Method for preparing high-purity phycocyanin by coupling supercritical extraction with aqueous two-phase extraction

Abstract

The invention discloses a method for preparing high-purity phycocyanin by coupling supercritical extraction with aqueous two-phase extraction, which comprises the following steps: pretreatment of raw materials: pretreating spirulina powder, and centrifuging to obtain a phycocyanin crude extract; carrying out supercritical CO2 impurity removal: filling the phycocyanin crude extract obtained in the previous step into a supercritical extraction device, carrying out static extraction, and collecting an orange extract rich in carotenoid; then pumping ethanol as a carrying agent, carrying out dynamic extraction, and respectively collecting chlorophyll components and wet algae residues; and two-aqueous-phase extraction purification and post-treatment: purifying the treated wet algae residues by adopting a two-aqueous-phase extraction method, dialyzing by adopting a dialysis bag, and carrying out vacuum freeze drying to obtain a pure product. The phycocyanin distribution selectivity is remarkably improved, and the method is environmentally friendly and efficient; the time is shortened by about 93% compared with that of a traditional method, the phycocyanin purity is stable and is larger than or equal to 4.0, the production cost is greatly reduced, and the method is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of phycocyanin separation and purification technology, and in particular to a method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction. Background Technology

[0002] Spirulina is widely used in human and animal health supplements due to its high nutritional value. Phycobiliproteins in spirulina are mainly classified into phycocyanin (C-PC), allophycocyanin (A-PC), and phycoerythrin (R-PC) based on differences in their absorption spectra. Phycocyanin accounts for 20% of the dry weight of spirulina and can be used as a natural pigment in food and cosmetics. Phycocyanin is a multi-chain whole protein and a widely developed natural blue compound. Its purity is classified according to the ratio of its characteristic peak absorbance at 620 nm to the protein absorbance at 280 nm: when A620 / A280 ≥ 0.70, C-PC is food grade; when A620 / A280 is 0.70-3.9, C-PC is reagent grade; and when A620 / A280 ≥ 4.0, C-PC is analytical grade. The higher the purity of C-PC, the higher its commercial value. High-purity phycocyanin possesses bioactivities such as anti-inflammatory, antioxidant, anti-tumor, and immunofluorescence properties. It is an ideal natural substance that can be used as a pharmaceutical ingredient for healthcare without toxic side effects. Therefore, the availability of large quantities of high-purity phycocyanin has become an urgent need to realize the high-value application of phycocyanin.

[0003] Currently, methods for extracting C-PC from Spirulina include physical, chemical, and biological techniques such as ultrasound, high-pressure homogenization, repeated freeze-thaw cycles, chemical solvents, and enzymatic treatment. Chinese patent application CN 115894669 A discloses a method for extracting and purifying phycocyanin from Spirulina, comprising three steps: swelling-based cell wall disruption, activated carbon adsorption extraction, and hydrophobic chromatography purification. The inventors investigated the impact of factors in the swelling-based cell wall disruption and activated carbon adsorption extraction steps on the product, using phycocyanin purity and recovery rate as indicators, and optimized the optimal process parameters using response surface methodology. The final step used hydrophobic chromatography to purify and obtain food-grade and pharmaceutical-grade phycocyanin (with a maximum purity of 3.2%). Existing phycocyanin separation and purification technologies suffer from problems such as low purity, poor process stability, unsuitability for large-scale applications, and low recovery rates. To promote the deep processing and high-value applications of phycocyanin, low-cost, efficient, and rapid production of high-purity phycocyanin has become an urgent priority. Summary of the Invention

[0004] To overcome the aforementioned shortcomings and deficiencies of existing technologies, the present invention aims to provide a method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction (AFE). This method employs AFE coupled with supercritical fluid extraction to extract and purify phycocyanin. The core principle lies in combining the complementary advantages of these two technologies, achieving efficient purification through a synergistic impurity removal → PEG-directed enrichment process. Specifically, SFE preferentially removes lipid-soluble pigments (such as chlorophyll), preventing them from competing with proteins for distribution in the ATPS phase and improving phycocyanin selectivity. After defatting, the hydrophilicity of the algae increases, significantly improving the partition coefficient of phycocyanin in the PEG phase. Furthermore, the coupling process between SFE and ATPS avoids high temperatures and special organic environments, ensuring that the phycocyanin does not undergo changes in its natural conformation. The phycocyanin powder obtained through this coupling process has a purity of over 4.0 (A620 / A280).

[0005] Another object of the present invention is to provide high-purity phycocyanin prepared by the above preparation method.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention provides a method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, comprising the following steps:

[0008] (1) Pretreatment of raw materials: Spirulina powder was pretreated and centrifuged to obtain crude phycocyanin extract;

[0009] (2) Supercritical CO2 purification: The crude extract of phycocyanin obtained in step (1) is loaded into a supercritical extraction device. Static extraction is performed for 1 to 3 hours to collect the orange extract rich in carotenoids. Then, ethanol is pumped in as a carrier to perform dynamic extraction for 0.5 to 1.5 hours to collect the chlorophyll component and wet algal residue respectively.

[0010] The process parameters of the supercritical extraction device are set as follows: pressure 250-300 bar, mass ratio of wet algae residue to CO2 1:15-20; CO2 flow rate 15-25 mL / min; extraction temperature 30-50℃.

[0011] (3) Aqueous two-phase extraction purification and post-treatment: The wet algal residue after step (2) was purified by aqueous two-phase extraction, then dialyzed with a dialysis bag, and then freeze-dried under vacuum to obtain pure product.

[0012] Preferably, the water content of the crude phycocyanin extract is 55-65%; the water content of the wet algal residue is 55-65%.

[0013] Preferably, in step (2), during the supercritical phase, the phosphate buffer is maintained at pH 6.5 to 7.0 to maintain protein stability.

[0014] Preferably, in step (2), the pumping in of ethanol as a carrier specifically involves:

[0015] Ethanol with a concentration of 8-12% was pumped in at a flow rate of 1.0-2.0 mL / min as a carrier.

[0016] Preferably, the pretreatment of spirulina powder in step (1) specifically involves freeze-thaw coupled microwave treatment:

[0017] (1-1) First, freeze in liquid nitrogen for 8-12 minutes, then transfer to -20℃ for 3.5-4.5 hours, and finally thaw in a water bath at 35-40℃. Repeat this freeze-thaw cycle 3-5 times.

[0018] (1-2) The algal solution after freeze-thaw is subjected to microwave-assisted treatment. The microwave treatment power is 800-1000W, pulse irradiation is performed at 48-52℃, each pulse lasts 8-12 seconds, with an interval of 25-30 seconds, and the pulse irradiation is performed 5-8 times.

[0019] Preferably, the components of the aqueous two-phase system in step (3) are: polyethylene glycol accounting for 15-20% of the total mass of the system, ammonium sulfate accounting for 8-10%, and crude phycocyanin extract accounting for 70-72%; the molecular weight of polyethylene glycol is 1000-2000.

[0020] Preferably, step (3) involves purifying the wet algal residue treated in step (2) using a two-phase aqueous extraction method, specifically as follows:

[0021] The supercritical treated algal residue was resuspended in a phosphate buffer solution at pH 7.0 and used to construct an aqueous two-phase system with polyethylene glycol oleate and ammonium sulfate. The mixture was shaken at 180–220 rpm for 8–12 minutes at 20–30 °C and allowed to stand for 8–15 minutes to complete natural phase separation.

[0022] Phycocyanin was selectively partitioned to the upper phase, while polysaccharides and other proteins were enriched in the lower phase; the collected upper phase solution was transferred to a dialysis bag and dialyzed at 4°C in the dark.

[0023] The dialysis used a dialysis bag with a molecular weight cutoff of 5000 Da, a dialysis solution with a pH of 6.0 to 7.5, and a phosphate buffer solution with a concentration of 8 to 10 mM.

[0024] Preferably, the phycocyanin retention rate in the wet algal residue obtained in step (2) is ≥95%.

[0025] Preferably, the pure product is obtained through vacuum freeze-drying, specifically as follows:

[0026] After dialysis, the phycocyanin solution was stabilized and then freeze-dried under vacuum at -15 to -10°C. The cold trap temperature was -50 to -56°C, the vacuum degree was 0.07 to 0.09 MPa, and the drying time was 10 to 12 hours. The resulting phycocyanin freeze-dried powder had a moisture content of ≤3.5% and a purity of ≥4.0.

[0027] The stabilizer consists of 0.08–0.12 M sodium citrate, 1.2–1.6% trehalose, and 4.8–5.2 mM EDTA.

[0028] The present invention also provides a high-purity phycocyanin, which is prepared by the supercritical extraction coupled with aqueous two-phase extraction method described above.

[0029] Specifically, in the algal residue, more than 98% of carotenoids and 93% of chlorophyll are removed, while the phycocyanin retention rate is ≥95%.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] (1) The method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction of the present invention employs a combination of aqueous two-phase extraction and supercritical fluid extraction to extract and purify phycocyanin. The core lies in combining the complementary advantages of the two technologies, achieving efficient purification through a synergistic impurity removal → PEG-directed enrichment process. Specifically, SFE preferentially removes lipid-soluble pigments (such as chlorophyll) to avoid their competition for distribution with proteins in ATPS, thereby improving the selectivity of phycocyanin. After defatting, the hydrophilicity of the algae is enhanced, and the partition coefficient of phycocyanin in the PEG phase is significantly increased. Furthermore, the coupling process of SFE and ATPS avoids high temperatures and special organic environments, ensuring that the phycocyanin does not undergo changes in its natural conformation. The phycocyanin powder obtained by this coupling process has a purity of over 4.0 (A620 / A280).

[0032] (2) The method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction of the present invention implements three-stage pH control throughout the process chain. The supercritical stage maintains pH 6.5-7.0 (phosphate buffer) to maintain protein stability; the aqueous two-phase extraction stage adjusts the pH to 7.0 to enhance partition selectivity.

[0033] (3) The method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction of the present invention purifies the crude liquid using aqueous two-phase extraction coupled with supercritical fluid extraction. This coupled process significantly improves efficiency through process integration: the supercritical impurity removal stage of 3.5 hours replaces traditional organic solvent extraction (which usually takes 12-24 hours); the aqueous two-phase system naturally separates phases in 12 minutes without the need for centrifugation (the traditional PEG / salt system requires 30 minutes of centrifugation); the overall process time is ≤8 hours, which is more than 93% shorter than the process reported in CN104844707A (10-20 days). In terms of energy consumption, a supercritical waste heat recovery system is innovatively adopted to use the heat energy generated by the compressor (about 45°C) for heating the sublimation section of the freeze dryer, thereby reducing the overall energy consumption by 40%. According to calculations, the total energy consumption per kilogram of phycocyanin powder is 82 kWh, which is significantly lower than the traditional salting-out chromatography process (143 kWh / kg).

[0034] (4) The method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction of the present invention has no organic solvent residue or emission, high PEG and CO2 recovery rates, and low cost. While reducing the impact on the environment, it greatly improves the added value of phycocyanin, providing technical support for the low-cost and high-efficiency production and high-value application of Spirulina phycocyanin. At the same time, the fluorescence emission spectrum has a strong characteristic peak at 650 nm, indicating that the protein has an intact spatial structure.

[0035] (5) The method for preparing high-purity phycocyanin using supercritical extraction coupled with aqueous two-phase extraction of the present invention has a supercritical CO2 recovery rate of ≥95%, which is recycled through condensation and reliquefaction; the PEG ester in the aqueous two-phase components is recovered by membrane separation (200Da nanofiltration membrane), with a reuse rate of up to 80%; the saline wastewater is concentrated by reverse osmosis and then crystallized to recover ammonium sulfate. The discharge of waste gas, wastewater, and solid waste is reduced by 75% compared with the traditional process, and there is no organic solvent residue. According to the pilot-scale calculation, the production cost of phycocyanin per kilogram is reduced to $1,850, which is significantly more competitive in the market than the chromatography purification method ($5,200 / kg). Detailed Implementation

[0036] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise stated, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0037] Example 1

[0038] A method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, the preparation method being as follows:

[0039] (1) Pretreatment of raw materials: Spirulina powder (particle size ≤ 0.15 mm) was added to deionized water at 4℃ at a mass ratio of 1:100 and stirred and soaked for 8 hours to ensure full hydration. Then, a three-step coupled cell wall disruption was carried out: first, the mixture was flash-frozen in liquid nitrogen at -196℃ for 10 minutes, then transferred to -20℃ for 4 hours, and finally thawed in a water bath at 37℃. This freeze-thaw cycle was repeated 3 times. The algal solution after freeze-thaw was treated with microwave assistance. After cell wall disruption, the mixture was centrifuged at 4℃ for 20 minutes, and the supernatant was collected as crude phycocyanin extract.

[0040] The microwave treatment involved a power of 900W, a 50°C pulse irradiation, 10 seconds per pulse, with a 30-second interval, for a total of 5 pulse irradiations.

[0041] (2) Supercritical CO2 purification: The phycocyanin obtained in step (1) was subjected to supercritical extraction. The wet algal residue (with a water content of about 60%) after cell wall disruption was loaded into the supercritical extraction vessel. The parameters were set as follows: pressure 250 bar, temperature 40℃, CO2 flow rate 15 mL / min, and the mass ratio of wet algal residue (with a water content of 60%) to CO2 was 1:15. Static extraction was first performed for 2 hours to collect the orange extract rich in carotenoids. Then, 10% ethanol was pumped in at a flow rate of 1.5 mL / min as a carrier and dynamic extraction was performed for 1.5 hours. The chlorophyll components and algal residue were collected separately.

[0042] During the supercritical phase, the pH is maintained at 6.5-7.0 (phosphate buffer) to maintain protein stability;

[0043] (3) Aqueous two-phase extraction purification and post-treatment: Aqueous two-phase extraction was used to purify and stabilize the phycocyanin after step (2). The supercritical algal residue was resuspended in phosphate buffer (pH 6.8) and mixed with polyethylene glycol (PEG2000) with a molecular weight of 2000 and ammonium sulfate in a ratio of 20% oleic acid polyethylene glycol ester, 8% ammonium sulfate, and 72% crude phycocyanin extract to form an aqueous two-phase system. The mixture was shaken at 200 rpm for 10 minutes at 30°C and allowed to stand for 15 minutes to complete natural phase separation. Phycocyanin selectively partitioned to the upper phase (partition coefficient K≈30), while polysaccharides and other proteins were enriched in the lower phase. The collected upper phase solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed for 24 hours at 4°C, pH 6.8, in the dark, with the dialysate changed every 4 hours. During dialysis, a 10 mM phosphate buffer with a pH of 7.0 was used as the dialysate.

[0044] The phycocyanin solution after dialysis was stabilized with a mixture of 0.1M sodium citrate, 1.5% trehalose, and 5mM EDTA, and then freeze-dried under vacuum at -10°C. The cold trap temperature was -55°C, the vacuum degree was 0.08 MPa, and the drying time was 12 hours. The moisture content of the freeze-dried powder was controlled to be ≤3.5%. The purity of the phycocyanin was 4.01, and the yield was 43.6%.

[0045] Example 2

[0046] A method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, the preparation method being as follows:

[0047] (1) Pretreatment of raw materials: Spirulina powder (particle size ≤ 0.15 mm) was added to deionized water at 4℃ at a mass ratio of 1:100 and stirred and soaked for 8 hours to ensure full hydration. Then, a three-step coupled cell wall disruption was carried out: first, the mixture was flash-frozen in liquid nitrogen at -196℃ for 10 minutes, then transferred to -20℃ for 4 hours, and finally thawed in a water bath at 37℃. This freeze-thaw cycle was repeated 3 times. The algal solution after freeze-thaw was treated with microwave assistance. After cell wall disruption, the mixture was centrifuged at 4℃ for 20 minutes, and the supernatant was collected as crude phycocyanin extract.

[0048] The microwave treatment involved a power of 1000W, a 50°C pulse irradiation, 8 seconds per pulse, with a 30-second interval, for a total of 5 pulses.

[0049] (2) Supercritical CO2 purification: The phycocyanin obtained in step (1) was subjected to supercritical extraction. The wet algal residue (with a water content of about 60%) after cell wall disruption was loaded into the supercritical extraction vessel. The parameters were set as follows: pressure 300 bar, temperature 45℃, CO2 flow rate 25 mL / min, and the mass ratio of wet algal residue (with a water content of 60%) to CO2 was 1:20. Static extraction was first performed for 2 hours, and the orange extract rich in carotenoids was collected. Then, 10% ethanol was pumped in at a flow rate of 1.5 mL / min as a carrier, and dynamic extraction was performed for 1 hour. The chlorophyll components and algal residue were collected separately.

[0050] During the supercritical phase, the pH is maintained at 6.5-7.0 (phosphate buffer) to maintain protein stability;

[0051] (3) Aqueous two-phase extraction purification and post-treatment: Aqueous two-phase extraction was used to purify and stabilize the phycocyanin after step (2). The supercritical algal residue was resuspended in phosphate buffer (pH 7.0) and mixed with polyethylene glycol (PEG1000) and ammonium sulfate at a ratio of 18% oleic acid polyethylene glycol ester, 10% ammonium sulfate, and 72% crude phycocyanin extract to form an aqueous two-phase system. The mixture was shaken at 200 rpm for 10 minutes at 25°C and allowed to stand for 12 minutes to complete natural phase separation. Phycocyanin selectively partitioned to the upper phase (partition coefficient K≈30), while polysaccharides and other proteins were enriched in the lower phase. The collected upper phase solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed for 24 hours at 4°C, pH 7.0, in the dark, with the dialysate changed every 4 hours. During dialysis, a phosphate buffer with a pH of 7.0 and a concentration of 8 mM was used as the dialysate.

[0052] The phycocyanin solution after dialysis was stabilized with 0.1M sodium citrate + 1.5% trehalose + 5mM EDTA and then freeze-dried under vacuum at -15℃, with a cold trap temperature of -55℃, a vacuum degree of 0.08MPa, and a drying time of 12 hours. The moisture content of the freeze-dried powder was controlled to be ≤3.5%. The purity of the phycocyanin was 4.18.

[0053] Example 3

[0054] A method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, the preparation method being as follows:

[0055] (1) Pretreatment of raw materials: Spirulina powder (particle size ≤ 0.15 mm) was added to deionized water at 4℃ at a mass ratio of 1:100 and stirred and soaked for 8 hours to ensure full hydration. Then, a three-step coupled cell wall disruption was carried out: first, the mixture was flash-frozen in liquid nitrogen at -196℃ for 10 minutes, then transferred to -20℃ for 4 hours, and finally thawed in a water bath at 37℃. This freeze-thaw cycle was repeated 3 times. The algal solution after freeze-thaw was treated with microwave assistance. After cell wall disruption, the mixture was centrifuged at 4℃ for 20 minutes, and the supernatant was collected as crude phycocyanin extract.

[0056] The microwave treatment involved a power of 800W, a pulse irradiation at 52°C, 12 seconds per pulse, with a 25-second interval, for a total of 5 pulses.

[0057] (2) Supercritical CO2 purification: The phycocyanin obtained in step (1) was subjected to supercritical extraction. The wet algal residue (with a water content of about 60%) after cell wall disruption was loaded into the supercritical extraction vessel. The parameters were set as follows: pressure 300 bar, temperature 40℃, CO2 flow rate 25 mL / min, and the mass ratio of wet algal residue (with a water content of 60%) to CO2 was 1:15. Static extraction was first performed for 2 hours to collect the orange extract rich in carotenoids. Then, 10% ethanol was pumped in at a flow rate of 1.5 mL / min as a carrier and dynamic extraction was performed for 1 hour. The chlorophyll components and algal residue were collected separately.

[0058] (3) Aqueous two-phase extraction purification and post-treatment: Aqueous two-phase extraction was used to purify and stabilize the phycocyanin after step (2). The supercritical algal residue was resuspended in phosphate buffer (pH 7.0) and mixed with polyethylene glycol (PEG2000) and ammonium sulfate at a ratio of 20% polyethylene glycol, 8% ammonium sulfate, and 72% crude phycocyanin extract to form an aqueous two-phase system. The mixture was shaken at 200 rpm for 10 minutes at 30°C and allowed to stand for 15 minutes to complete natural phase separation. Phycocyanin selectively partitioned to the upper phase (partition coefficient K≈30), while polysaccharides and other proteins were enriched in the lower phase. The collected upper phase solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed for 24 hours at 4°C, pH 7.0, in the dark, with the dialysate changed every 4 hours. During dialysis, a 9 mM phosphate buffer with a pH of 7.0 was used as the dialysate.

[0059] The phycocyanin solution after dialysis was stabilized with 0.1M sodium citrate + 1.5% trehalose + 5mM EDTA and then freeze-dried under vacuum at -15℃, with a cold trap temperature of -55℃, a vacuum degree of 0.08MPa, and a drying time of 12 hours. The moisture content of the freeze-dried powder was controlled to be ≤3.5%. The purity of the phycocyanin was 4.06.

[0060] Example 4

[0061] A method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, the preparation method being as follows:

[0062] (1) Pretreatment of raw materials: Spirulina powder (particle size ≤ 0.15 mm) was added to deionized water at 4℃ at a mass ratio of 1:100 and stirred and soaked for 8 hours to ensure full hydration. Then, a three-step coupled cell wall disruption was carried out: first, the mixture was flash-frozen in liquid nitrogen at -196℃ for 10 minutes, then transferred to -20℃ for 4 hours, and finally thawed in a water bath at 37℃. This freeze-thaw cycle was repeated 3 times. The algal solution after freeze-thaw was treated with microwave assistance. After cell wall disruption, the mixture was centrifuged at 4℃ for 20 minutes, and the supernatant was collected as crude phycocyanin extract.

[0063] The microwave treatment involved a power of 900W, a 48°C pulse irradiation, 12 seconds per pulse, with a 30-second interval, for a total of 5 pulses.

[0064] (2) Supercritical CO2 purification: The phycocyanin obtained in step (1) was subjected to supercritical extraction. The wet algal residue (with a water content of about 60%) after cell wall disruption was loaded into the supercritical extraction vessel. The parameters were set as follows: pressure 280 bar, temperature 42℃, CO2 flow rate 22 mL / min, and the mass ratio of wet algal residue (with a water content of 60%) to CO2 was 1:16. The total time was 2.5 hours. Static extraction was performed for 2 hours first, and the orange extract rich in carotenoids was collected. Then, 10% ethanol was pumped in at a flow rate of 1.5 mL / min as a carrier, and dynamic extraction was performed for 0.5 hours. The chlorophyll components and algal residue were collected separately.

[0065] During the supercritical phase, the pH is maintained at 6.5-7.0 (phosphate buffer) to maintain protein stability;

[0066] (3) Aqueous two-phase extraction purification and post-treatment: Aqueous two-phase extraction was used to purify and stabilize the phycocyanin after step (2). The supercritical algal residue was resuspended in phosphate buffer (pH 7.0) and mixed with polyethylene glycol oleate (PEG1500) and ammonium sulfate at a ratio of 17% oleate, 11% ammonium sulfate, and 72% crude phycocyanin extract to form an aqueous two-phase system. The mixture was shaken at 200 rpm for 10 minutes at 25°C and allowed to stand for 15 minutes to complete natural phase separation. Phycocyanin selectively partitioned to the upper phase (partition coefficient K≈30), while polysaccharides and other proteins were enriched in the lower phase. The collected upper phase solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed at 4°C, pH 6.8, and in the dark for 24 hours, with the dialysate being replaced every 4 hours. During dialysis, a phosphate buffer with a pH of 6.8 and a concentration of 8 mM was used as the dialysate.

[0067] The phycocyanin solution after dialysis was stabilized with 0.1M sodium citrate + 1.5% trehalose + 5mM EDTA and then freeze-dried under vacuum at -12℃, with a cold trap temperature of -55℃, a vacuum degree of 0.08MPa, and a drying time of 12 hours. The moisture content of the freeze-dried powder was controlled to be ≤3.5%. The purity of the phycocyanin was 4.04.

[0068] Example 5

[0069] A method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, the preparation method being as follows:

[0070] (1) Pretreatment of raw materials: Spirulina powder (particle size ≤ 0.15 mm) was added to deionized water at 4℃ at a mass ratio of 1:100 and stirred and soaked for 8 hours to ensure full hydration. Then, a three-step coupled cell wall disruption was carried out: first, the mixture was flash-frozen in liquid nitrogen at -196℃ for 10 minutes, then transferred to -20℃ for 4 hours, and finally thawed in a water bath at 37℃. This freeze-thaw cycle was repeated 3 times. The algal solution after freeze-thaw was treated with microwave assistance. After cell wall disruption, the mixture was centrifuged at 4℃ for 20 minutes, and the supernatant was collected as crude phycocyanin extract.

[0071] The microwave treatment involved a power of 800W, a 48°C pulse irradiation, 12 seconds per pulse, with a 25-second interval, for a total of 5 pulses.

[0072] (2) Supercritical CO2 purification: The phycocyanin obtained in step (1) was subjected to supercritical extraction. The wet algal residue (with a water content of about 60%) after cell wall disruption was loaded into the supercritical extraction vessel. The parameters were set as follows: pressure 270 bar, temperature 45℃, CO2 flow rate 20 mL / min, and the mass ratio of wet algal residue (with a water content of 60%) to CO2 was 1:15. The total time was 3 hours. First, static extraction was performed for 2 hours to collect the orange extract rich in carotenoids. Then, 10% ethanol was pumped in at a flow rate of 1.5 mL / min as a carrier and dynamic extraction was performed for 1 hour. The chlorophyll components and algal residue were collected separately.

[0073] During the supercritical phase, the pH is maintained at 6.5-7.0 (phosphate buffer) to maintain protein stability;

[0074] (3) Aqueous two-phase extraction purification and post-treatment: Aqueous two-phase extraction was used to purify and stabilize the phycocyanin after step (2). The supercritical algal residue was resuspended in phosphate buffer (pH 6.8) and mixed with polyethylene glycol (PEG1000) and ammonium sulfate at a ratio of 18% PEG, 10% ammonium sulfate, and 72% crude phycocyanin extract to form an aqueous two-phase system. The mixture was shaken at 250 rpm for 10 minutes at 25°C and allowed to stand for 12 minutes to complete natural phase separation. Phycocyanin selectively partitioned to the upper phase (partition coefficient K≈30), while polysaccharides and other proteins were enriched in the lower phase. The collected upper phase solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed at 4°C, pH 7.0, and in the dark for 24 hours, with the dialysate being replaced every 4 hours. During dialysis, a 9 mM phosphate buffer with a pH of 7.5 was used as the dialysate.

[0075] The phycocyanin solution after dialysis was stabilized with 0.1M sodium citrate + 1.5% trehalose + 5mM EDTA and then freeze-dried under vacuum at -13℃, with a cold trap temperature of -55℃, a vacuum degree of 0.08MPa, and a drying time of 12 hours. The moisture content of the freeze-dried powder was controlled to be ≤3.5%. The purity of the phycocyanin was 4.10.

[0076] Example 6

[0077] A method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, the preparation method being as follows:

[0078] (1) Pretreatment of raw materials: Spirulina powder (particle size ≤ 0.15 mm) was added to deionized water at 4℃ at a mass ratio of 1:100 and stirred and soaked for 8 hours to ensure full hydration. Then, a three-step coupled cell wall disruption was carried out: first, the mixture was flash-frozen in liquid nitrogen at -196℃ for 10 minutes, then transferred to -20℃ for 4 hours, and finally thawed in a water bath at 37℃. This freeze-thaw cycle was repeated 3 times. The algal solution after freeze-thaw was treated with microwave assistance. After cell wall disruption, the mixture was centrifuged at 4℃ for 20 minutes, and the supernatant was collected as crude phycocyanin extract.

[0079] The microwave treatment involved a power of 1000W, a 50°C pulse irradiation, 9 seconds per pulse, with a 25-second interval, for a total of 5 pulses.

[0080] (2) Supercritical CO2 purification: The phycocyanin obtained in step (1) was subjected to supercritical extraction. The wet algal residue (with a water content of about 60%) after cell wall disruption was loaded into the supercritical extraction vessel. The parameters were set as follows: pressure 270 bar, temperature 45℃, CO2 flow rate 20 mL / min, and the mass ratio of wet algal residue (with a water content of 60%) to CO2 was 1:15. The total time was 3 hours. First, static extraction was performed for 2 hours to collect the orange extract rich in carotenoids. Then, 10% ethanol was pumped in at a flow rate of 1.5 mL / min as a carrier and dynamic extraction was performed for 1 hour. The chlorophyll components and algal residue were collected separately.

[0081] During the supercritical phase, the pH is maintained at 6.5-7.0 (phosphate buffer) to maintain protein stability;

[0082] (3) Aqueous two-phase extraction purification and post-treatment: Aqueous two-phase extraction was used to purify and stabilize the phycocyanin after step (2). The supercritical algal residue was resuspended in phosphate buffer (pH 6.8) and mixed with polyethylene glycol oleate (PEG1000) and ammonium sulfate at a ratio of 18% oleate, 10% ammonium sulfate, and 72% crude phycocyanin extract to form an aqueous two-phase system. The mixture was shaken at 250 rpm for 10 minutes at 25°C and allowed to stand for 12 minutes to complete natural phase separation. Phycocyanin selectively partitioned to the upper phase (partition coefficient K≈30), while polysaccharides and other proteins were enriched in the lower phase. The collected upper phase solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed at 4°C, pH 6.8, and in the dark for 24 hours, with the dialysate being replaced every 4 hours. During dialysis, a 10 mM phosphate buffer with a pH of 6.8 was used as the dialysate.

[0083] The phycocyanin solution after dialysis was stabilized with 0.1M sodium citrate + 1.5% trehalose + 5mM EDTA and then freeze-dried under vacuum at -12℃, with a cold trap temperature of -55℃, a vacuum degree of 0.08MPa, and a drying time of 12 hours. The moisture content of the freeze-dried powder was controlled to be ≤3.5%. The purity of the phycocyanin was 4.09.

[0084] Example 7

[0085] A method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, the preparation method being as follows:

[0086] (1) Pretreatment of raw materials: Spirulina powder (particle size ≤ 0.15 mm) was added to deionized water at 4℃ at a mass ratio of 1:100 and stirred and soaked for 8 hours to ensure full hydration. Then, a three-step coupled cell wall disruption was carried out: first, the mixture was flash-frozen in liquid nitrogen at -196℃ for 10 minutes, then transferred to -20℃ for 4 hours, and finally thawed in a water bath at 37℃. This freeze-thaw cycle was repeated 3 times. The algal solution after freeze-thaw was treated with microwave assistance. After cell wall disruption, the mixture was centrifuged at 4℃ for 20 minutes, and the supernatant was collected as crude phycocyanin extract.

[0087] The microwave treatment involved a power of 1000W, a pulse irradiation at 52°C, 8 seconds per pulse, with a 30-second interval, for a total of 5 pulses.

[0088] (2) Supercritical CO2 purification: The phycocyanin obtained in step (1) was subjected to supercritical extraction. The wet algal residue (with a water content of about 60%) after cell wall disruption was loaded into the supercritical extraction vessel. The parameters were set as follows: pressure 280 bar, temperature 42℃, CO2 flow rate 22 mL / min, and the mass ratio of wet algal residue (with a water content of 60%) to CO2 was 1:16. The total time was 3.5 hours. First, static extraction was performed for 2.5 hours, and the orange extract rich in carotenoids was collected. Then, 10% ethanol was pumped in at a flow rate of 1.5 mL / min as a carrier, and dynamic extraction was performed for 1 hour. The chlorophyll components and algal residue were collected separately.

[0089] During the supercritical phase, the pH is maintained at 6.5-7.0 (phosphate buffer) to maintain protein stability;

[0090] (3) Aqueous two-phase extraction purification and post-treatment: Aqueous two-phase extraction was used to purify and stabilize the phycocyanin after step (2). The supercritical algal residue was resuspended in phosphate buffer (pH 6.5) and mixed with polyethylene glycol (PEG1500) and ammonium sulfate at a ratio of 16% PEG, 12% ammonium sulfate, and 72% crude phycocyanin extract to form an aqueous two-phase system. The mixture was shaken at 200 rpm for 10 minutes at 25°C and allowed to stand for 15 minutes to complete natural phase separation. Phycocyanin selectively partitioned to the upper phase (partition coefficient K≈30), while polysaccharides and other proteins were enriched in the lower phase. The collected upper phase solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed at 4°C, pH 6.5, and in the dark for 24 hours, with the dialysate being replaced every 4 hours. During dialysis, a phosphate buffer with a pH of 6.5 and a concentration of 8 mM was used as the dialysate.

[0091] The phycocyanin solution after dialysis was stabilized with 0.1M sodium citrate + 1.5% trehalose + 5mM EDTA and then freeze-dried under vacuum at -15°C, with a cold trap temperature of -55°C, a vacuum degree of 0.08MPa, and a drying time of 12 hours. The moisture content of the freeze-dried powder was controlled to be ≤3.5%. The purity of the phycocyanin was 4.04, but the yield was lower than in other examples.

[0092] Example 8

[0093] A method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, the preparation method being as follows:

[0094] (1) Pretreatment of raw materials: Spirulina powder (particle size ≤ 0.15 mm) was added to deionized water at 4℃ at a mass ratio of 1:100 and stirred and soaked for 8 hours to ensure full hydration. Then, a three-step coupled cell wall disruption was carried out: first, the mixture was flash-frozen in liquid nitrogen at -196℃ for 10 minutes, then transferred to -20℃ for 4 hours, and finally thawed in a water bath at 37℃. This freeze-thaw cycle was repeated 3 times. The algal solution after freeze-thaw was treated with microwave assistance. After cell wall disruption, the mixture was centrifuged at 4℃ for 20 minutes, and the supernatant was collected as crude phycocyanin extract.

[0095] The microwave treatment involved a power of 1000W, a 48°C pulse irradiation, 10 seconds per pulse, with a 25-second interval, for a total of 5 pulses.

[0096] (2) Supercritical CO2 purification: The phycocyanin obtained in step (1) was subjected to supercritical extraction. The wet algal residue (with a water content of about 60%) after cell wall disruption was loaded into the supercritical extraction vessel. The parameters were set as follows: pressure 300 bar, temperature 40℃, CO2 flow rate 25 mL / min, and the mass ratio of wet algal residue (with a water content of 60%) to CO2 was 1:16. The total time was 3 hours. First, static extraction was performed for 2 hours, and the orange extract rich in carotenoids was collected. Then, 10% ethanol was pumped in at a flow rate of 1.5 mL / min as a carrier, and dynamic extraction was performed for 1 hour. The chlorophyll components and algal residue were collected separately.

[0097] During the supercritical phase, the pH is maintained at 6.5-7.0 (phosphate buffer) to maintain protein stability;

[0098] (3) Aqueous two-phase extraction purification and post-treatment: Aqueous two-phase extraction was used to purify and stabilize the phycocyanin after step (2). The supercritical algal residue was resuspended in phosphate buffer (pH 6.5) and mixed with polyethylene glycol (PEG1000) and ammonium sulfate at a ratio of 18% PEG, 10% ammonium sulfate, and 72% crude phycocyanin extract to form an aqueous two-phase system. The mixture was shaken at 200 rpm for 10 minutes at 25°C and allowed to stand for 15 minutes to complete natural phase separation. Phycocyanin selectively partitioned to the upper phase (partition coefficient K≈30), while polysaccharides and other proteins were enriched in the lower phase. The collected upper phase solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed for 24 hours at 4°C, pH 7.0, and in the dark, with the dialysate changed every 4 hours. During dialysis, a 10 mM phosphate buffer with a pH of 7.5 was used as the dialysate.

[0099] The phycocyanin solution after dialysis was stabilized with a mixture of 0.1M sodium citrate, 1.5% trehalose, and 5mM EDTA, and then freeze-dried under vacuum at -13°C. The cold trap temperature was -55°C, the vacuum degree was 0.08 MPa, and the drying time was 12 hours. The moisture content of the freeze-dried powder was controlled to be ≤3.5%. The purity of the phycocyanin was 4.07, and the yield was higher than that of Example 7 but lower than that of other examples.

[0100] Phycocyanin content and purity determination: The absorbance of the samples obtained in Examples 1 to 8 was measured at 620, 280, and 652 nm.

[0101] Calculate the concentration, yield, and purity of phycocyanin using formulas (1)-(3):

[0102] Purity = A620 / A280 (1)

[0103] Volume concentration C (mg / mL) = (A 620 -0.474A 652 ) / 5.34 (2)

[0104] Phycocyanin yield (mg / L) = C * V / m (3)

[0105] In the formula: A620nm, A280nm, and A652nm are the absorbances of the phycocyanin solution at 620nm, 280nm, and 652nm, respectively; C is the volume concentration of phycocyanin (mg / mL); m is the mass of Spirulina powder (g); and V is the volume of the phycocyanin solution (mL).

[0106] The spirulina powder used in this invention contains 10-20% phycocyanin, with the remainder consisting of miscellaneous proteins, polysaccharides, chlorophyll, carotenoids, and insoluble cell walls. This invention uses a freeze-thaw microwave method to break down and roughly purify the phycocyanin, followed by supercritical CO2 extraction for further purification. Finally, an aqueous two-phase extraction method is used to further purify the supercritically extracted sample. The aqueous two-phase system is constructed using a phosphate-buffered saline (pH 7.0), polyethylene glycol oleate, and ammonium sulfate in a specific ratio to improve its selective distribution of phycocyanin. In this embodiment, the partition coefficient of phycocyanin reaches its maximum when the pH of the aqueous two-phase system is 7, resulting in high extraction rate and high purity phycocyanin (A620 / A280 > 4.0).

[0107] Compared to patents such as CN110872518A that use traditional methods to purify phycocyanin:

[0108]

[0109]

[0110] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing high-purity phycocyanin using supercritical fluid extraction coupled with aqueous two-phase extraction, characterized in that, Includes the following steps: (1) Pretreatment of raw materials: Spirulina powder was pretreated and centrifuged to obtain crude phycocyanin extract; (2) Supercritical CO2 purification: The crude extract of phycocyanin obtained in step (1) is loaded into a supercritical extraction device. Static extraction is performed for 1 to 3 hours to collect the orange extract rich in carotenoids. Then, ethanol is pumped in as a carrier to perform dynamic extraction for 0.5 to 1.5 hours to collect the chlorophyll component and wet algal residue respectively. The process parameters of the supercritical extraction device are set as follows: pressure 250-300 bar, mass ratio of wet algae residue to CO2 1:15-20; CO2 flow rate 15-25 mL / min; extraction temperature 30-50℃. (3) Aqueous two-phase extraction purification and post-treatment: The wet algal residue after step (2) was purified by aqueous two-phase extraction, then dialyzed with a dialysis bag, and then freeze-dried under vacuum to obtain pure product.

2. The method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction according to claim 1, characterized in that, The crude phycocyanin extract has a water content of 55-65%; the wet algal residue has a water content of 55-65%.

3. The method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction according to claim 1, characterized in that, In step (2), during the supercritical phase, the phosphate buffer is maintained at pH 6.5–7.0 to maintain protein stability.

4. The method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction according to claim 1, characterized in that, In step (2), the pumping in of ethanol as a carrier specifically involves: Ethanol with a concentration of 8-12% was pumped in at a flow rate of 1.0-2.0 mL / min as a carrier.

5. The method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction according to claim 1, characterized in that, The pretreatment of spirulina powder in step (1) specifically involves freeze-thaw coupled microwave treatment: (1-1) First, freeze in liquid nitrogen for 8-12 minutes, then transfer to -20℃ for 3.5-4.5 hours, and finally thaw in a water bath at 35-40℃. Repeat this freeze-thaw cycle 3-5 times. (1-2) The algal solution after freeze-thaw is subjected to microwave-assisted treatment. The microwave treatment power is 800-1000W, pulse irradiation is performed at 48-52℃, each pulse lasts 8-12 seconds, with an interval of 25-30 seconds, and the pulse irradiation is performed 5-8 times.

6. The method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction according to claim 1, characterized in that, The components of the aqueous two-phase system in step (3) are: polyethylene glycol accounts for 15-20% of the total mass of the system, ammonium sulfate accounts for 8-10%, and crude phycocyanin extract accounts for 70-72%; the molecular weight of polyethylene glycol is 1000-2000.

7. The method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction according to claim 5, characterized in that, Step (3) involves purifying the wet algal residue treated in step (2) using a two-phase aqueous extraction method, specifically as follows: The supercritical treated algal residue was resuspended in a phosphate buffer solution at pH 7.0 and used to construct an aqueous two-phase system with polyethylene glycol oleate and ammonium sulfate. The mixture was shaken at 180–220 rpm for 8–12 minutes at 20–30 °C and allowed to stand for 8–15 minutes to complete natural phase separation. Phycocyanin was selectively partitioned to the upper phase, while polysaccharides and other proteins were enriched in the lower phase; the collected upper phase solution was transferred to a dialysis bag and dialyzed at 4°C in the dark. The dialysis used a dialysis bag with a molecular weight cutoff of 5000 Da, a dialysis solution with a pH of 6.0 to 7.5, and a phosphate buffer solution with a concentration of 8 to 10 mM.

8. The method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction according to claim 1, characterized in that, The phycocyanin retention rate in the wet algal residue obtained in step (2) is ≥95%.

9. The method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction according to claim 1, characterized in that, The pure product was obtained by vacuum freeze-drying, specifically as follows: After dialysis, the phycocyanin solution was stabilized and then freeze-dried under vacuum at -15 to -10°C. The cold trap temperature was -50 to -56°C, the vacuum degree was 0.07 to 0.09 MPa, and the drying time was 10 to 12 hours. The resulting phycocyanin freeze-dried powder had a moisture content of ≤3.5% and a purity of ≥4.

0. The stabilizer consists of 0.08–0.12 M sodium citrate, 1.2–1.6% trehalose, and 4.8–5.2 mM EDTA.

10. High-purity phycocyanin, characterized in that, It is prepared by the method for preparing high-purity phycocyanin by supercritical extraction coupled with aqueous two-phase extraction as described in any one of claims 1 to 9.

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