially activated, initiating the hydrolysis of astaxanthin esters. During the heating stage, protease activity reaches its peak, catalyzing the degradation of the astaxanthin-protein complex and releasing astaxanthin.

[0012] S3: Add betaine aqueous solution to the material obtained in S2, mix well, and then dehydrate under an airflow of 25~35℃ and 10~20% relative humidity until the moisture content of the material is 28~32wt%, to obtain a semi-dry material. The dehydrated material is in loose granular form and can be directly used in step S4.

[0013] Betaine replaces water molecules and binds to the enzyme protein surface during dehydration, maintaining the enzyme's active conformation and preventing the endogenous enzyme from denaturing and becoming inactive during water loss. By controlling the water content at 28-32 wt%, the material is placed in a critical state of bound water, where free water is essentially removed while the hydration layer on the enzyme protein surface is retained.

[0014] S4: Add a hydrophobic deep eutectic solvent to the semi-dry material obtained in S3, mix for 40-60 minutes, let stand and separate into layers to obtain a deep eutectic solvent phase containing free astaxanthin.

[0015] The volume-to-weight ratio of the hydrophobic deep eutectic solvent to the semi-dry material is 5:1 to 10:1, that is, 5 to 10 ml of hydrophobic deep eutectic solvent is added per gram of semi-dry material. The hydrophobic deep eutectic solvent is composed of menthol, octanoic acid, and lauric acid, with a molar ratio of menthol to octanoic acid of 1:0.6 to 1:0.8 and a molar ratio of menthol to lauric acid of 1:0.2 to 1:0.4.

[0016] When the hydrophobic deep eutectic solvent comes into contact with semi-dry materials, it rapidly removes the hydration layer, causing the enzymatically cleaved protein fragments to denature and aggregate, exposing astaxanthin. Residual moisture in the semi-dry materials forms an interface upon contact with the hydrophobic deep eutectic solvent. Due to the protection of the enzyme protein by betaine in S3, the endogenous esterase maintains its catalytic activity at this interface, continuing to catalyze the hydrolysis of astaxanthin esters dissolved in the deep eutectic solvent phase into free astaxanthin, thus achieving a synergistic effect between the deep eutectic solvent and the natural catalyst.

[0017] The hydrophobic deep eutectic solvent exhibits good solubility for free astaxanthin while being immiscible with water, forming a distinct two-phase system. After mixing, the mixture is allowed to stand and separate into layers; the upper layer is the deep eutectic solvent phase containing free astaxanthin.

[0018] S5: The deep eutectic solvent phase obtained in S4 is allowed to stand at 2~8℃ for 8~10 hours to precipitate free astaxanthin. The astaxanthin product is obtained by solid-liquid separation.

[0019] Under low-temperature conditions, the solubility of free astaxanthin in menthol-fatty acid type hydrophobic deep eutectic solvents decreases significantly, precipitating as crystals or amorphous precipitates. After solid-liquid separation, the precipitate is washed with pre-cooled ethanol to remove residual fatty acids, and then vacuum dried to obtain the free astaxanthin product.

[0020] As a further improvement, the amount of citric acid powder added in S2 is 4-8 wt% of the dry weight of the shrimp and crab shell powder, and the particle size of the citric acid powder is smaller than that of the shrimp and crab shell powder. The smaller particle size of the citric acid powder preferentially dissolves and reacts with the exposed fine calcium carbonate particles, while the larger particle size of the shrimp and crab shell powder, with its internal calcium carbonate acting as a slow-release source, continues to react, naturally forming a reaction rate gradient, thus ensuring a continuous and stable release of carbon dioxide bubbles.

[0021] As a further improvement, the concentration of the betaine aqueous solution in S3 is 20-40 wt%, and the amount added based on the betaine solid is 1-3 wt% of the dry weight of the shrimp and crab shell powder.

[0022] As a further improvement, the hydrophobic deep eutectic solvent described in S4 is added to the semi-dry material in two stages. The first addition of the deep eutectic solvent accounts for 20-30% of the total volume of the deep eutectic solvent, and the mixture is stirred for 30-60 seconds. During this time, the hydration layer of the material is rapidly stripped away, the protein denatures and aggregates, and a large amount of astaxanthin is exposed. The remaining deep eutectic solvent is then added, and the mixture is stirred or agitated at 25-30°C for 40-60 minutes, allowing endogenous esterases to continuously catalyze the hydrolysis of astaxanthin esters at the interface.

[0023] Furthermore, after the second addition and mixing of the deep eutectic solvent, the system is heated to 35–40 °C and held for 5–10 minutes, then cooled to 8–15 °C at a rate of 0.5–1 °C / min. After standing and separating the phases, the deep eutectic solvent phase is separated. The heating phase causes the deep eutectic solvent and residual water to tend towards homogeneity, while the cooling phase causes the aqueous phase to precipitate again and form a new reaction interface, triggering secondary interfacial catalysis of the residual esterase.

[0024] As a further improvement, the cooling temperature in S5 is preferably 2~4℃. The precipitated astaxanthin is filtered at low temperature, washed with pre-cooled ethanol, and then vacuum dried.

[0025] As a further improvement, after solid-liquid separation in S5, an aqueous phase solid is obtained. The aqueous phase solid is then treated with water to dissolve the betaine, choline chloride, and glycerol. After concentration, it is reused in S1 to prepare the activation solution.

[0026] As a further improvement, after solid-liquid separation in S5, a deep eutectic solvent is obtained. After removing water by vacuum evaporation, at least one of octanoic acid or lauric acid is added to the deep eutectic solvent, and it is reused in S4.

[0027] The technical effects and advantages of this invention are as follows: (1) Using the endogenous proteases and esterases contained in the shrimp and crab shells as natural catalysts, no additional commercial enzymes are needed throughout the process, which significantly reduces the extraction cost and avoids the technical problem of the added enzymes being easily deactivated in non-aqueous deep eutectic solvents.

[0028] (2) By utilizing the in-situ reaction of calcium carbonate contained in the shrimp and crab shells with added citric acid, the self-regulation of pH, the enhancement of mass transfer by carbon dioxide microbubbles, and the in-situ immobilization of some endogenous enzymes by calcium citrate are simultaneously achieved. The entire self-hydrolysis process does not require external mechanical stirring, the process is simple, and the energy consumption is low.

[0029] (3) By pre-protecting the enzyme with betaine and combining the operation of multiple interventions with hydrophobic deep eutectic solvent, the hydration layer is first stripped off in a short time to denature the protein and expose the astaxanthin. Then, the protected endogenous esterase continuously catalyzes the hydrolysis of astaxanthin ester at the interface, realizing the synergistic effect of deep eutectic solvent and natural catalyst at the interface. The yield of free astaxanthin is high and the product does not require subsequent alkaline saponification.

[0030] (4) The entire process operates at a temperature not exceeding 40°C, which is mild and effectively avoids thermal degradation and oxidation loss of astaxanthin, resulting in high product purity.

[0031] (5) Both the hydrophobic deep eutectic solvent and the activator components can be recovered and recycled through simple operation. The process is green and environmentally friendly, with low wastewater discharge, and has good feasibility and economy for industrial scale-up. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the overall process flow of the present invention. Detailed Implementation

[0033] 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.

[0034] As attached Figure 1 This paper describes a method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst. In the following examples, the total astaxanthin content was determined by UV-Vis spectrophotometry at 474 nm, and the ratio of free astaxanthin to astaxanthin esters was analyzed by high-performance liquid chromatography. The yield of free astaxanthin was defined as the percentage of the mass of the obtained free astaxanthin relative to the total mass of astaxanthin in the raw material.

[0035] Raw material description: The raw shrimp and crab shells are fresh or frozen at -20℃. Before use, they should be rinsed with clean water to remove surface impurities. After draining the fresh shrimp and crab shells, the moisture content is measured, and then they are pulverized to 40-80 mesh to obtain shrimp and crab shell powder. All weights of shrimp and crab shell powder mentioned below are wet weights; the dry weight is calculated based on the moisture content. Unless otherwise specified, the proportions of all reagents added are based on the dry weight of the shrimp and crab shell powder.

[0036] If using frozen shrimp and crab shells, thaw them at 4°C for 12 hours after removal, drain, measure the moisture content, and then crush them. Subsequent operations are the same. The endogenous protease and esterase activities of frozen shrimp and crab shells decrease after freezing, so the S1 soaking time should be extended accordingly to 10-12 hours.

[0037] General operating conditions: The activating solution is prepared at room temperature by stirring and dissolving choline chloride, glycerol and water in a certain proportion, and then adding ascorbic acid and stirring to dissolve.

[0038] The carbon dioxide gas is food grade and is introduced by bubbling it into the mixture at a flow rate of 0.1~0.3L / min, while monitoring with a pH meter. The gas is then introduced until the pH stabilizes in the range of 5.0~5.5, at which point the gas supply is stopped and the container is sealed.

[0039] In S2, the citric acid powder is mixed manually or mechanically until uniform. The material is placed in an open or breathable container to allow carbon dioxide gas to escape and prevent pressure buildup, while maintaining a semi-solid state. The semi-solid state without water seepage refers to the absence of liquid seepage or accumulation on the surface of the material upon visual observation.

[0040] S3 dehydration uses a constant temperature and humidity chamber with an air velocity set at 0.5~2m / s. The material is spread thinly at a thickness of 1~3cm. During dehydration, the material is turned over every 5 minutes to ensure uniform dehydration. The moisture content is measured using an infrared moisture analyzer. After dehydration, the material is in a loose granular form. If clumps appear, they are appropriately broken up and dispersed before proceeding to step S4.

[0041] The hydrophobic deep eutectic solvent in S4 is prepared by mixing menthol, octanoic acid, and lauric acid at room temperature until a clear, homogeneous liquid is formed. Mixing is carried out in a jacketed glass or stainless steel container at a stirring speed of 50–150 rpm. The separation time is 20–60 minutes, and the upper deep eutectic solvent phase is removed by decantation or siphoning.

[0042] In S5, cooling and settling are carried out in a freezer or low-temperature circulating tank. Low-temperature filtration uses a Buchner funnel and filter paper pre-cooled to the same temperature as the settling point, followed by vacuum filtration. The pre-cooled ethanol concentration is 95% by volume, and it is pre-cooled at -20°C before use. The vacuum drying temperature is 25~30°C, the vacuum degree is -0.09~-0.095MPa, and the drying time is 4~8 hours.

[0043] Recycling operation: Deep eutectic solvent recovery: The S5 filtrate was subjected to vacuum rotary evaporation at 45℃ and -0.09MPa for 15~30 minutes to remove water. After removing water, samples were taken to analyze the content of octanoic acid and lauric acid. The solution was then added back to the initial molar ratio, stirred until homogeneous, and reused.

[0044] Aqueous phase solids recovery: Add twice the weight of the lower solids separated by S4 to deionized water, stir at 40℃ for 30 minutes, filter, and concentrate the filtrate under reduced pressure at 60℃ and -0.09MPa to 1 / 3 of the original volume. Take a sample to determine the concentrations of choline chloride, glycerol and betaine, and add it to the required concentration of S1 activation solution before reuse.

[0045] Example 1

[0046] Take fresh whiteleg shrimp shells, wash and drain them. The moisture content is measured to be 68 wt%, and the total astaxanthin content is 0.16 wt% on dry weight. Grind them to 60 mesh to obtain shrimp and crab shell powder.

[0047] Weigh 100g of shrimp and crab shell powder (wet weight, dry weight 32g). Weigh 100g of activation solution according to the ratio of wet weight of shrimp and crab shell powder to weight of activation solution of 1:1.

[0048] Activation solution preparation: Mix choline chloride and glycerol in a molar ratio of 1:1, add water to make the molar ratio of choline chloride to water 1:5, and add ascorbic acid to make the concentration 10 mmol / L.

[0049] Mix the shrimp and crab shell powder with the activation solution evenly, and introduce carbon dioxide at a flow rate of 0.2 L / min until the pH reaches 5.3. After sealing, soak at 4°C for 8 hours.

[0050] S2: Grind anhydrous citric acid to 200 mesh. Weigh 1.9g of citric acid powder (calculated as 6wt% of the dry weight of the shrimp and crab shell powder) and mix it evenly into the material obtained in S1. Place the material in a temperature-controlled environment, first maintaining it at 12℃ for 4 hours, then raising the temperature to 35℃ and maintaining it for 2 hours. The material should be a loose, wet powder with no flowing water separation.

[0051] S3: Prepare a 30wt% betaine aqueous solution. Based on the calculation that the betaine solid is 2wt% of the dry weight of shrimp and crab shell powder, weigh out 0.64g of betaine solid, which is equivalent to 2.1g of 30wt% betaine aqueous solution. Spray it onto the material obtained in S2 and stir to mix well.

[0052] The material was spread thinly to a thickness of 2cm and placed in a constant temperature and humidity chamber at 30℃ and 15% relative humidity for dehydration. The wind speed was 1m / s. After dehydration for 25 minutes, the moisture content was measured to be 30wt%, resulting in semi-dry material in the form of loose granules, weighing approximately 49.3g.

[0053] S4: Prepare a hydrophobic deep eutectic solvent with a molar ratio of menthol, octanoic acid, and lauric acid of 1:0.7:0.3.

[0054] Take 345 mL of deep eutectic solvent at a volume-to-weight ratio of 7:1 mL / g. First, add 86 mL of deep eutectic solvent (25% of the total volume) and mix rapidly with the semi-dry material for 45 seconds. Then add the remaining 259 mL and stir at 80 rpm for 50 minutes at 28°C. Allow to stand for 30 minutes to separate the phases, obtaining a deep red deep eutectic solvent phase.

[0055] S5: The deep eutectic solvent phase was placed at 3°C ​​and allowed to stand for 9 hours, resulting in the precipitation of a purplish-black precipitate. The precipitate was filtered through a Buchner funnel pre-cooled to 3°C, washed with 5 mL of pre-cooled ethanol, and dried under vacuum at 25°C and -0.095 MPa for 6 hours to obtain 0.046 g of free astaxanthin product.

[0056] Tests showed that the yield of free astaxanthin was 82%, and the product purity was 91%.

[0057] DES recovery: The S5 filtrate is subjected to rotary evaporation at 45℃ and -0.09MPa for 15 minutes to remove moisture. The sample is analyzed and lauric acid is added to bring the molar ratio to 1:0.7:0.3. The filtrate is then returned to S4 for recycling.

[0058] Aqueous phase solids recovery: Add twice the weight of deionized water to the aqueous phase solids separated in S4, stir at 40°C for 30 minutes, filter, concentrate the filtrate under reduced pressure at 60°C to 1 / 3 of the original volume, add choline chloride, glycerol and ascorbic acid to the initial concentration, and reuse it in S1.

[0059] Example 2

[0060] Take the shells of Antarctic krill, wash and drain them, with a moisture content of 65 wt% and a total astaxanthin content of 0.38 wt% on a dry weight basis, and grind them to 80 mesh.

[0061] Weigh 100g of shrimp and crab shell powder (wet weight, dry weight 35g). Take 110g of S1 activation solution at a wet weight ratio of 1:1.1.

[0062] The activating solution contained choline chloride to glycerol in a molar ratio of 1:1.2, choline chloride to water in a molar ratio of 1:4.5, and ascorbic acid at a concentration of 8 mmol / L. The solution was purged with carbon dioxide to pH 5.1 and incubated at 3°C ​​for 9 hours.

[0063] S2: The amount of citric acid powder added is 5 wt% of the dry weight of the shrimp and crab shell powder, i.e., 1.75 g. First, maintain the temperature at 10℃ for 5 hours, then raise the temperature to 38℃ and maintain it for 1.5 hours.

[0064] S3: The solid betaine is 2 wt% of the dry weight of the shrimp and crab shell powder, i.e., 0.70 g, and 2.8 g of a 25 wt% betaine aqueous solution is used. It is dehydrated to a moisture content of 29 wt% under an airflow at 28℃ and 12% relative humidity to obtain a semi-dry material in the form of loose granules, weighing about 52.7 g.

[0065] S4: The molar ratio of menthol, octanoic acid, and lauric acid in the hydrophobic deep eutectic solvent is 1:0.65:0.35. Take 370 mL of DES according to a deep eutectic solvent to semi-dry material volume-to-weight ratio of 7:1 mL / g. Add in two portions: first, add 93 mL of DES (25% of the total volume), mix rapidly for 30 seconds; second, add the remaining 277 mL, and stir at 30°C for 40 minutes. After mixing, heat the system to 38°C and hold for 8 minutes, then cool to 10°C at a rate of 0.8°C / min. After standing and separating the layers, separate the deep eutectic solvent phase.

[0066] S5: The deep eutectic solvent phase was allowed to stand at 2℃ for 10 hours, filtered at low temperature, washed with pre-cooled ethanol, and dried under vacuum to obtain 0.12g of free astaxanthin product.

[0067] Tests showed that the yield of free astaxanthin was 79%, and the product purity was 88%.

[0068] Example 3

[0069] Take the shell of the red swamp crayfish, wash and drain it. The moisture content is 62 wt%, and the total astaxanthin content is 0.13 wt% on dry weight. Grind it to 40 mesh.

[0070] Weigh 100g of shrimp and crab shell powder (wet weight, dry weight 38g). Take 90g of S1 activation solution at a wet weight ratio of 1:0.9.

[0071] The activating solution contained choline chloride to glycerol in a molar ratio of 1:0.9, choline chloride to water in a molar ratio of 1:5.5, and ascorbic acid at a concentration of 12 mmol / L. The solution was purged with carbon dioxide to pH 5.4 and incubated at 5°C for 7 hours.

[0072] S2: The amount of citric acid powder added is 7 wt% of the dry weight of the shrimp and crab shell powder, i.e., 2.66 g. First, maintain the temperature at 15℃ for 3.5 hours, then raise the temperature to 34℃ and maintain it for 2.5 hours.

[0073] S3: The solid betaine is 1.5 wt% of the dry weight of the shrimp and crab shell powder, i.e., 0.57 g, and 1.63 g of a 35 wt% betaine aqueous solution is used. It is dehydrated to a moisture content of 31 wt% under an airflow at 32℃ and 18% relative humidity to obtain a semi-dry material in the form of loose granules, weighing about 59.8 g.

[0074] S4: The molar ratio of menthol, octanoic acid, and lauric acid in the hydrophobic deep eutectic solvent is 1:0.75:0.25. Take 420 mL of DES according to a deep eutectic solvent to semi-dry material volume-to-weight ratio of 7:1 mL / g. Add in two portions: first, add 92 mL of DES (22% of the total volume), mix rapidly for 50 seconds; second, add the remaining 328 mL, and stir at 26°C for 55 minutes.

[0075] S5: The deep eutectic solvent phase was allowed to stand at 4°C for 8.5 hours, filtered at low temperature, washed with pre-cooled ethanol, and dried under vacuum to obtain 0.042g of free astaxanthin product.

[0076] Tests showed that the yield of free astaxanthin was 74%, and the product purity was 87%.

[0077] Example 4: Effect of DES Cyclic Use Following the operating steps of Example 1, the deep eutectic solvent recovered in S5 was reused in the next batch in step S4, and this cycle was repeated three times. The results are shown in the table below:

[0078] As can be seen from the table above, after three cycles of recycling, the yield of free astaxanthin in the deep eutectic solvent can still be maintained at over 75%, and the product purity can still be maintained at over 87%, indicating that the deep eutectic solvent of the present invention has good reusability.

[0079] Example 5: Recycling and reuse of activation solution Following the operating steps of Example 1, the activated liquid component recovered from the aqueous phase solids separated in S4 through water treatment was reused in S1, and this cycle was repeated three times. The results are shown in the table below:

[0080] As can be seen from the table above, after the activating liquid components are recycled three times, the yield of free astaxanthin decreases by no more than 5 percentage points, indicating that the present invention has good economic and environmental benefits.

[0081] Example 6: Frozen Shrimp Shell Raw Material 100g of Litopenaeus vannamei shells, frozen at -20℃ for 30 days, were taken. After thawing, the moisture content was 67wt%, the dry weight was 33g, and the total astaxanthin content (dry weight) was 0.15wt%. The procedure was the same as in Example 1, except that the soaking time (S1) was extended to 10 hours. Testing showed that the free astaxanthin yield was 79%, the product purity was 89%, and the product weight was 0.044g. This indicates that frozen raw materials are also applicable, requiring only a slightly longer activation soaking time.

[0082] Comparative Example 1: Conventional Deep Eutectic Solvent Extraction Method 100g of fresh shrimp and crab shell powder (wet weight, dry weight 32g) was taken, as in Example 1. The powder was boiled in 95℃ hot water for 10 minutes to inactivate endogenous enzymes. After drying, it was extracted with a choline chloride-glycerol deep eutectic solvent at 50℃ for 2 hours. The extract was then saponified using an alkaline method to obtain free astaxanthin. The yield of free astaxanthin was 51%, and the product purity was 76%.

[0083] Comparative Example 2: Direct DES extraction after enzyme inactivation, without saponification. 100g of shrimp and crab shell powder (wet weight, dry weight 32g) was taken as in Example 1. The powder was boiled in 95℃ hot water for 10 minutes to inactivate endogenous enzymes. After drying, it was directly extracted using the same hydrophobic deep eutectic solvent as in Example 1, following steps S4 and S5, but omitting steps S1-S3. The yield of free astaxanthin was 18%, and the product purity was 82%. This comparative example demonstrates that without endogenous enzyme participation in hydrolysis, relying solely on DES physical extraction results in an extremely low yield of free astaxanthin.

[0084] Comparative Example 3: Hydrolysis using only the aqueous phase system, without the use of DES 100g of shrimp and crab shell powder (wet weight, dry weight 32g) was taken, and the procedure was performed according to steps S1-S3 of Example 1. In step S4, the hydrophobic deep eutectic solvent was replaced with an equal volume of deionized water. After stirring for 50 minutes, the supernatant was collected by centrifugation and freeze-dried to obtain the extract. The yield of free astaxanthin was 31%, and the product purity was 45%. This comparative example shows that relying solely on endogenous enzyme hydrolysis without DES capture, free astaxanthin cannot be effectively transferred from the solid phase to the liquid phase, resulting in unsatisfactory yield and purity.

[0085] Comparative Example 4: No betaine added The procedure was the same as in Example 1, except that in S3, no betaine aqueous solution was added; dehydration was performed directly. Testing showed that the yield of free astaxanthin was 57%, and the product purity was 79%.

[0086] Comparative Example 5: DES added without multiple additions The procedure was the same as in Example 1, except that in step S4, all 345 mL of the hydrophobic deep eutectic solvent was added to the semi-dry material at once and mixed for 50 minutes. Testing revealed that the free astaxanthin yield was 68% and the product purity was 84%.

[0087] Comparative Example 6: No heating-cooling operation performed The procedure was the same as in Example 2, except that after the second addition of DES and mixing, the temperature was not raised to 38°C and then lowered to 10°C; instead, the mixture was allowed to stand and separate. Testing showed that the yield of free astaxanthin was 71%, and the product purity was 85%.

[0088] Parameter optimization experiment To determine the optimal amount of citric acid powder added in S2, based on Example 1 and keeping other conditions constant, the yield of free astaxanthin was investigated when the amount of citric acid added was 2 wt%, 4 wt%, 6 wt%, 8 wt%, and 10 wt% of the dry weight of shrimp and crab shell powder. The results are shown in the table below:

[0089] As can be seen from the table above, the optimal amount of citric acid to be added is 4 to 8 wt% of the dry weight of the shrimp and crab shell powder.

[0090] To determine the optimal amount of betaine added in S3, based on Example 1 and keeping other conditions constant, the yield of free astaxanthin was investigated when the amount of betaine added was 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, and 4 wt% of the dry weight of shrimp and crab shell powder. The results are shown in the table below:

[0091] As can be seen from the table above, the optimal amount of betaine added is 1 to 3 wt% of the dry weight of shrimp and crab shell powder.

[0092] To determine the optimal ratio of hydrophobic deep eutectic solvent in S4, based on Example 1 and keeping other conditions constant, the effects of different molar ratios of menthol, caprylic acid, and lauric acid on the yield of free astaxanthin were investigated. The results are shown in the table below:

[0093] As shown in the table above, the free astaxanthin yield reached over 75% when the molar ratio of menthol to caprylic acid was 1:0.6 to 1:0.8 and the molar ratio of menthol to lauric acid was 1:0.2 to 1:0.4.

[0094] All the above embodiments are operated under normal pressure conditions, and the equipment used is conventional stirring tank, constant temperature and humidity chamber, refrigerator, vacuum distillation device and vacuum drying oven. No high pressure, ultra-low temperature or special material equipment is required, and industrial scale-up is feasible.

[0095] Detection method: Determination of total astaxanthin content: Accurately weigh 0.5g of shrimp and crab shell powder or extract, add 10mL of acetone, extract by sonication in an ice bath for 15 minutes, centrifuge at 4000rpm for 10 minutes, take the supernatant, repeat the extraction until the residue is colorless, combine the supernatants, and make up to 50mL with acetone. Measure the absorbance at 474nm using a UV-Vis spectrophotometer, and calculate the content by plotting a standard curve using astaxanthin standards.

[0096] Determination of free astaxanthin purity: High-performance liquid chromatography (HPLC) was used. A C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was methanol:acetonitrile:dichloromethane = 70:20:10 (v / v), the flow rate was 1.0 mL / min, the detection wavelength was 474 nm, and the injection volume was 20 μL. The retention time of free astaxanthin was approximately 8.2 min, astaxanthin monoester approximately 12–15 min, and astaxanthin diester approximately 18–22 min. The purity of free astaxanthin was calculated using the peak area normalization method.

[0097] Protease activity verification: Take 2g of the material before and after S1 infiltration, add 10mL of phosphate buffer (pH 5.5), homogenize in an ice bath, centrifuge at 4℃, collect the supernatant, and determine the enzyme activity at 680nm using the Folin-phenol reagent method with tyrosine as the standard. Esterase activity verification: Use p-nitrophenol butyrate as the substrate, and determine the formation rate of p-nitrophenol at 405nm.

[0098] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Those skilled in the art should understand that the parameters in the specific embodiments can be modified or replaced without departing from the technical principles of the present invention, and such modifications and replacements should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting astaxanthin using a deep eutectic solvent in conjunction with a natural catalyst, characterized in that, Includes the following steps: S1: After crushing the shrimp and crab shells, mix them with the activation solution at a weight ratio of 1:0.8 to 1:1.

2. Introduce carbon dioxide into the mixture to maintain the pH at 5.0 to 5.

5. Soak the mixture at 2 to 6°C for 6 to 10 hours to activate the endogenous proteases and esterases in the shrimp and crab shells as natural catalysts. The activation solution is composed of choline chloride, glycerol, water, and ascorbic acid. The molar ratio of choline chloride to glycerol is 1:0.8 to 1:1.5, the molar ratio of choline chloride to water is 1:4 to 1:6, and the concentration of ascorbic acid is 5 to 15 mmol / L. S2: Citric acid powder is evenly mixed into the material obtained in S1. First, it is kept at 10~15℃ for 3~5 hours, and then the temperature is raised to 32~38℃ and kept for 1~3 hours. This allows the calcium carbonate contained in the shrimp and crab shells to react with citric acid in the reaction medium provided by the water contained in the material in S1 to generate carbon dioxide bubbles and calcium citrate precipitate. At the same time, endogenous proteases and esterases catalyze the degradation of astaxanthin-protein complex and the hydrolysis of astaxanthin ester. Throughout the process, the material is in a semi-solid state without the precipitation of flowing water and no external stirring is required. S3: Add betaine aqueous solution to the material obtained in S2, mix well, and dehydrate under an airflow of 25~35℃ and 10~20% relative humidity until the moisture content of the material is 28~32wt% to obtain semi-dry material; S4: Add a hydrophobic deep eutectic solvent to the semi-dry material obtained in S3. The volume-to-weight ratio of the hydrophobic deep eutectic solvent to the semi-dry material is 5:1~10:1 mL / g. After mixing for 40~60 minutes, allow the mixture to stand and separate into layers to obtain a deep eutectic solvent phase containing free astaxanthin. During the mixing process, the residual water in the semi-dry material comes into contact with the hydrophobic deep eutectic solvent to form an interface. The endogenous esterase protected by betaine in S3 continues to catalyze the hydrolysis of astaxanthin esters at this interface. The hydrophobic deep eutectic solvent is composed of menthol, octanoic acid, and lauric acid. The molar ratio of menthol to octanoic acid is 1:0.6~1:0.8, and the molar ratio of menthol to lauric acid is 1:0.2~1:0.

4. S5: The deep eutectic solvent phase obtained in S4 is allowed to stand at 2~8℃ for 8~10 hours to precipitate free astaxanthin. The astaxanthin product is obtained by solid-liquid separation.

2. The method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst according to claim 1, characterized in that, The amount of citric acid powder added in S2 is 4-8 wt% of the dry weight of the shrimp and crab shell powder, and the particle size of the citric acid powder is smaller than that of the shrimp and crab shell powder.

3. The method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst according to claim 1 or 2, characterized in that, The concentration of the betaine aqueous solution mentioned in S3 is 20~40wt%, and the amount added based on the betaine solid is 1~3wt% of the dry weight of the shrimp and crab shell powder.

4. The method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst according to claim 1, characterized in that, The hydrophobic deep eutectic solvent described in S4 is added to the semi-dry material in two parts: the first addition of deep eutectic solvent accounts for 20-30% of the total volume of deep eutectic solvent, and is mixed for 30-60 seconds; the second addition of the remaining deep eutectic solvent is added, and the mixture is stirred or turned at 25-30°C for 40-60 minutes.

5. The method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst according to claim 4, characterized in that, After the second addition of the deep eutectic solvent and mixing, the system is heated to 35-40℃ and held for 5-10 minutes, then cooled to 8-15℃ at a rate of 0.5-1℃ / min. After standing and separating the phases, the deep eutectic solvent phase is separated.

6. The method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst according to claim 1 or 2, characterized in that, The settling temperature in S5 is 2~4℃.

7. The method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst according to claim 1 or 2, characterized in that, The astaxanthin precipitated from the solid-liquid separation in S5 was filtered at low temperature, washed with pre-cooled ethanol, and then vacuum dried.

8. The method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst according to claim 1 or 2, characterized in that, After solid-liquid separation in S5, an aqueous solid phase is obtained. The aqueous solid phase is treated with water to dissolve betaine, choline chloride, and glycerol. After concentration, it is reused in S1 to prepare the activation solution.

9. The method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst according to claim 4, characterized in that, After solid-liquid separation in S5, an aqueous solid phase is obtained. The aqueous solid phase is treated with water to dissolve betaine, choline chloride, and glycerol. After concentration, it is reused in S1 to prepare the activation solution.

10. The method for extracting astaxanthin using a deep eutectic solvent and a natural catalyst according to claim 1 or 2, characterized in that, After solid-liquid separation in S5, a deep eutectic solvent is obtained. The deep eutectic solvent is then evaporated under reduced pressure to remove moisture, and at least one of octanoic acid or lauric acid is added. This solvent is then reused in S4.

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