Phycocyanin extraction microspheres and preparation method thereof

Abstract

The invention discloses a phycocyanin extraction microsphere, and discloses a structure and a preparation method of the phycocyanin extraction microsphere, and an application of the phycocyanin extraction microsphere in phycocyanin extraction. The polymer microspheres provided by the invention can be efficiently and conveniently used for extracting phycocyanin, and cost reduction and efficiency improvement in the phycocyanin extraction and purification process are facilitated.

Description

Technical Field

[0001] The invention relates to phycocyanin extraction microspheres and a preparation method thereof, belonging to the technical field of separation and purification materials. Background Art

[0002] Phycocyanins are a class of porphyrin-containing pigment proteins that are soluble in water but insoluble in alcohol and oil. They are widely found in blue-green algae. Phycocyanins have diverse potential applications in food colorants, fluorescent labels, cosmetics, nanotechnology, nutritional supplements, and the pharmaceutical industry, with the global market size projected to be approximately US$250 million by 2027. Phycocyanins are often considered a natural pigment, replacing synthetic dyes in the food and cosmetics industries, and are widely used due to their non-carcinogenic and non-toxic properties. Furthermore, studies have shown that phycocyanins possess health benefits such as hepatoprotective, antioxidant, and anti-inflammatory potential. Due to their high molar absorptivity, visible emission wavelength, high water solubility, high fluorescence quantum yield, and large extinction coefficient, phycobiliproteins are also used as fluorescent markers in various immunoassays in biochemical and bioengineering research. These properties make phycocyanins widely used as convenient labels in a variety of highly sensitive fluorescence techniques, such as isoelectric focusing, cell and macromolecule labeling, gel exclusion chromatography, gel electrophoresis, and fluorescence microscopy. Industrial demand for these biologically derived natural pigments is rapidly increasing.

[0003] The main producers of phycocyanin are Arthrospira platensis, Spirulina platensis, Cryptomonas, Nostoc commune var.sphaeroides and Oscillatoria okeni TISTR8549. The extraction methods of phycocyanin include traditional solvent extraction (such as maceration, soaking, filtration, etc.), repeated freeze-thaw processes, enzyme-assisted extraction and new extraction technologies such as ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), high-pressure processing (HPP), pulsed electric field (PEF) and supercritical fluid extraction. It can also include chemical treatment (organic acid and inorganic acid), physical treatment (freezing and thawing, ultrasonic treatment, homogenization and pulsed electric field) and enzyme treatment (lysozyme) and their combinations. The separation process of phycocyanin involves steps such as cell disruption, preliminary extraction and purification. These processes usually require large facilities and equipment, consume energy and generally require the addition of chemical additives, and the long processing time leads to low yield. Furthermore, high temperatures during drying not only affect the state of phycobilisomes but also lead to a loss of their total quantity. The extraction method, solvent, solvent to biomass ratio, and extraction time all affect the yield obtained.

[0004] Chinese invention patent CN119529065A discloses a method for extracting phycocyanin with a natural deep eutectic solvent. Phycocyanin is extracted by mixing a fixed ratio of hydrogen-bonding donors and hydrogen-bonding acceptors. No complex purification and extraction solvent removal steps such as precipitation, centrifugation, dialysis, and ion exchange chromatography are required, and the phycocyanin can be directly added to the preparation of cosmetics. This method is more environmentally friendly and safer. The glycerol-glucose solvent can protect the phycocyanin, making its structure less susceptible to light and heat damage and improving storage stability. This solves the problem of the existing method for extracting phycocyanin, in which the imidazole and pyridine ionic liquids used in the extraction process of phycocyanin are difficult to degrade and toxic. Chinese invention patent CN119951642A discloses a dynamic material-moving phycocyanin wall-breaking mechanism and its wall-breaking process, including a wall-breaking tube, a wall-breaking ring, and a dynamic material-moving component. When the movable barrel moves from the annular receiving groove to the side expansion chamber, the movable barrel will move the material originally blocked in the straight-through wall-breaking channel into the side expansion chamber, and then the ejecting motor drives the ejecting column to move downward into the inside of the movable barrel, squeezing the material blocked inside the movable barrel downward and discharging it from the discharge port, thereby effectively preventing the blockage of the straight-through wall-breaking channel. At the same time, the material that originally passed through the straight-through wall-breaking channel can be dispersed by the movable barrel, so that the straight-through wall-breaking channel and the discharge port can be discharged in coordination, thereby improving the discharge efficiency and production capacity; Chinese invention patent CN119869045A discloses a high-efficiency separation type phycocyanin extraction and processing mechanism and its processing technology, including a processing tank body, a positioning ring plate, a movable separation component, a feed pipe, and a power motor. Normal filtering and impurity removal operations are performed through the upper positioning filter cartridge. When a large amount of impurities are deposited inside the upper positioning filter cartridge, the sealing plug and the upper positioning filter cartridge are separated. At this time, the phycocyanin liquid fed into the inner end of the feed pipe will enter the lower floating filter cartridge through the telescopic sealing tube, and at the same time, the deposited impurities enter the lower floating filter cartridge. At this time, the phycocyanin liquid is filtered through the micropores around the lower floating filter cartridge to achieve continuous operation. In this way, the present invention can ensure the continuous operation of the phycocyanin liquid, and at the same time, it can achieve the purpose of cleaning impurities, thereby improving operation efficiency and cleaning convenience.

[0005] Chinese invention patent CN119285756A discloses a method for separating phycocyanin from spirulina platensis by extracting phycocyanin from spirulina platensis using a deep eutectic aqueous two-phase extraction method. The method comprises extracting spirulina platensis powder with a CaCl2 solution, subjecting the extracted spirulina platensis powder to freeze-thaw wall-breaking and centrifugation to obtain a crude extract containing phycocyanin. After ultrafiltration and desalination, a deep eutectic solvent and a potassium sodium tartrate solution are added, mixed to obtain a two-phase aqueous system, the pH is adjusted and mixed, vortexed, and the phases are allowed to separate. Centrifugation promotes phase separation, separates the upper phase, and ultrafiltration is performed on the upper phase to obtain a filtrate of the deep eutectic solvent component and a concentrated phycocyanin solution. After freeze-drying, phycocyanin powder is obtained. Chinese invention patent CN118420749A discloses a method for preparing high-purity phycocyanin. The method comprises extracting phycocyanin from spirulina platensis powder using an ultrasonically coupled high-pressure homogenization method, subjecting the extracted phycocyanin to salting-out and dialysis, and purifying the phycocyanin using a silicone-modified hydroxyapatite column chromatography method. High-purity phycocyanin can be obtained by selectively adsorbing phycocyanin onto organosiloxane-modified hydroxyapatite, followed by desorption with an eluent. Chinese utility model patent CN222034246U discloses a membrane separation device for extracting phycocyanin, comprising a separation box and a feed pipe fixedly connected to the side wall. A first electric valve is fixedly connected to one end of the feed pipe. A connecting tube is provided within the separation box, and a mounting ring and separation membrane are fixedly connected to the upper end of the connecting tube. The separation box has ample space for storing impurities, eliminating the need for frequent interruptions to clean the separation membrane and eliminating the need for disassembly and cleaning of the complex separation membrane assembly. Summary of the Invention

[0006] The purpose of the present invention is to provide a phycocyanin extraction microsphere that can efficiently adsorb and extract phycocyanin.

[0007] The technical solution adopted in the present invention is:

[0008] A phycocyanin extraction microsphere, the structure of which is shown in the following formula:

[0009]

[0010] Wherein at least two of m, n, and p are not zero, and the number average molecular weight of the microspheres is M n It is between 270,000 and 480,000.

[0011] The value of m ranges from 0 to 2000, the value of n ranges from 0 to 2000, and the value of p ranges from 0 to 2000.

[0012] Preferably, the particle size of the microspheres is 1 to 1000 μm, more preferably 110 to 160 μm.

[0013] Preferably, the polymer monomers are dispersed in an aqueous solution containing a surfactant, and an initiator is added to carry out a polymerization reaction to obtain the phycocyanin extraction microspheres, wherein the polymer monomers are two or three of styrene, 4-vinylpyridine, and 1-vinylimidazole.

[0014] Preferably, the initiator is azobisisobutyronitrile, potassium persulfate, ammonium persulfate or Fe 2+ -Hydrogen peroxide.

[0015] Preferably, the surfactant is one or more of vitamin E polyethylene glycol succinate, sorbitan fatty acid ester, sorbitan fatty acid ester, and polyoxyethylene-polyoxypropylene polymer.

[0016] The present invention also discloses a method for preparing the above-mentioned phycocyanin extraction microspheres, the specific steps of which are:

[0017] A. Add 1 to 5 wt % of a surfactant, preferably 1 wt % to water, stir well, then add a polymer monomer, the amount of the polymer monomer added is 10 to 50% by weight of the aqueous solution containing the surfactant, and stir the mixture at 20 to 50° C. for 0.1 to 1 h, preferably 25° C. for 0.5 h, at a stirring speed of 200 to 800 rpm, preferably 400 rpm, to obtain a microemulsion;

[0018] B. Add an initiator to the microemulsion in an amount of 0.1-2% by weight of the microemulsion, fully dissolve, and heat to 60-90° C. for 0.5-10 h, preferably 1% at 70° C. for 5 h;

[0019] C. After the reaction is completed, the reaction solution is centrifuged, the precipitate is collected, washed with water and then dried to obtain phycocyanin extraction microspheres.

[0020] Preferably, in the polymer monomers added in step A, the weight ratio of styrene to 4-vinylpyridine and 1-vinylimidazole is 0-50:0-50:0-50, and the ratio of at least two of styrene, 4-vinylpyridine and 1-vinylimidazole is not zero, preferably 20:15:10.

[0021] Preferably, the centrifuge speed in step C is 2000-8000 rpm, preferably 4000 rpm.

[0022] The invention also discloses the application of the phycocyanin extraction microspheres in extracting phycocyanin.

[0023] Preferably, 1 to 30% by weight of phycocyanin extraction microspheres, preferably 5%, are added to the crude product solution of phycocyanin to be extracted, and stirred at 20 to 50° C. for 0.1 to 5 hours, preferably 25° C. for 1.5 hours. After the stirring stops, the microspheres are separated and a solution with a pH of 0 to 5 of 0.1 to 3 times the total weight of the microspheres is added, preferably 2 times the weight, pH = 3. The pH value of the solution can be adjusted by sulfuric acid, hydrochloric acid, nitric acid, acetic acid, etc., preferably hydrochloric acid, and soaked for 0.1 to 3 hours, preferably 0.5 hours. After the microspheres are separated, the resulting solution is adjusted to a neutral pH, and purified phycocyanin is obtained by recrystallization or freeze-drying. The separated microspheres can be reused for the next extraction of phycocyanin.

[0024] The polymer microspheres provided by the present invention can be used to extract phycocyanin efficiently and conveniently, helping to reduce costs and increase efficiency in the phycocyanin extraction and purification process. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the use of the phycocyanin extraction microspheres of the present invention.

[0026] Figure 2 is the chemical reaction formula of the phycocyanin extraction microspheres of the present invention.

[0027] Figure 3 The microspheres obtained in Example 1 and photos of their extraction of phycocyanin are shown (A. microsphere product; B. microspheres and phycocyanin extraction solution are mixed and then settled; C. phycocyanin solution after elution from the microspheres). Specific implementation methods

[0028] The present invention will be further described below with reference to the examples, but the description of the examples does not limit the scope of protection of the present invention in any way.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. The terms used in the specification of the present invention are intended only to describe specific embodiments and are not intended to limit the present invention. Furthermore, while examples of parameters including specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but may be approximated to the corresponding values ​​within acceptable error tolerances or design constraints.

[0030] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0031] Example 1

[0032] To 100 mL of pure water, add 1 g of the surfactant VE-TPGS and stir at 25°C for 0.5 h. Sequentially add 20 g of styrene and 15 g of vinyl pyridine and stir at 400 rpm for 0.5 h. Then add 1 g of ammonium persulfate, dissolve thoroughly, and heat to 70°C for 5 h. After cooling to room temperature, centrifuge the emulsion to remove the precipitate at 4000 rpm. Rinse the resulting microspheres three times with pure water and soak in pure water or dry for storage. This results in polymer microsphere product 1, which can be used to extract phycocyanin.

[0033] The phycocyanin extracted microspheres are added to a crude phycocyanin solution 20 times its weight, stirred at 25°C for 1.5 hours, and the microspheres are filtered out. A pH 3 solution 2 times its total weight is then added, soaked for 0.5 hours, and the microspheres are filtered out. The resulting solution is adjusted to a neutral pH and recrystallized to obtain purified phycocyanin. The crude phycocyanin solution is obtained by breaking the cell wall of fresh spirulina or dried spirulina powder using commonly used industrial methods such as grinding.

[0034] Example 2

[0035] To 100 mL of pure water, add 1.5 g of the surfactant VE-TPGS and stir at 25°C for 0.5 h. Sequentially add 20 g of vinyl imidazole and 20 g of vinyl pyridine and stir at 400 rpm for 0.5 h. Then add 1.2 g of ammonium persulfate, dissolve thoroughly, and heat to 70°C for 5 h. After cooling to room temperature, centrifuge the emulsion to remove the precipitate at 4000 rpm. Rinse the resulting microspheres three times with pure water and soak in pure water or dry for storage. This yields polymer microsphere product 2, which can be used to extract phycocyanin.

[0036] The phycocyanin extracted microspheres were added to a crude phycocyanin solution 20 times its weight, stirred at 25°C for 1.5 hours, and then the microspheres were filtered out. A pH = 3 solution 2 times its total weight was added and soaked for 0.5 hours, and then the microspheres were filtered out. The resulting solution was adjusted to a neutral pH and recrystallized to obtain purified phycocyanin.

[0037] Example 3

[0038] To 100 mL of pure water, add 2.0 g of the surfactant VE-TPGS and stir at 25°C for 0.5 h. Then, add 10 g of styrene, 15 g of vinyl imidazole, and 15 g of vinyl pyridine in sequence and stir at 400 rpm for 0.5 h. Then, add 1.5 g of ammonium persulfate, dissolve thoroughly, and heat to 70°C for 5 h. After cooling to room temperature, centrifuge the emulsion to remove the precipitate at 4000 rpm. Rinse the resulting microspheres three times with pure water and soak in pure water or dry them for storage. This yields polymer microsphere product 3, which can be used to extract phycocyanin.

[0039] The phycocyanin extracted microspheres were added to a crude phycocyanin solution 20 times its weight, stirred at 25°C for 1.5 hours, and then the microspheres were filtered out. A pH = 3 solution 2 times its total weight was added and soaked for 0.5 hours, and then the microspheres were filtered out. The resulting solution was adjusted to a neutral pH and recrystallized to obtain purified phycocyanin.

[0040]

[0041] The yield is calculated as the percentage of phycocyanin extracted by microspheres to the total phycocyanin content in the crude product solution, and is quantified by Ni element analysis in phycocyanin.

[0042] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A phycocyanin extraction microsphere, characterized in that: Its structure is shown below: Among them, at least two of m, n, and p are not zero, and the number average molecular weight of the microspheres is M n It is between 270,000 and 480,000.

2. The phycocyanin extraction microspheres according to claim 1, characterized in that The particle size of the microspheres is 1 to 1000 μm.

3. The method for preparing phycocyanin extraction microspheres according to claim 1 or 2, characterized in that: The polymer monomers are dispersed in an aqueous solution containing a surfactant, and an initiator is added to carry out a polymerization reaction to obtain the phycocyanin extraction microspheres, wherein the polymer monomers are two or three of styrene, 4-vinylpyridine, and 1-vinylimidazole.

4. The preparation method according to claim 3, characterized in that The initiator is azobisisobutyronitrile, potassium persulfate, ammonium persulfate or Fe 2+ -Hydrogen peroxide.

5. The preparation method according to claim 3, characterized in that The surfactant is one or more of vitamin E polyethylene glycol succinate, sorbitan fatty acid ester, sorbitan fatty acid ester, and polyoxyethylene-polyoxypropylene polymer.

6. The preparation method according to any one of claims 3 to 5, characterized in that The specific steps are: A. Add 1 to 5 wt% of a surfactant to water, stir evenly, then add a polymer monomer, wherein the amount of the polymer monomer added is 10 to 50% by weight of the aqueous solution containing the surfactant, and stir the mixture at 20 to 50° C. for 0.1 to 1 hour to obtain a microemulsion; B. Add an initiator to the microemulsion in an amount of 0.1-2% by weight of the microemulsion, fully dissolve it, heat it to 60-90°C, and react for 0.5-10 hours; C. After the reaction is completed, the reaction solution is centrifuged, the precipitate is collected, washed with water and then dried to obtain phycocyanin extraction microspheres.

7. The preparation method according to claim 6, characterized in that In the polymer monomers added in step A, the weight ratio of styrene to 4-vinylpyridine and 1-vinylimidazole is 0-50:0-50:0-50, and the ratio of at least two of styrene, 4-vinylpyridine and 1-vinylimidazole is not zero.

8. The preparation method according to claim 6, characterized in that In step C, the centrifuge speed is 2000-8000 rpm.

9. Use of the phycocyanin extraction microspheres according to claim 1 or 2 in extracting phycocyanin.

10. The use according to claim 9, characterized in that A phycocyanin extraction microsphere is added at a weight ratio of 1 to 30% to a crude product solution of phycocyanin to be extracted, and the mixture is stirred at 20 to 50° C. for 0.1 to 5 hours. After the stirring stops, the microspheres are separated, and a solution with a pH of 0 to 5 in an amount of 0.1 to 3 times the total weight of the microspheres is added and soaked for 0.1 to 3 hours. After the microspheres are separated, the resulting solution is adjusted to a neutral pH and recrystallized or freeze-dried to obtain purified phycocyanin.

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