A method for preparing phycocyanin and related applications thereof

Abstract

The application discloses a preparation method of phycocyanin and related application thereof, relates to the field of active product extraction, and innovatively constructs a synergistic extraction system of a low-temperature environment and a specific extraction solvent, and through function adaptation and parameter matching, denaturation of target substances is inhibited from the source, directional and sufficient dissolution of the target substances is realized, and extraction efficiency is remarkably improved. Through disc centrifugation, tubular centrifugation and plate-and-frame filtration, cell fragments and macromolecular impurities are effectively removed. Then, the target substances with high purity and high activity are obtained through concentration and desalination of an ultrafiltration membrane. The function design and parameter setting of each step of the preparation process are highly matched, a stable and controllable closed-loop system is formed, efficient extraction and high purity of the product are ensured, and the system has the characteristics of continuous operation, easy control of parameters and reasonable energy consumption, and is completely suitable for industrial large-scale production requirements.

Description

Technical Field

[0001] This invention relates to the field of active product extraction, and more specifically, to a method for preparing phycocyanin and its related applications. Background Technology

[0002] Phycocyanin is an important natural food coloring and bioactive substance, widely used in food, cosmetics, medicine and diagnostic reagents.

[0003] However, the industrial production of phycocyanin has long faced bottlenecks in extraction processes. Currently used traditional methods, such as repeated freeze-thaw cycles, ultrasound-assisted extraction, or enzymatic hydrolysis, essentially rely on violent cell disruption mechanisms. These methods easily lead to the denaturation and inactivation of phycocyanin due to heat or mechanical shearing forces, and also result in low extraction efficiency, numerous impurities, and difficulties in subsequent purification, making it difficult to meet the high requirements for product purity and activity in industrial production.

[0004] Therefore, it is of great significance to develop a mild, efficient and easily scalable phycocyanin production process.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing phycocyanin and its related applications.

[0007] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a method for preparing phycocyanin, comprising the following steps: extracting spirulina powder with an extraction solvent at 0-20°C to obtain an extract; wherein the extraction solvent comprises one or more combinations of phosphate buffer, water, calcium chloride solution, and sodium chloride solution; centrifuging the extract using a disc centrifuge and a tube centrifuge to obtain a primary supernatant; filtering the primary supernatant using a plate and frame filter to obtain a purified filtrate; and ultrafiltration the purified filtrate to collect the retentate.

[0008] Secondly, embodiments of the present invention provide products prepared by the preparation method described in the foregoing embodiments.

[0009] Thirdly, embodiments of the present invention provide the application of the preparation method as described in the foregoing embodiments or the product obtained therefrom in the preparation of food additives or cosmetic additives.

[0010] The present invention has the following beneficial effects: This invention innovatively constructs a synergistic extraction system combining a low-temperature environment and a specific extraction solvent to extract spirulina components. This system effectively inhibits phycocyanin denaturation at the source and achieves targeted and complete dissolution of phycocyanin, significantly improving extraction efficiency. Further, a multi-stage progressive purification system combining disc centrifugation, tubular centrifugation, and plate and frame filtration is constructed. This combination is precisely adapted to the physical properties of cell debris and macromolecular impurities, with each stage working synergistically and targeting impurities for removal. Compared to conventional purification systems, this system exhibits superior compatibility and can efficiently remove target impurities, providing a high-purity material basis for subsequent processes. Finally, ultrafiltration membranes are used for concentration and desalting to obtain high-purity, highly active phycocyanin.

[0011] The preparation process of this invention is highly compatible in terms of functional design and parameter setting, forming a stable and controllable closed-loop system. This not only ensures the core advantages of high purity and high activity of the product, but also features continuous operation, easy parameter control, and reasonable energy consumption, making it fully suitable for the needs of large-scale industrial production. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0013] On one hand, embodiments of the present invention provide a method for preparing phycocyanin, which includes the following steps: Spirulina powder was extracted using an extraction solvent at 0-20°C to obtain an extract; wherein the extraction solvent included one or more combinations of phosphate buffer, purified water, calcium chloride, or sodium chloride solution. The extract was subjected to disc centrifugation and tube centrifugation to obtain a primary supernatant; The primary supernatant is subjected to plate and frame filtration to obtain a refined filtrate; The purified filtrate is subjected to ultrafiltration, and the retentate is collected.

[0014] In an optional embodiment, the 0~20℃ can specifically be any one or any two of 0, 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20℃.

[0015] In an optional embodiment, the extraction includes: mixing the spirulina powder and the extraction solvent at a mass ratio of 1:(15~25), and extracting until the phycocyanin concentration is ≥4.5mg / mL to obtain an extract.

[0016] In an optional embodiment, during the extraction step, the mass ratio of the spirulina powder to the extraction solvent is 1:15 to 25, specifically any one or any two of the following: 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24 and 1:25.

[0017] In an optional embodiment, the extraction time can be any one of 1, 2, 3, 4, 4, 4.5 (equivalent to 4h30min), 5, 5.5 and 6h or any range between two of them.

[0018] In an optional embodiment, the extraction solvent is selected from at least one of the following reagents: phosphate buffer solution with a concentration of 0.005~0.1M and a pH of 6.8~7.2; pure water at 0~25°C; calcium chloride solution with a mass fraction of 1%~4%; and sodium chloride solution with a mass fraction of 2%~4%.

[0019] In an optional embodiment, the concentration of the phosphate buffer solution can be any one or a range between any two of 0.005, 0.006, 0.008, 0.01, 0.012, 0.014, 0.016, 0.018, 0.02, 0.04, 0.06, 0.08, and 0.1M, and the pH is 6.5 to 7.5, specifically any one or a range between any two of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, and 7.5.

[0020] In an optional embodiment, the phosphate buffer contains hydrogen phosphate ions, dihydrogen phosphate ions, phosphate ions, and alkali metal ions. The alkali metal ions are selected from one or both of potassium and sodium ions.

[0021] In an optional embodiment, the purified water is purified water at 0~10°C. The temperature of the purified water can be any one or any combination of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10°C.

[0022] In an optional embodiment, the mass fraction (w / w) of the calcium chloride solution can specifically be any one or a range between two of 1%, 2%, 3% and 4%.

[0023] In an optional embodiment, the mass fraction (w / w) of the sodium chloride solution can specifically be any one or a range between two of 1%, 2%, 3% and 4%.

[0024] In an optional embodiment, the centrifugal force of the disc centrifuge is 4000~10000×g, specifically within the range of any one or any two of 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 and 10000×g; the outlet pressure of the disc centrifuge is ≤0.2MPa, specifically within the range of any one or any two of 0, 0.1, 0.15, 0.18 and 0.2MPa; the feed flow rate is within the range of any one or any two of 50, 100, 150, 200, 250, 280, 300, 320, 350 and 400L / h; and the slag discharge cycle is 5~10min, specifically within the range of any one or any two of 5, 6, 7, 8, 9 and 10min.

[0025] In an optional embodiment, the conditions for the tubular centrifugation are 12000~18000×g, specifically any one or any two of 12000, 13000, 14000, 15000, 16000, 17000 and 18000×g; the feed flow rate is 50~200L / h, specifically any one or any two of 50, 60, 80, 100, 120, 140, 160, 180 and 200 L / h; and the slag discharge cycle is 20~90 min, specifically any one or any two of 20, 25, 30, 35, 40, 50, 60, 70, 80 and 90 min.

[0026] In an optional embodiment, the operating pressure of the plate and frame filter is ≤0.4MPa, specifically 0~0.4MPa, for example, any one or any two of 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35 and 0.4MPa.

[0027] In an optional embodiment, the plate and frame filter includes two-stage filtration: the first-stage filtration uses a filter screen or filter bag with a pore size of 0.5~300μm; the second-stage filtration uses a filter screen or filter bag with a pore size of 0.1~1μm. The pore size of the filter screen or filter bag in the first-stage filtration can be any one or any two of the following: 0.5, 0.6, 0.8, 1, 10, 50, 100, 150, 180, 190, 200, 210, 220, 250, and 300μm. The pore size of the filter screen or filter bag in the second-stage filtration can be any one or any two of the following: 0.1, 0.2, 0.3, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, and 1μm.

[0028] In an optional embodiment, the first stage of filtration uses a filter screen or filter bag with a pore size of 0.8~200μm; the second stage of filtration uses a filter screen or filter bag with a pore size of 0.2~0.5μm.

[0029] In an optional embodiment, the ultrafiltration membrane has a molecular cutoff of 5 to 120 kDa, specifically any one or any two of the following: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, and 120 kDa.

[0030] In an optional embodiment, the preparation method further includes filtering and / or drying the retentate.

[0031] In an optional embodiment, the filtration uses a filter screen or filter bag with a mesh size of 100 to 300. Specifically, the mesh size can be any one or any two of the following: 100, 120, 140, 150, 160, 180, 200, 220, 240, 260, 280, and 300 mesh.

[0032] In an optional embodiment, the drying method is spray drying or freeze drying.

[0033] In an optional embodiment, the spray drying conditions are: an inlet air temperature of 180~210℃ and an exhaust air temperature of 75~110℃. The inlet air temperature can be any one or a range between any two of 180, 185, 190, 195, 200, 205, and 210℃; the exhaust air temperature can specifically be any one or a range between any two of 75, 80, 85, 90, 95, 100, 105, and 110℃.

[0034] On the other hand, embodiments of the present invention also provide products prepared by the preparation method described in any of the foregoing embodiments.

[0035] Furthermore, embodiments of the present invention also provide the application of the preparation method as described in any of the foregoing embodiments or the product obtained therefrom in the preparation of food additives or cosmetic additives.

[0036] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0037] Experimental materials: The 0.01M phosphate buffer (pH=7.0) used in Example 1 was diluted 10 times from the 0.1 M stock solution (61 mL solution A + 39 mL solution B / 200 mL). Solution A (0.2 M Na2HPO4), Solution B (0.2 M NaH2PO4).

[0038] Example 1 A method for preparing phycocyanin includes the following steps.

[0039] (1) Extraction Weigh 100 kg of spirulina powder and place it in an extraction tank. Add 20 times (2000 L) of extraction solvent (0.01 M phosphate buffer (pH=7.0)), turn on the cooling circulating water, control the temperature at 15 ± 2℃, and slowly stir for extraction. Take samples every hour, centrifuge at 5000 × g, and measure the absorbance at 620 and 650 nm. Calculate the concentration (mg / mL) according to the formula: Concentration (mg / mL) = (A... 620 -0.474×A 652 Calculate the concentration using 5.34. When the phycocyanin concentration is greater than 4.5 mg / mL, perform coarse filtration (80-mesh metal filter), and then pump the solution into a disc centrifuge.

[0040] (2) Butterfly centrifuge The disc centrifuge was set with an outlet pressure of 0.1 MPa, a centrifugal force of approximately 8000 × g, a feed flow rate of 300 L / h, and an automatic slag discharge cycle of 6 minutes. Manual slag discharge was performed when the machine load increased. The supernatant was collected.

[0041] (3) Tubular centrifuge The supernatant after butterfly centrifugation is pumped into a tubular centrifuge (7 cm rotor radius, 13,000 rpm, 13,200 × g, feed flow rate of 100 L / h, and discharge cycle of 30 min) at a controlled flow rate for further purification and separation to remove finer impurities and obtain a clear supernatant.

[0042] (4) Plate and frame filtration Add 0.5% (w / v) of filter aid (diatomaceous earth in this example) to the supernatant obtained after tubular centrifugation and stir for 25 minutes. First, use a relatively coarse pre-filter (200 μm filter screen) to trap any large aggregates that may be present, with a feed pressure of 0.1 MPa to protect the main filter membrane. Then, use the main filter membrane (pore size 0.45 μm) and control the feed pressure to ≤0.4 MPa for filtration (starting from 0.1 MPa, gradually increasing it, and stopping filtration at 0.4 MPa), and collect the clear filtrate.

[0043] (5) Ultrafiltration concentration The filtrate collected in step (4) is pumped into an ultrafiltration system (molecular weight cutoff 30kDa). The permeate (containing small molecule impurities and salts) is discharged, and the retentate (phycocyanin concentrate) is collected. When the concentrate is reduced to about 1 / 10 of its original volume, an appropriate amount of purified water is added to perform a "water top" operation to displace the residual salts and further improve the purity of the product.

[0044] (6) Spray drying The concentrated liquid was filtered through a 200-mesh filter bag and then fed into a spray drying tower. The inlet air temperature was set to 195℃, and the exhaust air temperature was controlled at 85±5℃ by adjusting the feed pump speed. The powdery product at the bottom of the drying tower was collected, sealed in double-layer PE bags, yielding approximately 5.2 kg of phycocyanin powder (based on dry powder).

[0045] The purity (A) was determined to be... 620 / A 280 The value is 3.06.

[0046] Example 2 A method for preparing phycocyanin includes the following steps.

[0047] (1) Extraction Take 100 kg of spirulina powder and add 18 times (1800 L) of extraction solvent (pre-cooled purified water at 4°C in this example) for extraction. The temperature should be controlled below 10°C (4°C in this example). Stir at high speed (but avoid foaming) during extraction. Take samples every hour, centrifuge at 5000 × g, and measure the absorbance at 620 and 650 nm. The concentration (mg / mL) is calculated using the formula: Concentration (mg / mL) = (A... 620 -0.474×A 652 Calculate the concentration using 5.34. When the phycocyanin concentration is greater than or equal to 4.5 mg / mL, perform coarse filtration (80-mesh metal filter), and then pump the solution into a disc centrifuge.

[0048] (2) Butterfly centrifugation: Same as in Example 1.

[0049] (3) Tubular centrifuge: Same as in Example 1.

[0050] (4) Plate and frame filtering: Same as in Example 1.

[0051] (5) Ultrafiltration concentration: Same as in Example 1.

[0052] (6) Spray drying: Same as in Example 1.

[0053] The purity (A) was determined to be... 620 / A 280 The degree is 3.86.

[0054] Example 3 A method for preparing phycocyanin includes the following steps.

[0055] (1) Extraction: The steps are the same as in Example 1.

[0056] (2) Butterfly centrifugation: The steps are the same as in Example 1.

[0057] (3) Tube centrifugation: The steps are the same as in Example 1.

[0058] (4) Plate and frame filtration In plate and frame filtration, a two-stage filtration process is adopted. First, a filter membrane with a pore size of 0.8 μm (0.1 MPa) is used, and then a microporous filter membrane with a pore size of 0.45 μm (0.4 MPa) is used for terminal sterilization filtration to remove impurities and microorganisms to the maximum extent.

[0059] (5) Ultrafiltration concentration Using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa can more effectively enrich phycocyanin (whose molecular weight is approximately 140 kDa or higher) while more thoroughly removing small molecule proteins.

[0060] (6) Spray drying The steps are largely the same as in Example 1, but the inlet air temperature is reduced to 185°C to mitigate the thermal effect. The purity (A) was determined to be... 620 / A 280 The degree is 4.15.

[0061] Example 4 To verify the effect of extraction solvent on extraction efficiency.

[0062] Based on the preparation method of phycocyanin provided in Example 1, multiple experimental groups were set up, which were roughly the same as in Example 1, except that the extraction solvent was different.

[0063] The extracts obtained from different extraction solvents in step (1) were centrifuged at 10000×g for 5 minutes, diluted 40 times with 0.1M phosphate buffer, and scanned at 200~700nm using a UV-Vis spectrophotometer.

[0064] Table 1 Comparison results of multiple experimental groups

[0065] Note: 0.1 M stock solution (61 mL solution A + 39 mL solution B / 200 mL) is diluted 10 times. Solution A is 0.2 M Na2HPO4, and solution B is 0.2 M NaH2PO4.

[0066] Example 5 To verify the effect of extraction temperature on extraction efficiency.

[0067] According to the preparation method of phycocyanin provided in Example 1, multiple experimental groups were set up, which were roughly the same as in Example 1, except that the extraction temperature was different. 10000×g of the extracts extracted at different extraction temperatures in step (1) were centrifuged for 5 minutes, diluted 40 times with 0.1M phosphate buffer, and scanned at 200~700nm using a UV-Vis spectrophotometer.

[0068] Table 2 Comparison results of multiple experimental groups

[0069] Example 6 To verify the effect of centrifugation after one-step extraction on the extraction efficiency.

[0070] According to the preparation method of phycocyanin provided in Example 1, multiple experimental groups were set up, which were roughly the same as in Example 1, except that: (2) butterfly centrifugation, (3) tube centrifugation and / or (4) plate and frame filtration were different. The supernatant of group 1 to group 3 was collected after plate and frame filtration, and the supernatant of group 4 was collected after tube centrifugation. The above samples were centrifuged at 10000×g for 5 minutes, diluted 40 times with 0.1M phosphate buffer, and scanned with a UV-Vis spectrophotometer at 200~700nm.

[0071] Table 3 Comparison results of multiple experimental groups

[0072] Example 7 To verify the effect of the extraction time in the stepwise extraction process on the extraction efficiency.

[0073] According to the preparation method of phycocyanin provided in Example 1, multiple experimental groups were set up, which were roughly the same as in Example 1, except that the extraction time was different. 10000×g of the extract obtained from different extraction times in step (1) was taken, centrifuged for 5 minutes, diluted 40 times with 0.1M phosphate buffer, and scanned at 200~700nm using a UV-Vis spectrophotometer.

[0074] Table 4 Comparison results of multiple experimental groups

[0075] Example 8 To verify the effect of ultrafiltration membrane on the purity of phycocyanin.

[0076] According to the preparation method of phycocyanin provided in Example 1, multiple experimental groups were set up, which were roughly the same as in Example 1, except that the molecular cutoff of the ultrafiltration membrane was different. The filtrate after ultrafiltration was taken at 10000×g, centrifuged for 5 minutes, diluted 40 times with 0.1M phosphate buffer, and scanned at 200~700nm using a UV-Vis spectrophotometer.

[0077] Table 5 Comparison results of multiple experimental groups

[0078] Comparative Example 1 A method for preparing phycocyanin includes the following steps.

[0079] Take 100 kg of spirulina powder, add 20 times its weight of tap water, and extract by stirring at 50°C for 4 hours. Subsequent processing is similar to that in Example 1.

[0080] The final phycocyanin was dark in color (possibly due to denaturation), and its purity (A) was low. 620 / A 280 The yield was only around 0.3, and the yield was significantly lower than that of Example 1.

[0081] In summary, the low-temperature one-step extraction combined with multi-stage purification process provided by this invention can significantly improve the extraction efficiency and product purity of phycocyanin, and effectively protect its biological activity. The process parameters are clear, easy to control and scale up, and have significant industrial application value.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing phycocyanin, characterized in that, It includes the following steps: Spirulina powder was extracted using an extraction solvent at 0-20°C to obtain an extract; wherein the extraction solvent included one or more combinations of phosphate buffer, water, calcium chloride solution and sodium chloride solution; The extract was subjected to disc centrifugation and tube centrifugation to obtain a primary supernatant; The primary supernatant is subjected to plate and frame filtration to obtain a refined filtrate; The purified filtrate is subjected to ultrafiltration, and the retentate is collected.

2. The preparation method according to claim 1, characterized in that, The extraction process includes: mixing the spirulina powder and the extraction solvent at a mass ratio of 1:(15~25), and extracting until the phycocyanin concentration is ≥4.5mg / mL to obtain an extract; Optionally, the extraction temperature is 0~17℃.

3. The preparation method according to claim 1, characterized in that, The extraction solvent is selected from at least one of the following reagents: phosphate buffer solution with a concentration of 0.005~0.1M and a pH of 6.8~7.2; pure water; calcium chloride solution with a mass fraction of 1%~4%; A sodium chloride solution with a mass fraction of 2% to 4%.

4. The preparation method according to claim 1, characterized in that, The centrifugal force of the disc centrifuge is 4000~10000×g, the outlet pressure of the disc centrifuge is ≤0.2MPa, the feed flow rate is 50~400 L / h, and the slag discharge cycle is 5~10min; Optionally, the outlet pressure of the disc centrifuge is 0~0.2MPa; Optionally, the conditions for the tubular centrifuge are 12000~18000×g, feed flow rate is 50~200L / h, and slag discharge cycle is 20~90 min.

5. The preparation method according to claim 1, characterized in that, The operating pressure of the plate and frame filter is ≤0.4MPa; Optionally, the plate and frame filter includes two-stage filtration: the first stage filtration uses a filter screen or filter bag with a pore size of 0.5~300μm; the second stage filtration uses a filter screen or filter bag with a pore size of 0.1~1μm. Optionally, the first stage of filtration uses a filter screen or filter bag with a pore size of 0.8~200μm; the second stage of filtration uses a filter screen or filter bag with a pore size of 0.2~0.5μm.

6. The preparation method according to claim 1, characterized in that, The ultrafiltration membrane has a molecular cutoff of 5~120 kDa.

7. The preparation method according to claim 1, characterized in that, The preparation method further includes: filtering and / or drying the retentate; Optionally, the filtration uses a 100-300 mesh filter screen or filter bag.

8. The preparation method according to claim 7, characterized in that, The drying method is either spray drying or freeze drying; Optionally, the spray drying conditions are: inlet air temperature of 180~210℃ and outlet air temperature of 75~110℃.

9. The product prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the preparation method according to any one of claims 1 to 8 or the product obtained therefrom in the preparation of food additives or cosmetic additives.

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