Method for extracting phycocyanin from biogas slurry

Abstract

The invention discloses a method for extracting phycocyanin from biogas slurry, and belongs to the field of solid waste recycling and high value-added development and utilization thereof. The extraction method comprises the following steps: (1) centrifuging biogas slurry, taking a precipitate, mixing the precipitate with acetone, and stirring and centrifuging the mixed solution to obtain a cell precipitate; (2) dissolving the net cell precipitate of the biogas slurry system in water, adjusting the pH value to obtain an acidic net cell suspension, heating, stirring and centrifuging the acidic net cell suspension, collecting a supernatant, adjusting the pH value to obtain an alkaline phycocyanin suspension, and centrifuging the alkaline phycocyanin suspension to obtain a crude phycocyanin extract; and (3) dissolving the crude phycocyanin extract in hydrochloric acid, dialyzing to obtain a pure phycocyanin solution, adjusting the pure phycocyanin solution to be alkaline to obtain an alkaline phycocyanin suspension, centrifuging, collecting precipitates, and freeze-drying to obtain the phycocyanin. According to the method, the cyanophycin is extracted from the biogas slurry, a promising application is explored for resource utilization of the biogas slurry, and a new thought is provided for improving the efficiency and economic benefits of resource utilization of the biogas slurry.

Description

Technical Field

[0001] The present invention belongs to the field of solid waste resource utilization and its high-value development and utilization, and relates to a method for extracting cyanophycin from biogas slurry. Background Art

[0002] Biogas slurry is the liquid product of the anaerobic digestion process of organic solid waste, rich in organic matter, nutrient elements and bioactive substances. As an environmentally friendly resource, it has multiple application values in the agricultural field: it can be used as a high-quality organic fertilizer to promote crop growth and improve soil fertility; the plant hormones and trace elements required by plants contained in it can enhance the stress resistance of crops; at the same time, it can also be used for pest and disease control, reducing the use of chemical pesticides. However, at present, biogas slurry is mainly used as a liquid organic fertilizer, and its economic value is relatively low. Compared with the high treatment and transportation costs, directly selling biogas slurry often fails to obtain ideal economic returns. To improve economic feasibility, it is necessary to expand its high-value utilization ways and extract high-value components.

[0003] Cyanophycin granule peptide (CGP), as an important biopolymer compound, is the third type of polyamino acid substance found in nature after poly-γ-glutamic acid and poly-ε-lysine. This substance has unique molecular structural characteristics. Its main chain is composed of aspartic acid residues, and the side chain contains arginine groups. This amino acid polymer synthesized through the non-ribosomal pathway shows broad application prospects in multiple industrial fields. From the perspective of the biosynthesis mechanism, CGP can not only be naturally produced by various cyanobacteria, but some heterotrophic bacteria also have the ability to biosynthesize it. Given the excellent physical and chemical properties and material science performance of CGP, it has obtained important applications in the food industry, pharmaceutical research and development, cosmetics manufacturing, nutritional supplements, and agricultural production. At present, introducing the cphA gene into engineering strains through genetic engineering technology to achieve heterologous expression has become the main way for large-scale production of CGP. Research shows that a variety of genetically modified microbial systems, such as Escherichia coli, Pseudomonas putida, Rhizopus oryzae, and Saccharomyces cerevisiae, have been successfully used for the industrial production of CGP.

[0004] However, regarding the separation and purification technology of CGP, the existing methods are mainly limited to the extraction of single microbial systems or genetically engineered strains, and are difficult to apply to complex mixed systems such as biogas slurry. At the same time, these traditional processes have limitations such as complex operating procedures, harsh environmental conditions, and high economic costs. In addition, in the prior art, Chinese patents CN115746292 A (a method for extracting phycocyanin from activated sludge) and CN 119320668 A (a method and device for dual recovery of carbon and nitrogen by collecting oil and phycocyanin) respectively disclose methods for extracting phycocyanin from activated sludge and algae-bacteria symbiotic sewage treatment systems, but the extraction amount of phycocyanin in the above methods still has room for improvement.

[0005] So far, there is still no efficient extraction scheme for the mixed system of biogas slurry. Developing a new CGP extraction method suitable for this system has become an important problem that needs to be solved urgently. Summary of the invention

[0006] Technical issues

[0007] Some of the existing phycocyanin extraction methods are only applicable to pure bacterial strains or genetically engineered strains, and have problems such as complex operating procedures and harsh environmental conditions. They cannot be directly applied to biogas slurry systems. In addition, the phycocyanin extraction yield of some phycocyanin extraction methods is not high, and there is still room for improvement.

[0008] Technical Solution

[0009] In order to solve the above technical problems, the object of the present invention is to provide a method for extracting phycocyanin from biogas slurry, which can extract phycocyanin from a biogas slurry mixture system and realize high added value utilization of biogas slurry. The method is to obtain a cell precipitate by high-speed centrifugation after pre-treatment with acetone from biogas slurry; then the cell precipitate is resuspended in a buffer solution, and the cell precipitate is repeatedly washed several times and then centrifuged to obtain a clean cell precipitate. The clean cell precipitate is resuspended in ultrapure water and the pH is adjusted to obtain an acidic cell suspension and vigorously stirred so that the cells in the acidic suspension release phycocyanin, thereby obtaining an acidic cell-phycocyanin mixed solution. The mixed solution is centrifuged, and the supernatant is collected and adjusted to a weak alkaline state so that phycocyanin precipitates to obtain a phycocyanin suspension. The alkaline phycocyanin suspension is centrifuged at high speed to separate and obtain a crude phycocyanin extract; the crude phycocyanin extract is then redissolved under acidic conditions, and the solution is dialyzed to obtain a pure phycocyanin solution. The pH of the pure phycocyanin solution is adjusted to weak alkalinity, and centrifuged after standing to obtain phycocyanin particle precipitation; the phycocyanin particle precipitation is dried to obtain phycocyanin powder. The method of the present invention makes it possible to extract phycocyanin from biogas slurry, thereby improving the economic value of biogas slurry resource utilization.

[0010] The present invention provides a method for extracting phycocyanin from biogas slurry, and the extraction method comprises the following steps:

[0011] (1) Take biogas slurry and acetone to mix to obtain a mixed solution, stir the mixed solution, and then centrifuge to obtain cell precipitates. Wash the obtained cell precipitates with Tris-HCl buffer solution, and then centrifuge to obtain the net cell precipitates of the biogas slurry system;

[0012] (2) Dissolve the net cell precipitates of the biogas slurry system prepared in step (1) in water, then adjust the pH to obtain an acidic net cell suspension. Heat and stir the acidic net cell suspension, and then centrifuge the suspension after heating and stirring. Collect the supernatant and adjust the pH value of the supernatant to alkaline to obtain an alkaline phycocyanin suspension. Finally, centrifuge the alkaline phycocyanin suspension to obtain a crude phycocyanin extract;

[0013] (3) Dissolve the crude phycocyanin extract prepared in step (2) in hydrochloric acid aqueous solution for dialysis to obtain a pure phycocyanin solution, then adjust the pH value of the pure phycocyanin solution to alkaline to obtain an alkaline phycocyanin suspension. Then centrifuge the alkaline phycocyanin suspension, collect the precipitate and freeze-dry it to obtain phycocyanin.

[0014] Further, the biogas slurry in step (1) is a liquid formed after anaerobic fermentation.

[0015] Further, the raw material used for the anaerobic fermentation is the excess sludge of a sewage treatment plant; the time for the anaerobic fermentation is 20 to 40 days, and the temperature is 30 to 40 °C.

[0016] Further, the excess sludge refers to the activated sludge discharged from the secondary sedimentation tank or the sedimentation area in the activated sludge system outside the system.

[0017] Further, the volume concentration of the biogas slurry in the mixed solution in step (1) is 30 to 40%.

[0018] Further, the conditions for the centrifugation in step (1) are centrifugation for 10 to 30 min under the conditions of 20 to 30 °C and 15000 to 20000×g.

[0019] Further, the conditions for the stirring in step (1) are stirring for 30 to 60 min under the conditions of 20 to 30 °C and 1000 to 2000 rpm.

[0020] Further, the concentration of the Tris-HCl buffer solution in step (1) is 0.5 to 1.5 mol / L, and the pH is 7.5 to 8.5.

[0021] Further, the pH of the acidic net cell suspension in step (2) is 1 to 2.

[0022] Further, the conditions for heating and stirring in step (2) are stirring for 0.5 - 2 h under the conditions of 60 - 70 °C and 1000 - 2000 rpm.

[0023] Further, the conditions for centrifugation in step (2) are centrifuging for 10 - 30 min under the conditions of 20 - 30 °C and 15000 - 20000 × g.

[0024] Further, the pH of the alkaline phycocyanin suspension in step (2) is 7.5 - 8.

[0025] Further, the concentration of the hydrochloric acid aqueous solution in step (3) is 0.1 - 0.5 mol / L.

[0026] Further, the ratio of the crude phycocyanin extract to the hydrochloric acid aqueous solution in step (3) is 1:400 - 600.

[0027] Further, the conditions for dialysis in step (3) are dialyzing with a dialysis bag of 3000 - 4000 Da for 12 - 48 h.

[0028] Further, the pH of the alkaline phycocyanin suspension in step (3) is 7.5 - 8.

[0029] Further, the conditions for centrifugation in step (3) are centrifuging for 10 - 30 min under the conditions of 20 - 30 °C and 15000 - 20000 × g.

[0030] The present invention also provides a method for extracting phycocyanin with a high extraction amount, and the extraction method includes the following steps:

[0031] (1) Centrifuge the biogas slurry, collect the precipitate, take the precipitate and mix it with acetone to obtain a mixed solution, stir the mixed solution, then centrifuge to obtain a cell precipitate, wash the obtained cell precipitate with Tris-HCl buffer solution, and then centrifuge to obtain a net cell precipitate of the biogas slurry system;

[0032] (2) Dissolve the net cell precipitate of the biogas slurry system prepared in step (1) in water, then adjust the pH to obtain an acidic net cell suspension, heat and stir the acidic net cell suspension, then centrifuge the heated and stirred suspension, collect the supernatant and adjust the pH value of the supernatant to alkaline to obtain an alkaline phycocyanin suspension, and finally centrifuge the alkaline phycocyanin suspension to obtain a crude phycocyanin extract;

[0033] (3) Dissolve the crude phycocyanin extract prepared in step (2) in a hydrochloric acid aqueous solution for dialysis to obtain a pure phycocyanin solution, then adjust the pH value of the pure phycocyanin solution to alkaline to obtain an alkaline phycocyanin suspension, then centrifuge the alkaline phycocyanin suspension, collect the precipitate and freeze-dry it to obtain phycocyanin.

[0034] In one embodiment of the present invention, the biogas slurry described in step (1) is the liquid formed after anaerobic fermentation.

[0035] In one embodiment of the present invention, the raw material used for the anaerobic fermentation is the excess sludge from a sewage treatment plant; the time for the anaerobic fermentation is 20 to 40 days, and the temperature is 30 to 40 °C.

[0036] In one embodiment of the present invention, the excess sludge refers to the activated sludge discharged from the secondary sedimentation tank or the sedimentation area in the activated sludge system outside the system.

[0037] In one embodiment of the present invention, the volume concentration of the biogas slurry in the mixed liquid in step (1) is 30 to 40%.

[0038] In one embodiment of the present invention, the conditions for centrifugation in step (1) are centrifugation for 10 to 30 min at 20 to 30 °C and 15000 to 20000 × g; the conditions for stirring are stirring for 30 to 60 min at 20 to 30 °C and 1000 to 2000 rpm.

[0039] In one embodiment of the present invention, the concentration of the Tris-HCl buffer solution in step (1) is 0.5 to 1.5 mol / L, and the pH is 7.5 to 8.5.

[0040] In one embodiment of the present invention, the pH of the acidic net cell suspension in step (2) is 1 to 2.

[0041] In one embodiment of the present invention, the conditions for heating and stirring in step (2) are stirring for 0.5 to 2 h at 60 to 70 °C and 1000 to 2000 rpm; the conditions for centrifugation are centrifugation for 10 to 30 min at 20 to 30 °C and 15000 to 20000 × g.

[0042] In one embodiment of the present invention, the pH of the alkaline phycocyanin suspension in step (2) is 7.5 to 8.

[0043] In one embodiment of the present invention, the concentration of the hydrochloric acid aqueous solution in step (3) is 0.1 to 0.5 mol / L; the ratio of the crude phycocyanin extract to the hydrochloric acid aqueous solution is 1:400 to 600.

[0044] In one embodiment of the present invention, the conditions for dialysis in step (3) are dialysis for 12 to 48 h using a dialysis bag with a molecular weight cut-off of 3000 to 4000 Da.

[0045] In one embodiment of the present invention, the pH of the alkaline phycocyanin suspension in step (3) is 7.5 to 8.

[0046] In one embodiment of the present invention, the centrifugation conditions in step (3) are centrifugation at 20 - 30 °C and 15000 - 20000 × g for 10 - 30 min.

[0047] In one embodiment of the present invention, the high extraction amount means that the extraction amount of phycocyanin can reach 130 mg / g of dry sludge.

[0048] Beneficial effects

[0049] (1) Simple and efficient: The present invention extracts phycocyanin from biogas slurry, achieving a yield of 140 ± 5.7 mg / g of dry sludge. The entire process is simple, with mild conditions and good reproducibility, and does not involve harsh conditions or expensive raw materials.

[0050] (2) Environmentally friendly and sustainable: The present invention uses biogas slurry to extract phycocyanin, exploring a highly promising application for the resource utilization of biogas slurry, and providing new ideas for improving the efficiency and economic benefits of biogas slurry resource utilization. Description of the drawings

[0051] Figure 1 It is a diagram of a phycocyanin suspension under weakly alkaline conditions.

[0052] Figure 2 It is an appearance diagram of freeze-dried phycocyanin.

[0053] Figure 3 It is an infrared spectrum diagram of phycocyanin powder.

[0054] Figure 4 It is a microscope diagram of phycocyanin precipitate. Detailed implementation manners

[0055] Source of raw materials

[0056] The biogas slurry is taken from the liquid formed after anaerobic fermentation of the excess sludge in the sewage treatment plant. The anaerobic fermentation time is 30 days and the temperature is 35 °C.

[0057] Example 1: Extraction of phycocyanin from biogas slurry

[0058] Take 30 mL of biogas slurry in a 150 mL conical flask, add 60 mL of acetone and stir to mix to obtain a mixed solution (concentration 33.3 v / v%). Use a magnetic stirrer to stir the above mixed solution at high speed, and the stirring conditions are: 1600 rpm, 50 min. After the stirring is completed, place the mixed solution in a centrifuge tube and use a high-speed centrifuge to centrifuge to obtain cell precipitates, and the separation conditions are: at room temperature, centrifuge at 18000×g for 20 min. Subsequently, resuspend the obtained cell precipitates in Tris-HCl buffer solution with a concentration of 1 mol / L and pH = 8 for washing, and use a high-speed centrifuge to centrifuge at 18000×g for 20 min at room temperature. Repeat the washing and centrifugation 3 times to obtain the net cell precipitates of the biogas slurry system.

[0059] Resuspend the net cell precipitates of the biogas slurry system in 50 mL of ultrapure water, and adjust the pH value to 1 with an aqueous hydrochloric acid solution to obtain an acidic net cell suspension. Place the above acidic net cell suspension in a magnetic stirring constant temperature water bath, and continuously stir at 65 °C and 1400 rpm for 1 h to promote cell rupture in the system and release phycocyanin. Subsequently, centrifuge the heated and stirred suspension at room temperature and 18000×g for 20 min, collect the supernatant and adjust the pH value to 7.5 with an aqueous NaOH solution to precipitate the phycocyanin in the solution to obtain an alkaline phycocyanin suspension. It is observed that the precipitated phycocyanin appears yellowish-green, as Figure 1 shown. Finally, after standing the alkaline phycocyanin suspension for 20 min, centrifuge it at room temperature and 18000×g for 20 min to separate and obtain the crude phycocyanin extract.

[0060] Dissolve the above crude phycocyanin extract in 0.1 mol / L aqueous hydrochloric acid solution at a ratio of 1:500. After complete dissolution, dialyze it with a dialysis bag of 3500 Da for 24 h to obtain a relatively pure phycocyanin solution. Use an aqueous NaOH solution to re-adjust the pH value of the pure phycocyanin solution to 7.5 to precipitate the phycocyanin again to obtain an alkaline phycocyanin suspension. Subsequently, use a high-speed centrifuge to centrifuge the alkaline phycocyanin suspension at room temperature and 18000×g for 20 min, and collect the phycocyanin precipitate. Freeze-dry the obtained precipitate for 24 h to finally obtain light yellow powdery phycocyanin, as Figure 2 shown.

[0061] Example 2: Calculation of phycocyanin extraction amount

[0062] In this example, the phycocyanin content extracted from the biogas slurry system in Example 1 was calculated. Weigh the powdery phycocyanin obtained in Example 1 and repeat it 3 times, and it is found that the phycocyanin extraction amount for the biogas slurry system can reach 140 ± 5.7 mg / g dry sludge.

[0063] Example 3: Characterization of phycocyanin

[0064] The obtained cyanophycin in Example 1 was characterized by infrared spectroscopy, Figure 3 and its infrared spectrum was shown. As can be seen from the figure, the absorption peak near 2926 cm -1 corresponds to the anti-stretching vibration and stretching vibration of the methylene (–CH2–) in the cyanophycin molecule. The absorption peak at 2291 cm -1 is related to the combined frequency of the stretching vibration of –OH and the bending vibration of C–H in the carboxyl group (–COOH). In the range of 1777–1880 cm -1 , multiple sawtooth absorption peaks were observed, which are similar to the absorption peaks appearing in the range of 1667–1820 cm -1 for diisobutylamine. Therefore, it is speculated that these peaks may be related to the –NH2 group connecting two carbon atoms in cyanophycin. The absorption peak at 1631 cm -1 can be attributed to the vibration absorption of –C=NH. In addition, the absorption peaks at 1263 cm -1 and 1008 cm -1 are derived from the stretching vibrations of C–N and C–O, respectively.

[0065] In summary, the cyanophycin extracted from biogas slurry has similar chemical groups and structural compositions to the reported cyanophycin, indicating that the cyanophycin in biogas slurry was successfully extracted by this method.

[0066] Comparative Example 1: Influence of extraction conditions on the extraction of cyanophycin from biogas slurry

[0067] Take 30 mL of biogas slurry in a 150 mL conical flask, add 30 mL of acetone and stir to mix to obtain a mixed solution (concentration 50 v / v%). Use a magnetic stirrer to stir the above mixed solution at high speed, and the stirring conditions are: 1600 rpm, 30 min. After stirring, place the mixed solution in a centrifuge tube and centrifuge using a high-speed centrifuge to obtain cell precipitates, and the separation conditions are: at room temperature, centrifuge at 18000×g for 20 min. Subsequently, resuspend the obtained cell precipitates in a 1 mol / L, pH = 8 Tris-HCl buffer solution for washing, and centrifuge using a high-speed centrifuge at room temperature at 18000×g for 20 min. Repeat the washing and centrifugation 3 times to obtain the net cell precipitates of the biogas slurry system.

[0068] Resuspend the purified cell precipitate of the biogas slurry system in 50 mL of ultrapure water, and adjust the pH value to 1 using an aqueous hydrochloric acid solution to obtain an acidic purified cell suspension. Place the above acidic purified cell suspension in a magnetic stirring constant temperature water bath, and continuously stir for 30 min at 65 °C and 1400 rpm to promote cell rupture in the system and release phycocyanin. Subsequently, centrifuge the heated and stirred suspension at room temperature and 18000×g for 20 min, collect the supernatant, and adjust the pH value to 7.5 using an aqueous NaOH solution to precipitate phycocyanin in the solution, obtaining an alkaline phycocyanin suspension. It is observed that the precipitated phycocyanin appears yellowish-green. Finally, after allowing the alkaline phycocyanin suspension to stand for 20 min, centrifuge it at room temperature and 18000×g for 20 min to separate and obtain the crude phycocyanin extract.

[0069] Dissolve the above crude phycocyanin extract in an aqueous hydrochloric acid solution of 0.1 mol / L at a ratio of 1:500. After complete dissolution, dialyze it using a dialysis bag with a molecular weight cut-off of 3500 Da for 24 h to obtain a relatively pure phycocyanin solution. Adjust the pH value of the pure phycocyanin solution to 7.5 using an aqueous NaOH solution to precipitate phycocyanin again, obtaining an alkaline phycocyanin suspension. Subsequently, centrifuge the alkaline phycocyanin suspension at room temperature and 18000×g for 20 min using a high-speed centrifuge, and collect the phycocyanin precipitate. Freeze-dry the obtained precipitate for 24 h to finally obtain light yellow powdery phycocyanin.

[0070] Comparative Example 2: Effect of agent optimization on the extraction of phycocyanin from biogas slurry

[0071] Take 30 mL of biogas slurry in a 150 mL conical flask, add 60 mL of ethanol solution and stir to mix to obtain a mixed solution (concentration 33.3 v / v%). Use a magnetic stirrer to stir it at high speed, and the stirring conditions are: 1600 rpm, 50 min. After stirring, place the mixed solution in a centrifuge tube and centrifuge it using a high-speed centrifuge to obtain a cell precipitate, and the separation conditions are: at room temperature, centrifuge at 18000×g for 20 min. Subsequently, resuspend the obtained cell precipitate in PBS buffer with a pH of 8 for washing, and centrifuge it at 18000×g at room temperature for 20 min using a high-speed centrifuge. Repeat the washing and centrifugation 3 times to obtain the purified cell precipitate of the biogas slurry system.

[0072] Resuspend the net cell precipitate of the biogas slurry system in 50 mL of ultrapure water, and adjust the pH value to 1 using an aqueous hydrochloric acid solution to obtain an acidic net cell suspension. Place the above acidic net cell suspension in a magnetic stirring constant temperature water bath, and continuously stir at 65 °C and 1400 rpm for 50 min to promote cell rupture in the system and release phycocyanin. Subsequently, centrifuge the heated and stirred suspension at room temperature and 18000×g for 20 min, collect the supernatant, and adjust the pH value to 7.5 using an aqueous NaOH solution to precipitate phycocyanin in the solution, obtaining an alkaline phycocyanin suspension. It is observed that the precipitated phycocyanin appears yellowish-green. Finally, after allowing the alkaline phycocyanin suspension to stand for 20 min, centrifuge it at room temperature and 18000×g for 20 min to separate and obtain a crude phycocyanin extract.

[0073] Dissolve the above crude phycocyanin extract in an aqueous hydrochloric acid solution of 0.1 mol / L at a ratio of 1:500, and dialyze it in a dialysis bag with a molecular weight cut-off of 3500 Da for 24 h to obtain a relatively pure phycocyanin solution. Adjust the pH value of this solution to 7.5 using an aqueous NaOH solution to precipitate phycocyanin again, obtaining an alkaline phycocyanin suspension. Subsequently, centrifuge the alkaline phycocyanin suspension at room temperature and 18000×g for 20 min using a high-speed centrifuge to collect the phycocyanin precipitate. Freeze-dry the precipitate for 24 h to finally obtain light yellow powdery phycocyanin.

[0074] Comparative Example 3: Extraction of phycocyanin from aerobic activated sludge

[0075] Take 30 mL of the mixture of mud and water from the aerobic tank of a sewage treatment plant in a 150 mL conical flask, add 60 mL of acetone solution and stir to mix to obtain a mixed solution (concentration 33.3 v / v%). Use a magnetic stirrer to stir the above mixed solution at 1600 rpm at high speed for 50 min. After stirring, place the mixed solution in a centrifuge tube and centrifuge it using a high-speed centrifuge to obtain a cell precipitate. The separation conditions are: at room temperature, centrifuge at 18000×g for 20 min. Subsequently, resuspend the obtained cell precipitate in a Tris-HCl buffer solution with a pH of 8 for washing, and centrifuge it at room temperature at 18000×g for 20 min using a high-speed centrifuge. Repeat the washing 3 times to obtain a net cell precipitate of the sludge system.

[0076] The clean cell sediment was resuspended in 50 mL of ultrapure water, and the pH value was adjusted to 1 with an aqueous hydrochloric acid solution to obtain an acidic clean cell suspension. The above acidic clean cell suspension was then placed in a magnetic stirring constant temperature water bath, and stirred continuously for 50 min at 65 ° C and 1400 rpm to cause the cells in the system to rupture and release phycocyanin. The heated and stirred suspension was then centrifuged at room temperature and 18000 × g for 20 min, the supernatant was collected and the pH value was adjusted to 7.5 with an aqueous NaOH solution to precipitate the phycocyanin in the solution to obtain an alkaline phycocyanin suspension. The obtained phycocyanin is light brown. Finally, the alkaline phycocyanin suspension was allowed to stand for 20 min, and then centrifuged at room temperature and 18000 × g for 20 min to separate and obtain a crude phycocyanin extract.

[0077] The above-mentioned phycocyanin crude extract was dissolved in a 0.1 mol / L hydrochloric acid aqueous solution at a ratio of 1:500. After complete dissolution, it was dialyzed for 24 hours using a 3500Da dialysis bag to obtain a relatively pure pure phycocyanin solution. The pH value of the solution was adjusted to 7.5 using an aqueous NaOH solution so that the phycocyanin was reprecipitated to obtain an alkaline phycocyanin suspension. Subsequently, the alkaline phycocyanin suspension was centrifuged at room temperature and 18000×g for 20 minutes using a high-speed centrifuge to collect the phycocyanin precipitate. The resulting precipitate was freeze-dried for 24 hours to finally obtain a light brown powdered phycocyanin.

[0078] The powdered phycocyanin obtained in Comparative Example 1, Comparative Example 2 and Comparative Example 3 was weighed and the extraction amount of phycocyanin in each comparison group was calculated. Each comparative example was repeated 3 times. The results are shown in Table 1.

[0079] Table 1 Phycocyanin extraction amount

[0080]

[0081] From the comparison between Comparative Example 1 and Example 1 in Table 1, it can be seen that the concentration of the initial mixed solution has an impact on the extraction amount of phycocyanin in the biogas slurry, and an overly concentrated mixed solution will reduce the final extraction amount of phycocyanin. In addition, from the comparison between Comparative Example 2 and Example 1, it can be seen that the extraction agent is also crucial to the extraction amount of phycocyanin in the biogas slurry, and the use of ethanol does not bring good results. Finally, from the comparison between Comparative Example 3 and Example 1, it can be seen that different extraction raw materials have different extraction effects, and phycocyanin cannot be extracted in large quantities from the aerobic pool mud and water mixture.

[0082] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for extracting phycocyanin with a high extraction yield, characterized in that, The extraction method includes the following steps: (1) Take biogas slurry and acetone to mix to obtain a mixed solution, stir the mixed solution, and then centrifuge to obtain cell precipitates. Wash the obtained cell precipitates with Tris-HCl buffer solution, and then centrifuge to obtain the net cell precipitates of the biogas slurry system; the volume concentration of biogas slurry in the mixed solution is 30-40%; the biogas slurry is the liquid formed after anaerobic fermentation of the excess sludge in the sewage treatment plant; (2) Dissolve the net cell precipitates of the biogas slurry system prepared in step (1) in water, then adjust the pH to obtain an acidic net cell suspension, heat and stir the acidic net cell suspension, then centrifuge the heated and stirred suspension, collect the supernatant and adjust the pH value of the supernatant to alkaline to obtain an alkaline phycocyanin suspension, and finally centrifuge the alkaline phycocyanin suspension to obtain the crude phycocyanin extract; (3) Dissolve the crude phycocyanin extract prepared in step (2) in hydrochloric acid aqueous solution for dialysis to obtain a pure phycocyanin solution, then adjust the pH value of the pure phycocyanin solution to alkaline to obtain an alkaline phycocyanin suspension, then centrifuge the alkaline phycocyanin suspension, collect the precipitate and freeze-dry to obtain phycocyanin.

2. The extraction method according to claim 1, wherein The conditions of the centrifugation in step (1) are centrifugation for 10-30 min under the conditions of 20-30 °C and 15000-20000×g; the conditions of the stirring are stirring for 30-60 min under the conditions of 20-30 °C and 1000-2000 rpm.

3. The extraction method according to claim 1, wherein The concentration of the Tris-HCl buffer solution in step (1) is 0.5-1.5 mol / L, and the pH is 7.5-8.

5.

4. The extraction method according to claim 1, characterized in that, The pH of the acidic net cell suspension in step (2) is 1-2.

5. The extraction method according to claim 1, wherein The conditions of the heating and stirring in step (2) are stirring for 0.5-2 h under the conditions of 60-70 °C and 1000-2000 rpm; the conditions of the centrifugation are centrifugation for 10-30 min under the conditions of 20-30 °C and 15000-20000×g.

6. The extraction method according to claim 1, wherein The pH of the alkaline phycocyanin suspension in step (2) is 7.5-8.

7. According to the extraction method described in claim 1, characterized in that, The concentration of the hydrochloric acid aqueous solution in step (3) is 0.1-0.5 mol / L; the ratio of the crude phycocyanin extract to the hydrochloric acid aqueous solution is 1:400-600.

8. The extraction method according to claim 1, characterized in that, The pH of the alkaline phycocyanin suspension in step (3) is 7.5-8.

9. The extraction method according to claim 1, wherein The conditions of the centrifugation in step (3) are centrifugation for 10-30 min under the conditions of 20-30 °C and 15000-20000×g.

10. The extraction method according to claim 1, characterized in that, The high extraction amount means that the extraction amount of phycocyanin reaches 130 mg / g dry sludge.

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