A method for preparing phycocyanin

Abstract

This invention discloses a method for preparing phycocyanin, relating to the field of separation and purification technology. The method employs ammonium sulfate fractional crystallization for purification. The crystallization step utilizes precise control of the ammonium sulfate concentration and crystallization stirring time to optimize product purity and recovery rate. This method is simple, low-cost, easily scaled up, and can effectively purify phycocyanin. 620 / A 280 Purify food-grade raw materials of grades 1-3 to A 620 / A 280 It meets reagent-grade standards greater than 4.0 and effectively removes allophycocyanin. The crystallization process results in minimal loss of the target protein, a high overall recovery rate, and produces stable phycocyanin crystals suitable for industrial production, as well as long-term storage and transportation.

Description

Technical Field

[0001] This invention relates to the field of separation and purification technology, and more specifically, to a method for preparing phycocyanin. Background Technology

[0002] Phycocyanin is an important water-soluble fluorescent protein widely used in biomarking, immunoassay, food coloring, and health products. Reagent-grade phycocyanin requires extremely high purity, typically requiring A4 grade purity. 620 / A 280 The ratio is greater than 4.0.

[0003] Currently, the preparation of high-purity phycocyanin mainly relies on chromatographic techniques such as ion exchange chromatography, gel filtration chromatography, and hydroxyapatite chromatography. Although these methods can obtain high-purity products, they have significant drawbacks: (1) the chromatographic media are expensive, resulting in high production costs; (2) the operation steps are cumbersome, the cycle is long, and it is difficult to carry out continuous production; (3) the purified protein has poor stability in solution and is easily degraded. These factors lead to the high price of reagent-grade phycocyanin, which limits its large-scale application.

[0004] Therefore, it is of great significance to develop a purification method that is low-cost, high-efficiency, easy to scale up, and has good product stability.

[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.

[0007] This invention is implemented as follows: An embodiment of the present invention provides a method for preparing phycocyanin, which includes the following steps: Primary crystallization: Add saturated ammonium sulfate solution to the phycocyanin solution to be purified for crystallization until the ammonium sulfate concentration in the system reaches the set concentration, and stir for 8-120 hours; the set concentration is 15%-20% (w / v); filter and / or wash the crystallized solution.

[0008] The present invention has the following beneficial effects: The preparation method provided in the embodiments of the present invention uses ammonium sulfate fractional crystallization for purification. The crystallization step, through precise control of the ammonium sulfate concentration and crystallization stirring time, optimizes the purity and recovery rate of the product. This method is simple, low-cost (using only ammonium sulfate, an inexpensive raw material), easily scaled up, and can process A... 620 / A 280 Purify food-grade raw materials of grades 1-3 to A 620 / A 280It meets reagent-grade standards greater than 4.0 and effectively removes allophycocyanin. The crystallization process results in minimal loss of the target protein, a high overall recovery rate, and produces stable phycocyanin crystals suitable for industrial production, as well as long-term storage and transportation. Attached Figure Description

[0009] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 The crystal morphology of phycocyanin is shown under an optical microscope; where a~f are the crystal morphologies of phycocyanin after stirring for 8h, 24h, 48h, 72h, 96h and 120h respectively in Example 3. Detailed Implementation

[0011] 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.

[0012] An embodiment of the present invention provides a method for preparing phycocyanin, which includes the following steps: Primary crystallization: Add saturated ammonium sulfate solution to the phycocyanin solution to be purified for crystallization until the ammonium sulfate concentration in the system reaches the set concentration, and stir for 8-120 hours; the set concentration is 15%-20% (w / v); filter and / or wash the crystallized solution.

[0013] In an optional embodiment, the method for obtaining the phycocyanin solution to be purified includes: Phycocyanin powder is dissolved in an ammonium sulfate aqueous solution; wherein the concentration of the ammonium sulfate aqueous solution is 1%~12% (w / v). The dissolved solution is filtered to obtain the phycocyanin to be purified.

[0014] In an optional embodiment, during the dissolution of phycocyanin powder, the liquid-to-solid ratio of the ammonium sulfate aqueous solution to the phycocyanin powder is any one or any two of 6, 6.5, 7, 7.5 and 8 mL / g, and the concentration of the ammonium sulfate aqueous solution can be any one or any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% and 12% (w / v).

[0015] In an optional embodiment, the phycocyanin powder A 620 / A 280 The ratio can be any one or any two of 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 and 3.0, and the phycocyanin content can be any one or any two of 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48% and 50% (percentage of the dry weight of the finished phycocyanin powder).

[0016] In an optional embodiment, the phycocyanin powder is spirulina phycocyanin powder.

[0017] In an optional embodiment, the primary crystallization further includes: preparing the purified phycocyanin solution.

[0018] In an optional implementation, the set concentration can specifically be any one or a range between any two of 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% and 20% (w / v).

[0019] In an optional embodiment, the crystallization stirring time can be any one or a range between any two of the following: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120 h.

[0020] In an optional embodiment, the stirring speed for crystallization can be any one or a range between any two of the following: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120 r / min.

[0021] In an optional embodiment, during the crystallization, the addition rate of the saturated ammonium sulfate solution can be any one or a range between any two of 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 and 3.0 mL / h.

[0022] In an optional embodiment, the washing is performed using an ammonium sulfate solution; optionally, the number of washings is any one of 1, 2, 3, 4, and 5 times, or any range between two of them.

[0023] In an optional embodiment, the concentration of the ammonium sulfate solution used for washing is consistent with or independent of the set concentration and is any one or any two of 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% and 20% (w / v).

[0024] In an optional embodiment, the primary crystallization step further includes: filtering the crystallized product and / or drying the washed product.

[0025] In an optional embodiment, the filtration employs filter paper, filter membrane, or filter screen with a pore size of 10-30 micrometers. Specifically, the pore size can be any one or any combination of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 micrometers. In an optional embodiment, the drying conditions are: 0.08-0.1 MPa, 25-40°C, and 8-12 h. Specifically, the pressure can be 0.08, 0.09, or 0.1 MPa; the temperature can be any one or any combination of 25, 26, 28, 30, 32, 34, 36, 38, and 40°C; and the time can be any one or any combination of 8, 9, 10, 11, and 12 h.

[0026] In an optional implementation, the drying method is vacuum drying and / or freeze drying.

[0027] In an optional embodiment, after the primary crystallization, the preparation method further includes recrystallizing the product from the primary crystallization, wherein the recrystallization step is consistent with the primary crystallization step.

[0028] In an optional implementation, the number of recrystallizations is any one of 1, 2, or 3, or a range between any two.

[0029] In an optional embodiment, the product A obtained by the preparation method is... 620 / A 280The value is greater than 4.0, the phycocyanin content is greater than 90%, and the phycocyanin content of other substances is less than 1%.

[0030] In an optional embodiment, the product A obtained by the preparation method is... 620 / A 280 The value is greater than 4.2, the phycocyanin content is greater than 95%, and the phycocyanin content is less than 0.1%.

[0031] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0032] The formula for calculating the recovery rate in this embodiment is as follows: Phycocyanin recovery rate = Phycocyanin content in the product (g) / Phycocyanin content in the raw material (g) × 100%.

[0033] Detection methods for phycocyanin and allophycocyanin: "SNT 1113-2002 Determination of phycocyanin and chlorophyll content in imported and exported Spirulina".

[0034] Example 1 Raw materials: Food-grade spirulina phycocyanin powder, A 620 / A 280 =1.96, phycocyanin content 45.6% (w / w), allophycocyanin content 10.4% (w / w).

[0035] A method for producing reagent-grade phycocyanin includes the following steps.

[0036] Primary crystallization: (1) Dissolve Weigh 50.0 g of raw material, add 300 mL of 8% (w / v) ammonium sulfate solution, and stir magnetically until completely dissolved.

[0037] (2) Filtration Centrifuge the solution obtained in step (1) at 8000 rpm for 10 minutes and take the supernatant.

[0038] (3) Crystallization With stirring at 60 r / min, saturated ammonium sulfate solution was added dropwise to the supernatant at a rate of 2.0 mL / h until the ammonium sulfate concentration in the system reached 17% (w / v). After the addition was complete, stirring was continued at room temperature (20±5℃) for 4 hours (100 rpm).

[0039] (4) Filtration and washing Filter using a 250 mL, 80 mm Buchner funnel with qualitative filter paper of 20-25 micrometers pore size placed on the filter plate of the Buchner funnel. Collect the blue crystals and wash three times with 100 mL of 17% (w / v) ammonium sulfate solution.

[0040] (5) Drying The crystals were placed in a vacuum drying oven and dried at 0.09 MPa and 30°C for 10 hours to obtain 30.2g of the primary crystallized product.

[0041] The primary crystallization product A was measured. 620 / A 280 =3.86, phycocyanin content 72.1% (w / w), allophycocyanin content 3.57% (w / w), and raw material recovery rate 95.6%.

[0042] Recrystallization: The first-crystallized product obtained in step (5) was completely dissolved in 300 mL of 8% (w / v) ammonium sulfate solution, and steps (1) to (5) were repeated to obtain 22.5 g of the final product.

[0043] Measure the A of the final product 620 / A 280 =4.24, phycocyanin content 95.2% (w / w), allophycocyanin content 0.10% (w / w). The recovery rate of the recrystallization step was 98.2%, and the overall recovery rate of the raw materials for the process (two crystallizations) was 93.9%.

[0044] Example 2: Comparison Experiment of Different Final Concentrations of Ammonium Sulfate The production method of phycocyanin is basically the same as in Example 1, except that the target final concentration of ammonium sulfate in the system is changed during the first crystallization. The results are shown in Table 1 (all data are after the first crystallization).

[0045] Table 1 Test Results

[0046] The results showed that the purification effect was better when the concentration was between 15% and 18%, and the best combination of purity and recovery rate was achieved at 17%. The purity decreased when the concentration exceeded 16%, possibly due to increased co-precipitation of impurities.

[0047] Example 3: Comparison Experiment of Different Crystallization Stirring Times The production method of phycocyanin is basically the same as in Example 1 (single crystallization), with the final ammonium sulfate concentration fixed at 17%, only the crystallization stirring time after salt addition being changed. The results are shown in Table 2 (all data are after single crystallization). Crystal morphology is shown in the appendix. Figure 1 Observation and photography were performed using an optical microscope with a scale bar of 50 μm, and images were taken at the same magnification.

[0048] Table 2 Test Results

[0049] The results showed that the product purity and recovery rate were optimal when the stirring time was between 48 and 72 hours. Too short a stirring time resulted in incomplete crystallization, while too long a stirring time might lead to impurities being trapped in the crystals or changes in their physical properties.

[0050] Example 4: Effect of Crystallization Times on the Purity of the Final Product The experiment was conducted using the raw materials and conditions of Example 1 (17%, 48h) to investigate the number of crystallization cycles. Samples were taken and tested after each crystallization, and the results are shown in Table 3.

[0051] Table 3 Test Results

[0052] Note: 1st crystallization corresponds to the first crystallization in Example 1, 2nd crystallization corresponds to the recrystallization in Example 1, and 3rd crystallization is the recrystallization of Example 1 increased to 2 times.

[0053] The results showed that the purity was significantly improved after the first crystallization, but did not reach reagent grade. After the second crystallization, the main indicators (A) were improved. 620 / A 280 The purity (>4.2, APC <0.1%) fully meets the requirements with minimal loss in recovery. While tertiary crystallization offers limited improvement in purity, it further reduces the recovery rate; therefore, secondary crystallization is the optimal choice.

[0054] Example 5: Product Stability Study The secondary crystallization (recrystallization) phycocyanin crystal product (A) prepared in Example 1 was used. 620 / A 280 =4.24, purity 95.2%) was dispensed into brown glass bottles, sealed, and stored at room temperature (25±3℃) in the dark. Samples were taken at 0, 1, 2, and 3 months, redissolved, and their phycocyanin content was determined to assess stability. The results are as follows.

[0055] Table 4 Results

[0056] Data shows that when the crystal product is stored at room temperature for three months, the content of its main active ingredients remains stable and the degradation rate is extremely low, proving that the product obtained by the preparation method provided in this embodiment of the invention has excellent storage stability.

[0057] Example 6: Suitability Experiment of Different Raw Materials Using food-grade phycocyanin raw materials from another source (A) 620 / A 280 =2.5, phycocyanin content 40.1%, other phycocyanin content 15.0%) 50.0g, processed entirely according to the production method (secondary crystallization process) of Example 1.

[0058] Result: 46.2g of product was obtained. Final Product A 620 / A 280 =4.18, phycocyanin content 94.5%, allophycocyanin content 0.12%, total recovery rate 92.4%.

[0059] Conclusion: The method of this invention has good purification effect on food-grade raw materials of different qualities and has wide applicability.

[0060] 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: Primary crystallization: Add saturated ammonium sulfate solution to the phycocyanin solution to be purified for crystallization until the ammonium sulfate concentration in the system reaches the set concentration, and stir for 8-120 hours; the set concentration is 15%-20% (w / v); filter and / or wash the crystallized solution.

2. The preparation method according to claim 1, characterized in that, The set concentration is 15%~18% (w / v); Optionally, the set concentration is 15~17% (w / v).

3. The preparation method according to claim 1, characterized in that, The stirring time for crystallization is 24~96 h, and the rotation speed is 40~120 r / min; Optionally, the stirring time for crystallization is 48-72 h.

4. The preparation method according to claim 1, characterized in that, During the crystallization process, the saturated ammonium sulfate solution is added at a rate of 0.5 to 3.0 mL / h.

5. The preparation method according to claim 1, characterized in that, The washing is carried out using ammonium sulfate solution, and the number of washing cycles is 1 to 5. Optionally, the concentration of the ammonium sulfate solution used for washing is the same as the set concentration.

6. The preparation method according to claim 1, characterized in that, The primary crystallization step further includes: filtering the crystallized product and / or drying the washed product.

7. The preparation method according to claim 6, characterized in that, The drying method is vacuum drying and / or freeze drying; Optionally, the drying conditions are: 0.08~0.1 MPa, 25~40℃, 8~12 h.

8. The preparation method according to claim 1, characterized in that, The method for obtaining the phycocyanin solution to be purified includes: Phycocyanin powder is dissolved in an ammonium sulfate aqueous solution; wherein the concentration of the ammonium sulfate aqueous solution is 1%~12% (w / v). The dissolved solution is filtered to obtain the phycocyanin solution to be purified; Optionally, the filtration process uses filter paper, filter membrane, or filter screen with a pore size of 10-30 micrometers. Optionally, the liquid-to-solid ratio of the ammonium sulfate aqueous solution to the phycocyanin powder is 6-8 mL / g; Optionally, the concentration of the ammonium sulfate aqueous solution is 7% to 9% (w / v); Optionally, the A of the phycocyanin powder 620 / A 280 The ratio is 1.0~3.0, and the phycocyanin content is 30%~50%.

9. The preparation method according to claim 8, characterized in that, The phycocyanin powder is Spirulina phycocyanin powder.

10. The preparation method according to any one of claims 1 to 9, characterized in that, After the first crystallization, the preparation method further includes: recrystallizing the product of the first crystallization, wherein the recrystallization step is the same as the first crystallization step, and the recrystallization is performed 1 to 3 times; Optionally, the product A obtained by the preparation method 620 / A 280 Greater than 4.0, phycocyanin content greater than 90%, and other phycocyanin content less than 1%; Optionally, the product A obtained by the preparation method 620 / A 280 The value is greater than 4.2, the phycocyanin content is greater than 95%, and the phycocyanin content is less than 0.1%.

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