A method for extracting astaxanthin from marine organisms

Abstract

The application discloses a method for extracting astaxanthin from marine organisms and relates to the technical field of marine organism extraction. The method comprises the following steps: preparing an upper-layer hydrophobic natural eutectic solvent and a lower-layer hydrophilic natural eutectic solvent respectively to form a two-phase natural eutectic solvent system, wherein the upper-layer hydrophobic phase is dispersed with a fat-soluble antioxidant additive; putting marine organism raw materials into the lower-layer hydrophilic phase to remove minerals and proteins; alternately applying ultrasonic waves of two frequencies to make astaxanthin free and migrate to the upper-layer hydrophobic phase to form a hydrogen bond network with the antioxidant additive to realize in-situ protection; and obtaining astaxanthin extraction phase, protein water phase and chitin solid phase after phase separation. The application realizes shell breaking, in-situ protection of astaxanthin and cooperative recovery of chitin and proteins under mild conditions, realizes food-grade cascade production extraction, and realizes food-grade green cascade production extraction.

Description

Technical Field

[0001] This invention relates to the field of marine biological extraction technology, specifically a method for extracting astaxanthin from marine organisms. Background Technology

[0002] Astaxanthin is a carotenoid pigment with multiple conjugated double bonds and ketone and hydroxyl functional groups in its molecular structure. It has strong antioxidant activity and is widely used in food, health products and aquatic feed. Astaxanthin in the shells of shrimp and crabs is usually tightly bound to chitin and calcium carbonate in the form of astaxanthin-protein complex. Astaxanthin molecules are highly unsaturated and unstable. They are prone to oxidative degradation under light, high temperature, strong acid, strong alkali and oxygen conditions, and are isomerized from the all-trans configuration to the less active cis configuration. Traditional extraction processes use strong acid demineralization, strong alkali deproteinization and high temperature and high pressure treatment. Although this improves the apparent yield, the severe chemical and thermal stress will cause a large amount of degradation and isomerization of astaxanthin, resulting in an inflated yield and insufficient actual active substances.

[0003] The closest prior art to this invention is a method for extracting astaxanthin from shrimp shells using a single hydrophilic natural eutectic solvent combined with single-frequency ultrasound. This method involves washing, drying, and pulverizing the shrimp shell raw material, mixing it with the solvent, and then performing ultrasonic treatment and centrifugation to obtain a liquid phase containing astaxanthin. An organic solvent is then added for liquid-liquid extraction and concentration to obtain the astaxanthin product. In this method, astaxanthin remains in the single-phase system for a long time, lacking in-situ protection and timely separation mechanisms, making it prone to degradation and isomerization. The cavitation energy generated by single-frequency ultrasound is concentrated, easily forming local micro-hot spots, which exacerbates astaxanthin damage. The final separation of astaxanthin depends on the additional organic solvent, posing a risk of solvent residue, and failing to achieve the synergistic recovery of chitin and protein. Therefore, we propose a method for extracting astaxanthin from marine organisms. Summary of the Invention

[0004] The purpose of this invention is to provide a method for extracting astaxanthin from marine organisms.

[0005] The technical problem to be solved by this invention is that in the existing marine astaxanthin extraction process, it is difficult to balance the cell wall breaking efficiency with the protection of the natural configuration and bioactivity of astaxanthin. Traditional strong acid and strong alkali high temperature process leads to a large amount of degradation and cis-trans isomerization of astaxanthin. The existing green extraction method of single-phase eutectic solvent combined with single-frequency ultrasound still has problems such as long exposure time of astaxanthin during the extraction process, easy degradation, dependence on additional organic solvent separation, and inability to simultaneously produce chitin and protein.

[0006] To solve the above-mentioned technical problems, the present invention provides a method for extracting astaxanthin from marine organisms, comprising the following steps:

[0007] A hydrophobic natural eutectic solvent with an upper layer and a hydrophilic natural eutectic solvent with a lower layer were prepared separately. The upper hydrophobic natural eutectic solvent and the lower hydrophilic natural eutectic solvent were added to a reaction vessel and allowed to stand to separate into layers, forming a biphase natural eutectic solvent system in which the upper hydrophobic phase and the lower hydrophilic phase coexist. The upper hydrophobic natural eutectic solvent was formed by mixing menthol and medium-chain fatty acids, and a fat-soluble antioxidant was added to it. The lower hydrophilic natural eutectic solvent was formed by mixing choline chloride and L-lactic acid.

[0008] The cleaned, dried, and pulverized marine biological raw materials are put into the lower hydrophilic phase. Under low temperature conditions and with mechanical stirring, the acidic components in the lower hydrophilic phase react with the calcium carbonate in the marine biological raw materials to dissolve and loosen the bound proteins in the marine biological raw materials, thus exposing the astaxanthin and chitin fibers in the marine biological raw materials to free.

[0009] First-frequency ultrasound and second-frequency ultrasound are alternately applied to the liquid phase and the two-phase interface region in the reactor. The frequency of the first-frequency ultrasound is higher than that of the second-frequency ultrasound. Through asymmetric cavitation microjets, the complex composed of astaxanthin, chitin and protein is sheared and impacted, which causes astaxanthin molecules to become free and migrate across the two-phase interface to the upper hydrophobic phase. The astaxanthin molecules that migrate to the upper hydrophobic phase form a hydrogen bond network with the fat-soluble antioxidant.

[0010] After the application of ultrasound was stopped, the reaction system was allowed to separate into two phases, resulting in an upper extract phase containing astaxanthin and a hydrophobic natural eutectic solvent, a lower aqueous phase containing protein and calcium salt, and a solid chitin residue.

[0011] Compared with the prior art, the beneficial effects of the present invention by adopting the above technical solution are as follows:

[0012] 1. This invention constructs a biphase natural eutectic solvent system in which the upper hydrophobic phase and the lower hydrophilic phase coexist, enabling the demineralization and deproteinization reaction and astaxanthin extraction and separation to be completed synchronously and in situ in space. Astaxanthin is extracted and transferred by the upper hydrophobic phase in real time as soon as it is released, which shortens the exposure time of astaxanthin in the unstable microenvironment and improves the retention rate of all-trans astaxanthin in the extraction product.

[0013] 2. This invention introduces a lipid-soluble antioxidant in situ into the upper hydrophobic phase, which forms a hydrogen bond network with the astaxanthin molecules that migrate to the phase. This creates a spatial confinement effect on the configuration at both ends of the astaxanthin molecules, inhibiting the cis-trans isomerization of astaxanthin under physical and chemical stress. This achieves in situ protection of the natural all-trans configuration of astaxanthin, and the directional enrichment of astaxanthin can be completed without the need for additional organic solvents.

[0014] 3. By alternately applying two different frequencies of ultrasound, this invention avoids the continuous concentration of cavitation energy near the cavitation threshold corresponding to a single frequency, thus preventing the formation of local micro-hot spots. This achieves efficient dissociation of the astaxanthin-chitoxin-protein complex while reducing secondary damage to astaxanthin caused by local overheating.

[0015] 4. This invention simultaneously achieves astaxanthin extraction and separation, configuration protection, and synergistic recovery of chitin and protein within the same reaction system. The natural eutectic solvent used is recyclable, the overall process conditions are mild, there is no risk of toxic organic solvent residue, and it meets the requirements for food-grade green co-production extraction. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the overall complete process flow in an embodiment of the present invention;

[0017] Figure 2 This is a schematic diagram of the detailed preparation process of the two-phase solvent system in an embodiment of the present invention;

[0018] Figure 3 This is a schematic diagram of the core process of ultrasonic extraction and astaxanthin protection in an embodiment of the present invention. Detailed Implementation

[0019] The following is in conjunction with the appendix Figure 1-3 The specific embodiments of the present invention will be further described below. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0020] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0021] Example 1:

[0022] Step 1, raw material pretreatment: Take fresh shrimp shell raw materials, wash them to remove surface impurities and residual soft tissue, dry them under low temperature conditions, and then crush them into particles with a particle size of 0.5 mm to 2 mm to obtain pretreated raw materials;

[0023] Step 2, Construction of the biphase natural eutectic solvent system: Menthol is used as a hydrogen bond acceptor and octanoic acid is used as a hydrogen bond donor. They are stirred at 40°C in a molar ratio of 1:2 until completely miscible to form an upper hydrophobic natural eutectic solvent. Tocopherol, accounting for 1% of the mass of the solvent, is added as a lipid-soluble antioxidant auxiliary and stirred to disperse evenly. Choline chloride is used as a hydrogen bond acceptor and L-lactic acid is used as a hydrogen bond donor. They are mixed and stirred in a molar ratio of 1:1.5 until completely miscible to form a lower hydrophilic natural eutectic solvent. The upper hydrophobic natural eutectic solvent and the lower hydrophilic natural eutectic solvent are added to the reaction vessel in a volume ratio of 1:2 and allowed to stand to allow them to separate naturally, forming a biphase system in which the upper hydrophobic phase and the lower hydrophilic phase coexist.

[0024] Step 3, Feeding and Gentle Demineralization and Deproteinization: The pretreated raw material obtained in Step 1 is fed into the lower hydrophilic phase of the reactor at a solid-liquid ratio of 1:10. The temperature inside the reactor is controlled at 30°C. With slow mechanical stirring at a speed of 100 rpm, the reaction is carried out for 240 minutes. This allows the acidic components in the lower hydrophilic phase to dissolve and react with the calcium carbonate in the raw material, and the bound proteins are gradually dissolved and removed, while astaxanthin and chitin fibers are gradually released and exposed.

[0025] Step 4, Multi-frequency ultrasonic cavitation-assisted in-situ extraction: After the reaction described in Step 3 has been carried out for 120 minutes, the dual-frequency ultrasonic transducer is started, and ultrasonic waves with frequencies of 40kHz and 20kHz are alternately output at 20-second intervals to act on the liquid phase and the interface region between the two phases in the reactor. The treatment is continued for 120 minutes. During this process, the free astaxanthin molecules migrate across the interface between the two phases to the upper hydrophobic phase and form a hydrogen bond network with the tocopherol molecules pre-dispersed in the phase.

[0026] Step 5, phase separation and product separation: After ultrasonic treatment, stop stirring and ultrasonic output, let the reaction system stand and separate into layers, and obtain the upper extract phase containing astaxanthin and hydrophobic natural eutectic solvent, the lower aqueous phase containing dissolved protein and calcium salt, and the solid chitin residue insoluble in both phases.

[0027] Step 6, purification of astaxanthin extract phase: The upper extract phase obtained in step 5 is subjected to vacuum distillation at a temperature of 40℃ and a pressure of 0.01 MPa to remove the hydrophobic natural eutectic solvent carrier component, and the astaxanthin concentrate is obtained. The separated hydrophobic natural eutectic solvent is recycled for step 2 after being replenished with fresh senna alcohol, caprylic acid and tocopherol components.

[0028] Step 7, Chitosan and Protein Recovery: The solid residue obtained in Step 5 is washed to remove residual natural eutectic solvent and soluble impurities, and then dried to obtain chitosan product. The lower aqueous phase obtained in Step 5 is recovered by isoelectric point precipitation to recover the dissolved protein components. The hydrophilic natural eutectic solvent obtained is regenerated and recycled for Step 2.

[0029] In this embodiment, the extraction effect is quantitatively characterized as follows;

[0030] The total yield of astaxanthin is calculated as follows:

[0031] ;

[0032] in, This indicates the total yield of astaxanthin. This indicates the actual mass of astaxanthin obtained by this method, expressed in milligrams. This indicates the theoretical total astaxanthin content in the raw material, expressed in milligrams.

[0033] The all-trans configuration retention rate is used to characterize the protective effect of this invention on the active configuration of astaxanthin, and it is calculated as follows:

[0034] ;

[0035] in, Indicates the retention rate of the all-trans configuration. This indicates the concentration of all-trans astaxanthin in the extracted product as determined by high-performance liquid chromatography. This indicates the concentration of cis-astaxanthin in the extracted product obtained by high performance liquid chromatography.

[0036] The partition efficiency of astaxanthin in a two-phase system is characterized by the partition coefficient, which is calculated as follows:

[0037] ;

[0038] in, This represents the partition coefficient of astaxanthin in a two-phase natural eutectic solvent system. This indicates the equilibrium concentration of astaxanthin in the upper hydrophobic phase. This indicates the equilibrium concentration of astaxanthin in the lower hydrophilic phase. The higher the value, the stronger the in-situ directional enrichment ability of the biphasic system of the present invention for astaxanthin;

[0039] The calculation method for the recycling rate of natural eutectic solvents is as follows:

[0040] ;

[0041] in, This indicates the recycling rate of natural eutectic solvents. This indicates the quality of a natural eutectic solvent that can be reused after regeneration. This indicates the total mass of the natural eutectic solvent initially used;

[0042] The test results showed that the total yield of astaxanthin obtained in this embodiment was 78.6%, and the all-trans configuration retention rate was 92.3%. The total yield and retention rate obtained by the control method were 62.1% and 76.8%, respectively. The chitin product obtained in this embodiment had a complete structure, and the DES recycling rate was 85.2%. The obtained chitin product had a complete structure, and the protein product can be further utilized. The recycling rate test of the natural eutectic solvent showed that it can be reused in multiple batches.

[0043] Example 2:

[0044] Based on Example 1, the medium-chain fatty acids in the upper hydrophobic natural eutectic solvent are replaced with lauric acid, and the fat-soluble antioxidant is replaced with ascorbyl palmitate. The remaining steps and conditions are the same as in Example 1, which can also achieve in-situ protection of astaxanthin and synergistic recovery of chitin and protein.

[0045] Example 3:

[0046] Based on Example 1, the ultrasonic frequencies applied alternately in step four are adjusted to 45kHz and 15kHz, and the alternation interval is adjusted to 40 seconds. The remaining steps and conditions are the same as in Example 1, which can achieve a composite structure dissociation effect and astaxanthin protection effect similar to that in Example 1.

[0047] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, any modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting astaxanthin from marine organisms, characterized in that, Includes the following steps: An upper layer of hydrophobic natural eutectic solvent and a lower layer of hydrophilic natural eutectic solvent were prepared separately. The upper layer of hydrophobic natural eutectic solvent and the lower layer of hydrophilic natural eutectic solvent were added to a reaction vessel and allowed to stand to separate into layers, forming a biphase natural eutectic solvent system in which the upper hydrophobic phase and the lower hydrophilic phase coexist. The upper layer of hydrophobic natural eutectic solvent was formed by mixing menthol and medium-chain fatty acids, and a fat-soluble antioxidant was added to it. The lower layer of hydrophilic natural eutectic solvent was formed by mixing choline chloride and L-lactic acid. The cleaned, dried, and pulverized marine biological raw materials are added to the lower hydrophilic phase. Under the condition of 25°C to 40°C, mechanical stirring is carried out to cause the acidic components in the lower hydrophilic phase to react with the calcium carbonate in the marine biological raw materials to dissolve and loosen the bound proteins in the marine biological raw materials, thereby exposing the astaxanthin and chitin fibers in the marine biological raw materials to be free. A first frequency ultrasonic wave and a second frequency ultrasonic wave are alternately applied to the liquid phase and the interface region between the upper hydrophobic phase and the lower hydrophilic phase in the reactor. The frequency of the first frequency ultrasonic wave is higher than that of the second frequency ultrasonic wave. The shear impact intensity generated by the asymmetric cavitation microjets on the astaxanthin-chitoxin-protein complex changes periodically, avoiding the continuous concentration of single-frequency cavitation energy at a fixed cavitation threshold to form local micro-hot spots. This promotes the astaxanthin molecules to become free and migrate across the interface to the upper hydrophobic phase. The astaxanthin molecules that migrate to the upper hydrophobic phase form a hydrogen bond network with the lipid-soluble antioxidant. After the application of ultrasound was stopped, the reaction system was allowed to separate into two phases, yielding an upper extract phase containing astaxanthin and a hydrophobic natural eutectic solvent, a lower aqueous phase containing protein and calcium salt, and a solid chitin residue.

2. The method for extracting astaxanthin from marine organisms according to claim 1, characterized in that: In the upper hydrophobic natural eutectic solvent, the molar ratio of menthol to medium-chain fatty acid is 1:1 to 1:3, and the medium-chain fatty acid is one of caprylic acid and lauric acid.

3. The method for extracting astaxanthin from marine organisms according to claim 1, characterized in that: The lipid-soluble antioxidant is one of tocopherol and ascorbyl palmitate, and the amount of the lipid-soluble antioxidant added is 0.1% to 2% of the mass of the upper hydrophobic natural eutectic solvent.

4. The method for extracting astaxanthin from marine organisms according to claim 1, characterized in that: In the lower layer of hydrophilic natural eutectic solvent, the molar ratio of choline chloride to L-lactic acid is 1:1 to 1:

2.

5. The method for extracting astaxanthin from marine organisms according to claim 1, characterized in that: The first frequency ultrasound has a frequency of 35 kHz to 45 kHz, the second frequency ultrasound has a frequency of 15 kHz to 25 kHz, and the time interval between the alternation of the first frequency ultrasound and the second frequency ultrasound is 10 seconds to 60 seconds.

6. The method for extracting astaxanthin from marine organisms according to claim 1, characterized in that: The low-temperature condition refers to controlling the temperature inside the reactor within the range of 25°C to 40°C.

7. The method for extracting astaxanthin from marine organisms according to claim 1, characterized in that, It also includes vacuum distillation of the upper extract phase to remove the hydrophobic natural eutectic solvent therein, to obtain astaxanthin concentrate, and the separated hydrophobic natural eutectic solvent is recycled for the preparation of the upper hydrophobic natural eutectic solvent after being replenished with fresh components.

8. The method for extracting astaxanthin from marine organisms according to claim 1, characterized in that: It also includes protein recovery treatment of the lower aqueous phase to obtain protein products, and washing and drying of the solid chitin residue to obtain chitin products.

9. A biphasic natural eutectic solvent system for extracting astaxanthin, characterized in that, It comprises an upper layer of hydrophobic natural eutectic solvent and a lower layer of hydrophilic natural eutectic solvent, wherein the upper layer of hydrophobic natural eutectic solvent and the lower layer of hydrophilic natural eutectic solvent are immiscible and spontaneously separate into layers. The upper layer of hydrophobic natural eutectic solvent is formed by mixing menthol and medium-chain fatty acids, wherein a fat-soluble antioxidant is dispersed. The lower layer of hydrophilic natural eutectic solvent is formed by mixing choline chloride and L-lactic acid.

10. A biphasic natural eutectic solvent system for extracting astaxanthin according to claim 9, characterized in that: The fat-soluble antioxidant is one of tocopherol and ascorbyl palmitate.

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