Method for efficiently separating and purifying reagent-grade phycocyanin

Abstract

The invention relates to the technical field of phycocyanin separation and purification, in particular to a method for efficiently separating and purifying reagent-grade phycocyanin. Comprising the following steps: 1) mixing a spirulina raw material with a phosphate buffer solution, soaking, stirring, centrifuging, and collecting a supernatant to obtain a phycocyanin crude extract; (2) adding activated carbon into the phycocyanin crude extract obtained in the step (1), stirring, centrifuging and collecting supernate; (3) adding an aqueous two-phase extraction system into the supernate obtained in the step (2), centrifuging to layer the system, and respectively absorbing the upper phase and the lower phase of the aqueous two-phase extraction system; and 4) dialyzing the upper phase solution obtained in the step 3), and after ultrafiltration, removing the solvent and impurities to obtain the reagent-grade phycocyanin solution. The method provided by the invention solves the problems of tedious operation steps, long operation period and high cost in the traditional technology, has the advantages of mild conditions, good purification effect, short period and easy amplification, and provides reference for efficient preparation of reagent-grade phycocyanin.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and purification of phycocyanin, in particular to a method for efficiently separating and purifying reagent-grade phycocyanin. Background Art

[0002] Phycocyanin (PC) is widely found in cyanobacteria, red algae and cryptomonads. It is a natural pigment-protein complex that is non-toxic and harmless, has good water solubility, and can be used as a natural pigment in food and cosmetics. Due to its own fluorescence, it can be used as a fluorescent molecular probe. It also has biological activities such as improving the body's resistance to radiation, antioxidant, anti-aging, anti-cancer, anti-inflammatory, antibacterial, protecting nerve tissue from damage, enhancing the body's immunity, and protecting the liver. It has been widely used in various fields such as health products, medicine, molecular probes, disease treatment, etc., and has a very broad application prospect.

[0003] According to the spectroscopic properties of phycocyanin, A 620 / A 280 Its purity is characterized by the ratio of . A purity of ≥0.7 is food grade and can be used as a natural blue pigment; a purity of ≥3.0 is pharmaceutical grade; and a purity of ≥4.0 is reagent grade. Its application and value depend on its purity; the higher the purity, the higher its commercial value. Currently, there is extensive research on the extraction and purification processes of phycocyanin, and its extraction and purification processes are relatively mature. Traditional methods for the separation and purification of phycocyanin generally use techniques such as salting out and column chromatography. The salting out method is simple but the purification effect is not obvious, and it is suitable for phycocyanin pretreatment. The column chromatography method has good purification effects, but the fillers are expensive and often require the combination or repeated use of two or more chromatography columns. The steps are cumbersome, time-consuming, and the recovery rate is low. Existing phycocyanin purification processes are mostly suitable for laboratory operations, which seriously limits its large-scale application. Therefore, the development of a low-cost, efficient, rapid, and easily scaled-up method for the separation and purification of high-purity phycocyanin is of great significance for the expanded development and large-scale application of phycocyanin. Summary of the Invention

[0004] In view of the shortcomings of the above-mentioned traditional technologies, the purpose of the present invention is to provide a method for efficiently separating and purifying reagent-grade phycocyanin, which is used to solve the problems of cumbersome operation steps, long operation cycle and high cost in traditional technologies, and has the advantages of mild conditions, good purification effect, short cycle and easy scale-up.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for efficiently separating and purifying reagent-grade phycocyanin, comprising the following steps:

[0007] 1) mixing spirulina raw material with phosphate buffer solution, stirring, centrifuging to collect supernatant, obtaining crude phycocyanin extract;

[0008] 2) adding activated carbon to the crude phycocyanin extract obtained in step 1), stirring, centrifuging to collect supernatant;

[0009] 3) adding aqueous two-phase extraction system to the supernatant obtained in step 2), centrifuging to make the system stratify, respectively sucking the upper and lower phases of the aqueous two-phase extraction system;

[0010] 4) dialyzing and ultrafiltering the upper phase solution obtained in step 3), removing solvent and impurities to obtain reagent-grade phycocyanin solution.

[0011] Preferably, the mass ratio of spirulina raw material to phosphate buffer solution in step 1) is 1:5-7; the pH of the phosphate buffer solution is 6-8, and the concentration is 0.01-0.02 mol / L; the stirring time is 13-17 min; the centrifugation speed is 7500-8500 r / min, the environmental temperature is 3-5℃, and the time is 18-22 min.

[0012] Preferably, the amount of activated carbon added in step 2) is 0.04-0.06 g / mL; the stirring time is 8-12 min.

[0013] Preferably, the ratio of supernatant to aqueous two-phase extraction system in step 3) is 5-10 ml:25-35 ml; the aqueous two-phase extraction system is a PEG 4000 aqueous two-phase extraction system with a mass concentration of 10-14% and a potassium phosphate salt aqueous two-phase extraction system with a mass concentration of 13-17%.

[0014] Preferably, the centrifugation speed in steps 2) and 3) is 4000-4400 rpm, and the time is 8-12 min.

[0015] Preferably, the ultrafiltration method in step 4) is: diluting the dialyzed and filtered solution 8-12 times with phosphate buffer solution, then ultrafiltering with an ultrafiltration tube with MW 10KD, 4000-4400 rpm ultrafiltration for 18-22 min, repeated 3-4 times.

[0016] Preferably, the soaking time in step 1) is 10-14 h, and the temperature is 3-5℃.

[0017] Preferably, the dialysis time in step 4) is 10-14 h, and the temperature is 3-5℃.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] (1) This method uses PBS buffer to extract phycocyanin, which is simple to pre-treat, does not require an ultrasonic disruptor or high-pressure homogenizer, and consumes little energy;

[0020] (2) The present invention uses powdered activated carbon with low price and wide source to replace the traditional salting-out method. The present invention can carry out the next step of purification without dialysis, introduces less impurities, is simple to operate, and the material can be recycled;

[0021] (3) The present invention uses a two-phase aqueous extraction technology to replace the traditional column chromatography method, which has a short extraction time, high recovery efficiency, simple experimental operation, and easy process scale-up, which is conducive to the large-scale industrial preparation of phycocyanin;

[0022] (4) This method can prepare reagent-grade phycocyanin in just 2 days, with a purity of 4.47 and a recovery rate of 41.77%. It not only has a short production cycle but also has simple equipment requirements, making it easy to prepare phycocyanin in large quantities, thereby reducing production costs.

[0023] (5) The present invention soaks the spirulina raw material in PBS buffer overnight, collects the supernatant by centrifugation, and obtains a crude phycocyanin extract. The crude phycocyanin extract is separated and purified using a three-step method of powdered activated carbon adsorption-two-phase aqueous extraction-dialysis ultrafiltration. Compared with the traditional purification process, the present invention can simultaneously separate and purify the reagent-grade phycocyanin while taking the recovery rate into consideration. The process has the advantages of mild conditions, good purification effect, short cycle, and easy scale-up, thereby achieving high value-added comprehensive utilization.

[0024] The present invention solves the problems of complicated operation steps, long operation cycle and high cost in traditional technologies. It has the advantages of mild conditions, good purification effect, short cycle and easy scale-up. It provides a reference for the efficient preparation of reagent-grade phycocyanin and has guiding significance for improving the pilot production process of phycocyanin and promoting the marketization of phycocyanin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0026] Figure 1 The present invention is a flow chart of the method for efficiently separating and purifying reagent-grade phycocyanin.

[0027] Figure 2 This is the UV-visible absorption spectrum of the crude extract of Spirulina phycocyanin, the sample after activated carbon adsorption treatment, two-phase aqueous extraction, and dialysis ultrafiltration. The absorption peak at 620nm is the characteristic absorption peak of phycocyanin.

[0028] Figure 3 The SDS-PAGE electrophoresis diagram of phycocyanin prepared in each purification step. DETAILED DESCRIPTION

[0029] The present invention provides a method for efficiently separating and purifying reagent-grade phycocyanin, comprising the following steps:

[0030] 1) mixing the spirulina raw material with phosphate buffer, stirring, centrifuging, and collecting the supernatant to obtain a crude phycocyanin extract;

[0031] 2) adding activated carbon to the crude phycocyanin extract obtained in step 1), stirring, centrifuging, and collecting the supernatant;

[0032] 3) adding a two-phase aqueous extraction system to the supernatant obtained in step 2), centrifuging the system to separate the layers, and respectively aspirating the upper and lower phases of the two-phase aqueous extraction system;

[0033] 4) The upper phase solution obtained in step 3) is dialyzed and ultrafiltered to remove the solvent and impurities to obtain a reagent-grade phycocyanin solution.

[0034] In the present invention, the mass ratio of the spirulina raw material to the phosphate buffer in step 1) is 1:5-7, preferably 1:6.

[0035] In the present invention, the pH of the phosphate buffer in step 1) is 6-8, and the concentration is 0.01-0.02 mol / L; preferably, the pH of the phosphate buffer is 7, and the concentration is 0.015 mol / L.

[0036] In the present invention, the stirring time in step 1) is 13 to 17 minutes, preferably 14 to 16 minutes, and more preferably 15 minutes.

[0037] In the present invention, the centrifugal speed in step 1) is 7500-8500 r / min, the ambient temperature is 3-5°C, and the time is 18-22 min; preferably, the centrifugal speed is 7700-8300 r / min, the ambient temperature is 4°C, and the time is 19-21 min; more preferably, the centrifugal speed is 7900-8100 r / min, the ambient temperature is 4°C, and the time is 20 min; more preferably, the centrifugal speed is 8000 r / min, the ambient temperature is 4°C, and the time is 20 min.

[0038] In the present invention, the amount of activated carbon added in step 2) is 0.04-0.06 g / mL, preferably 0.05 g / mL.

[0039] In the present application, the stirring time of step 2) is 8-12 min; preferably 9-11 min; further preferably 10 min.

[0040] In the present application, the ratio of the supernatant to the aqueous two-phase extraction system in step 3) is 5-10 ml:25-35 ml; preferably 6-9 ml:27-33 ml; further preferably 7-8 ml:29-31 ml; more preferably 8 ml:30 ml.

[0041] In the present application, the aqueous two-phase extraction system in step 3) is a PEG 4000 with a mass concentration of 10-14% and a potassium phosphate salt with a mass concentration of 13-17%; preferably the aqueous two-phase extraction system is a PEG 4000 with a mass concentration of 11-13% and a potassium phosphate salt with a mass concentration of 14-16%; further preferably the aqueous two-phase extraction system is a PEG 4000 with a mass concentration of 12% and a potassium phosphate salt with a mass concentration of 15%.

[0042] In the present application, the centrifugal speed in steps 2) and 3) is 4000-4400 rpm, and the time is 8-12 min; preferably the centrifugal speed is 4100-4300 rpm, and the time is 9-11 min; further preferably the centrifugal speed is 4200 rpm, and the time is 10 min.

[0043] In the present application, the method of ultrafiltration in step 4) is that the dialysis filtered solution is diluted 8-12 times with phosphate buffer and then ultrafiltrated with an ultrafiltration tube with MW 10KD, 4000-4400 rpm for 18-22 min, repeated 3-4 times; preferably the dialysis filtered solution is diluted 9-11 times with phosphate buffer and then ultrafiltrated with an ultrafiltration tube with MW 10KD, 4100-4300 rpm for 19-21 min, repeated 3 times; further preferably the dialysis filtered solution is diluted 10 times with phosphate buffer and then ultrafiltrated with an ultrafiltration tube with MW 10KD, 4200 rpm for 20 min, repeated 3 times.

[0044] In the present application, the soaking time in step 1) is 10-14 h, and the temperature is 3-5℃; preferably the soaking time is 11-13 h, and the temperature is 4℃; further preferably the soaking time is 12 h, and the temperature is 4℃.

[0045] In the present application, the dialysis time in step 4) is 10-14 h, and the temperature is 3-5℃; preferably the dialysis time is 11-13 h, and the temperature is 4℃; further preferably the dialysis time is 12 h, and the temperature is 4℃.

[0046] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] A method for efficiently separating and purifying reagent-grade phycocyanin, comprising the following steps:

[0049] (1) Take 50 g of Spirulina powder and add 300 mL of phosphate buffer solution (PBS) with pH = 7 and 0.01 mol / L, soak at 4°C overnight; place on a magnetic oscillating stirrer and stir at high speed (4000 rpm) for 15 min, centrifuge at 8000 r / min and 4°C for 20 min, take the supernatant to obtain a crude phycocyanin extract, determine the purity of the phycocyanin in the solution, and calculate the recovery rate, while performing UV-visible full wavelength scanning [the purity of phycocyanin is the ratio of the absorbance values ​​at 620 nm and 280 nm (A 620 / A 280 )】

[0050] (2) adding an appropriate amount of powdered activated carbon to the crude phycocyanin extract obtained in step (1), controlling the amount of powdered activated carbon added to the phycocyanin solution to be 0.05 g / mL, placing the solution on a magnetic stirrer and stirring uniformly (2000 rpm) for 10 min, centrifuging at 4200 rpm for 10 min, collecting the supernatant, determining the purity of the phycocyanin in the solution, and calculating the recovery rate, while performing UV-visible full wavelength scanning;

[0051] (3) 10 ml of the phycocyanin solution obtained in step (2) was added to a two-phase aqueous extraction system containing 12% PEG 4000 and 15% potassium phosphate, and the total mass of the system was adjusted to 40 g with PBS buffer. The mixture was fully mixed on a vortex mixer to dissolve all the PEG and potassium phosphate. The mixture was centrifuged at 4200 rpm for 10 min to accelerate the separation of the system. The upper and lower phases of the two-phase aqueous system were respectively aspirated, and the volumes of the upper and lower phases, the purity and concentration of the phycocyanin in the upper and lower phases were measured. The recovery rate of phycocyanin and the partition coefficient of the two-phase aqueous system were calculated, and a UV-visible full-wavelength scan was performed simultaneously.

[0052] (4) The upper phase solution obtained in step (3) was dialyzed at 4°C overnight, diluted 10-fold with PBS buffer, and then ultrafiltered using a MW10KD ultrafiltration tube at 4200 rpm for 20 min. The ultrafiltration process was repeated three times. The purity of the phycocyanin in the solution was determined, and the recovery rate was calculated to be 41.77%. UV-visible full wavelength scanning was also performed;

[0053] (5) Pass Figure 2It can be clearly seen that after two-phase extraction and dialysis ultrafiltration treatment, the absorbance at 280nm is almost close to zero, indicating that the impurities are well removed; at 620nm, the characteristic absorption peak of phycocyanin still appears, indicating that the extraction and purification process has not destroyed the structure of phycocyanin and the phycocyanin structure is intact. The purity of the phycocyanin obtained in steps (1), (2), (3) and (4) was further tested by SDS-PAGE method. The results are as follows Figure 3 As shown (b, c, d, e correspond to steps (1), (2), (3), and (4) respectively), compared with the crude extract, after treatment in steps (3) and (4), the miscellaneous bands shown on SDS-PAGE gradually decrease, the molecular weight range becomes smaller, and are consistent with the structure of phycocyanin. The above spectral characteristics of phycocyanin and denaturing electrophoresis results prove that the phycocyanin prepared by the method of the present invention is reagent-grade phycocyanin.

[0054] In order to compare the method of the present invention with the traditional method for separating and purifying Spirulina phycocyanin, various methods for purifying Spirulina phycocyanin that have been disclosed are summarized as shown in Table 1.

[0055] As can be seen from Table 1, compared with various traditional phycocyanin separation and purification methods, the method of the embodiment of the present invention has the advantages of mild conditions, good purification effect, low cost, short cycle and easy amplification, and the extracted phycocyanin has high purity. Since the present invention does not require traditional ultrasonic crushers and high-pressure homogenization equipment, the pre-treatment operation is simple, energy consumption is low, and the effect on the biological activity of phycocyanin is small. In addition, the present invention uses low-cost, widely available powdered activated carbon adsorption treatment combined with two-phase aqueous extraction technology to increase the purity of phycocyanin to more than 3.6 in a treatment time of less than 2 hours, greatly shortening the production cycle and effectively controlling production costs.

[0056] Table 1 Comparison of different technical solutions

[0057]

[0058]

[0059]

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for efficiently separating and purifying reagent-grade phycocyanin, characterized in that: The steps include: 1) mixing the spirulina raw material with phosphate buffer, stirring, centrifuging, and collecting the supernatant to obtain a crude phycocyanin extract; 2) adding activated carbon to the crude phycocyanin extract obtained in step 1), stirring, centrifuging, and collecting the supernatant; 3) adding a two-phase aqueous extraction system to the supernatant obtained in step 2), centrifuging the system to separate the layers, and respectively aspirating the upper and lower phases of the two-phase aqueous extraction system; 4) The upper phase solution obtained in step 3) is dialyzed and ultrafiltered to remove the solvent and impurities to obtain a reagent-grade phycocyanin solution.

2. The method for efficiently separating and purifying reagent-grade phycocyanin according to claim 1, characterized in that: In step 1), the mass ratio of the spirulina raw material to the phosphate buffer is 1:5-7; the pH of the phosphate buffer is 6-8, and the concentration is 0.01-0.02 mol / L; the stirring time is 13-17 minutes; the centrifugation speed is 7500-8500 r / min, the ambient temperature is 3-5°C, and the time is 18-22 minutes.

3. The method for efficiently separating and purifying reagent-grade phycocyanin according to claim 1, characterized in that: In the step 2), the amount of activated carbon added is 0.04 to 0.06 g / mL; and the stirring time is 8 to 12 minutes.

4. The method for efficiently separating and purifying reagent-grade phycocyanin according to claim 1, characterized in that: The ratio of the supernatant in step 3) to the aqueous two-phase extraction system is 5-10 ml: 25-35 ml; the aqueous two-phase extraction system is an aqueous two-phase extraction system with a mass concentration of 10-14% PEG 4000 and a mass concentration of 13-17% potassium phosphate.

5. The method for efficiently separating and purifying reagent-grade phycocyanin according to claim 1, characterized in that: The centrifugal speed in steps 2) and 3) is 4000-4400 rpm, and the time is 8-12 minutes.

6. The method for efficiently separating and purifying reagent-grade phycocyanin according to claim 1, characterized in that: The ultrafiltration method in step 4) is as follows: the diafiltration solution is diluted 8 to 12 times with phosphate buffer and then ultrafiltered using a MW 10KD ultrafiltration tube at 4000 to 4400 rpm for 18 to 22 minutes, and repeated 3 to 4 times.

7. The method for efficiently separating and purifying reagent-grade phycocyanin according to claim 1, characterized in that: The soaking time in step 1) is 10 to 14 hours, and the temperature is 3 to 5°C.

8. The method for efficiently separating and purifying reagent-grade phycocyanin according to claim 1, characterized in that: The dialysis time in step 4) is 10 to 14 hours, and the temperature is 3 to 5°C.

Images