Method for extracting cianidins in biogas slurry

Abstract

The application discloses a method for extracting cianidins from biogas slurry, and belongs to the field of solid waste resource and high added value development and utilization. The method comprises the following steps: (1) taking the biogas slurry, centrifuging to obtain the precipitate and mixing with acetone, stirring and centrifuging the mixed liquid to obtain the cell precipitate; (2) dissolving the cell precipitate in water, adjusting the pH to obtain the acidic cell suspension, heating and stirring, centrifuging to collect the supernatant, adjusting the pH to obtain the alkaline cianidin suspension, and then centrifuging to obtain the cianidin crude extract; (3) dissolving the cianidin crude extract in hydrochloric acid, dialyzing to obtain the pure cianidin solution, adjusting the pure cianidin solution to be alkaline to obtain the alkaline cianidin suspension, centrifuging, collecting the precipitate, and then freeze-drying to obtain the cianidin. The application extracts cianidins from biogas slurry, explores a promising application for the resource utilization of biogas slurry, and provides a new idea for improving the efficiency and economic benefits of the resource utilization of biogas slurry.

Description

Technical Field

[0001] This invention belongs to the field of solid waste resource utilization and its high-value-added development and utilization, and relates to a method for extracting phycocyanin from biogas slurry. Background Technology

[0002] Biogas slurry is a liquid product of the anaerobic digestion of organic solid waste, rich in organic matter, nutrients, and bioactive substances. As an environmentally friendly resource, it has multiple applications in agriculture: it can be used as a high-quality organic fertilizer to promote crop growth and improve soil fertility; the plant hormones and micronutrients it contains can enhance crop resistance; and it can also be used for pest and disease control, reducing the use of chemical pesticides. However, currently, biogas slurry is mainly used as liquid organic fertilizer, and its economic value is relatively low. Compared with the high processing and transportation costs, direct sales of biogas slurry often fail to yield ideal economic returns. To improve economic viability, it is necessary to expand its high-value-added utilization pathways and extract high-value components.

[0003] Cyanocyanin granule peptide (CGP), an important biopolymer, is the third type of polyamino acid discovered in nature, following poly-γ-glutamic acid and poly-ε-lysine. This substance possesses unique molecular structural characteristics; its main chain consists of aspartic acid residues, while its side chains contain arginine groups. This amino acid polymer synthesized via a non-ribosomal pathway shows broad application prospects in multiple industrial fields. From a biosynthetic mechanism perspective, CGP can be naturally produced by various cyanobacteria, and some heterotrophic bacteria also possess the ability to biosynthesize it. Given CGP's excellent physicochemical properties and materials science performance, it has found important applications in the food industry, pharmaceutical research and development, cosmetic manufacturing, nutritional supplements, and agricultural production. Currently, heterologous expression of the cphA gene by introducing it into engineered strains through genetic engineering has become the main approach for large-scale CGP production. Studies have shown that various 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, existing methods for separating and purifying CGP are mainly limited to extraction from single microbial systems or genetically engineered strains, making them difficult to apply to complex mixed systems like biogas slurry. Furthermore, these traditional processes suffer from limitations such as complex operating procedures, stringent environmental conditions, and high economic costs. In addition, existing Chinese patents CN115746292 A (a method for extracting phycocyanin from activated sludge) and CN 119320668 A (a method and apparatus for dual carbon and nitrogen recovery by collecting oil and phycocyanin) disclose methods for extracting phycocyanin from activated sludge and algae-bacteria symbiotic wastewater treatment systems, respectively, but there is still room for improvement in the extraction yield of phycocyanin using these methods.

[0005] To date, there is still a lack of efficient extraction solutions for biogas slurry mixed systems, and developing a new CGP extraction method suitable for this system has become an important problem that urgently needs to be solved. Summary of the Invention

[0006] Technical issues

[0007] Existing phycocyanin extraction methods are only applicable to pure bacterial strains or genetically engineered strains, and have problems such as complex operation procedures and harsh environmental conditions. They cannot be directly applied to biogas slurry systems. In addition, some phycocyanin extraction methods have low phycocyanin extraction yields, and there is still room for improvement.

[0008] Technical solution

[0009] To address the aforementioned technical problems, the present invention aims to provide a method for extracting phycocyanin from biogas slurry. This method can extract phycocyanin from a biogas slurry mixture system, achieving high-value utilization of biogas slurry. The method involves pretreating biogas slurry with acetone, followed by high-speed centrifugation to obtain cell precipitate. The cell precipitate is then resuspended in a buffer solution and repeatedly washed before centrifugation to obtain a net cell precipitate. The net cell precipitate is resuspended in ultrapure water, and the pH is adjusted to obtain an acidic cell suspension. This suspension is then vigorously stirred to release phycocyanin from the cells, resulting in an acidic cell-phycocyanin mixture. This mixture is centrifuged, and the supernatant is collected and adjusted to a weakly alkaline state to precipitate phycocyanin, obtaining a phycocyanin suspension. The alkaline phycocyanin suspension is then centrifuged at high speed to obtain a crude phycocyanin extract. This 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 was adjusted to weakly alkaline, and after standing, it was centrifuged to obtain phycocyanin particle precipitate. The phycocyanin particle precipitate was then dried to obtain phycocyanin powder. This method makes it possible to extract phycocyanin from biogas slurry, thereby enhancing the economic value of biogas slurry resource utilization.

[0010] This invention provides a method for extracting phycocyanin from biogas slurry, the extraction method comprising the following steps:

[0011] (1) Take biogas slurry and acetone to mix to obtain a mixture, stir the mixture, then centrifuge to obtain cell precipitate, wash the obtained cell precipitate with Tris-HCl buffer, and then centrifuge to obtain net cell precipitate of biogas slurry system.

[0012] (2) Dissolve the cell precipitate of the biogas slurry system obtained in step (1) in water, then adjust the pH to obtain an acidic cell suspension. Heat and stir the acidic cell suspension, then centrifuge the heated and stirred suspension, collect the supernatant and adjust the pH of the supernatant to alkaline to obtain an alkaline phycocyanin suspension. Finally, centrifuge the alkaline phycocyanin suspension to obtain crude phycocyanin extract.

[0013] (3) Dissolve the crude phycocyanin extract obtained in step (2) in hydrochloric acid aqueous solution and dialyze to obtain pure phycocyanin solution. Then adjust the pH value of the pure phycocyanin solution to alkaline to obtain alkaline phycocyanin suspension. Then centrifuge the alkaline phycocyanin suspension, collect the precipitate and freeze dry to obtain phycocyanin.

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

[0015] Furthermore, the raw material used for the anaerobic fermentation is residual sludge from a wastewater treatment plant; the anaerobic fermentation time is 20 to 40 days, and the temperature is 30 to 40°C.

[0016] Furthermore, the excess sludge refers to the activated sludge discharged from the secondary sedimentation tank or sedimentation zone of the activated sludge system.

[0017] Furthermore, the volume concentration of biogas slurry in the mixture described in step (1) is 30-40%.

[0018] Furthermore, the centrifugation conditions described in step (1) are centrifugation at 20-30℃ and 15000-20000×g for 10-30 minutes.

[0019] Furthermore, the stirring conditions described in step (1) are stirring at 20-30°C and 1000-2000 rpm for 30-60 minutes.

[0020] Furthermore, 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.

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

[0022] Furthermore, the heating and stirring conditions described in step (2) are stirring at 60-70°C and 1000-2000 rpm for 0.5-2 hours.

[0023] Furthermore, the centrifugation conditions described in step (2) are centrifugation at 20–30°C and 15,000–20,000 × g for 10–30 min.

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

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

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

[0027] Furthermore, the dialysis conditions described in step (3) are dialysis for 12 to 48 hours using a dialysis bag with a capacity of 3000 to 4000 Da.

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

[0029] Furthermore, the centrifugation conditions described in step (3) are centrifugation at 20-30℃ and 15000-20000×g for 10-30 minutes.

[0030] This invention also provides a method for extracting phycocyanin with high extraction yield, the extraction method comprising the following steps:

[0031] (1) Centrifuge the biogas slurry, collect the precipitate, and mix the precipitate with acetone to obtain a mixture. Stir the mixture and then centrifuge to obtain cell precipitate. Wash the obtained cell precipitate with Tris-HCl buffer and then centrifuge to obtain the net cell precipitate of the biogas slurry system.

[0032] (2) Dissolve the cell precipitate of the biogas slurry system obtained in step (1) in water, then adjust the pH to obtain an acidic cell suspension. Heat and stir the acidic cell suspension, then centrifuge the heated and stirred suspension, collect the supernatant and adjust the pH of the supernatant to alkaline to obtain an alkaline phycocyanin suspension. Finally, centrifuge the alkaline phycocyanin suspension to obtain crude phycocyanin extract.

[0033] (3) Dissolve the crude phycocyanin extract obtained in step (2) in hydrochloric acid aqueous solution and dialyze to obtain pure phycocyanin solution. Then adjust the pH value of the pure phycocyanin solution to alkaline to obtain alkaline phycocyanin suspension. Then centrifuge the alkaline phycocyanin suspension, collect the precipitate and freeze dry to obtain phycocyanin.

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

[0035] In one embodiment of the present invention, the raw material used for anaerobic fermentation is residual sludge from a sewage treatment plant; the anaerobic fermentation time 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 sedimentation zone of the activated sludge system.

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

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

[0039] In one embodiment of the present invention, the concentration of Tris-HCl buffer 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 cell suspension in step (2) is 1 to 2.

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

[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 crude phycocyanin extract to hydrochloric acid aqueous solution is 1:400 to 600.

[0044] In one embodiment of the present invention, the dialysis conditions in step (3) are dialysis for 12 to 48 hours using a dialysis bag with a capacity 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 for 10 to 30 minutes at 20 to 30°C and 15,000 to 20,000 × g.

[0047] In one embodiment of the present invention, the high extraction rate refers to the extraction rate of phycocyanin reaching 130 mg / g dry sludge.

[0048] Beneficial effects

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

[0050] (2) Environmental protection and sustainability: This invention utilizes biogas slurry to extract phycocyanin, exploring a promising application for the resource utilization of biogas slurry and providing a new approach to improve the efficiency and economic benefits of biogas slurry resource utilization. Attached Figure Description

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

[0052] Figure 2 This is a picture of the appearance of phycocyanin after freeze-drying.

[0053] Figure 3 The image shows the infrared spectrum of phycocyanin powder.

[0054] Figure 4 Microscopic image of phycocyanin precipitation. Detailed Implementation

[0055] Source of raw materials

[0056] The biogas slurry is taken from the liquid formed by the anaerobic fermentation of the residual sludge from the sewage treatment plant. The anaerobic fermentation time is 30 days and the temperature is 35℃.

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

[0058] Take 30 mL of biogas slurry and place it in a 150 mL Erlenmeyer flask. Add 60 mL of acetone and stir to obtain a mixture (concentration 33.3 v / v%). Use a magnetic stirrer to stir the mixture at high speed: 1600 rpm for 50 min. After stirring, transfer the mixture to a centrifuge tube and centrifuge at high speed to obtain cell pellet. Separation conditions: room temperature, 18000 × g for 20 min. Subsequently, resuspend the obtained cell pellet in 1 mol / L Tris-HCl buffer (pH = 8) for washing, and centrifuge at 18000 × g for 20 min at room temperature. Repeat the washing and centrifugation three times to obtain a net cell pellet from the biogas slurry system.

[0059] The cell precipitate from the biogas slurry system was resuspended in 50 mL of ultrapure water, and the pH was adjusted to 1 using hydrochloric acid solution to obtain an acidic cell suspension. This acidic cell suspension was placed in a magnetically stirred constant-temperature water bath and stirred continuously at 65°C and 1400 rpm for 1 hour to induce cell rupture and release phycocyanin. The heated and stirred suspension was then centrifuged at room temperature and 18000 × g for 20 minutes. The supernatant was collected, and the pH was adjusted to 7.5 using NaOH solution to precipitate phycocyanin, resulting in an alkaline phycocyanin suspension. The precipitated phycocyanin was observed to be yellow-green, as shown in the image. Figure 1 As shown. Finally, after the alkaline phycocyanin suspension was allowed to stand for 20 minutes, it was centrifuged at room temperature and 18000×g for 20 minutes to obtain the crude phycocyanin extract.

[0060] The crude phycocyanin extract was dissolved in 0.1 mol / L hydrochloric acid solution at a ratio of 1:500. After complete dissolution, the solution was dialyzed through a 3500 Da dialysis bag for 24 hours to obtain a relatively pure phycocyanin solution. The pH of the pure phycocyanin solution was readjusted to 7.5 using NaOH aqueous solution, causing phycocyanin to redefine and form 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, and the phycocyanin precipitate was collected. The obtained precipitate was freeze-dried for 24 hours to finally obtain a light yellow powdered phycocyanin, as shown in the image. Figure 2 As shown.

[0061] Example 2: Calculation of Phycocyanin Extraction Amount

[0062] This embodiment calculates the phycocyanin content extracted from the biogas slurry system in Example 1. The powdered phycocyanin obtained in Example 1 is weighed and repeated 3 times. It is found that the phycocyanin extraction amount for the biogas slurry system can reach 140±5.7mg / g dry sludge.

[0063] Example 3: Characterization of Phycocyanin

[0064] The phycocyanin obtained in Example 1 was characterized by infrared spectroscopy. Figure 3 Its infrared spectrum is shown. The image shows that 2926 cm⁻¹ -1 The nearby absorption peaks correspond to the anti-stretching and stretching vibrations of the methylene group (–CH2–) in the phycocyanin molecule. 2291 cm⁻¹ -1 The absorption peak at 1777–1880 cm⁻¹ is correlated with the combined frequency of the stretching vibration of the –OH group in the carboxyl group (–COOH) and the bending vibration of C–H. -1 Multiple sawtooth absorption peaks were observed within the range, which is consistent with the absorption peaks of diisobutylamine in the range of 1667–1820 cm⁻¹. -1 The absorption peaks appearing within this range are similar, suggesting that these peaks may be related to the –NH2 group connecting two carbon atoms in phycocyanin. (1631 cm⁻¹) -1 The absorption peak at 1263 cm⁻¹ can be attributed to the vibrational absorption of –C=NH. Furthermore, the absorption peak at 1263 cm⁻¹... -1 and 1008cm -1 The absorption peaks at these locations originate from the stretching vibrations of C–N and C–O, respectively.

[0065] In summary, the phycocyanin extracted from biogas slurry has similar chemical groups and structural composition to the previously reported phycocyanin, indicating that this method successfully extracted phycocyanin from biogas slurry.

[0066] Comparative Example 1: Effect of extraction conditions on the extraction of phycocyanin from biogas slurry

[0067] Take 30 mL of biogas slurry and place it in a 150 mL Erlenmeyer flask. Add 30 mL of acetone and stir to obtain a mixture (concentration 50 v / v%). Use a magnetic stirrer to stir the mixture at high speed: 1600 rpm for 30 min. After stirring, transfer the mixture to a centrifuge tube and centrifuge at high speed to obtain cell pellet. Separation conditions: room temperature, 18000 × g for 20 min. Subsequently, resuspend the obtained cell pellet in 1 mol / L Tris-HCl buffer (pH = 8) for washing, and centrifuge at 18000 × g for 20 min at room temperature. Repeat the washing and centrifugation three times to obtain a net cell pellet from the biogas slurry system.

[0068] The purified cell precipitate from the biogas slurry system was resuspended in 50 mL of ultrapure water, and the pH was adjusted to 1 using hydrochloric acid to obtain an acidic purified cell suspension. This acidic suspension was placed in a magnetically stirred constant-temperature water bath and stirred continuously at 65°C and 1400 rpm for 30 min to induce cell 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 was adjusted to 7.5 using NaOH solution to precipitate the phycocyanin, resulting in an alkaline phycocyanin suspension. The precipitated phycocyanin was observed to be yellow-green. 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 obtain a crude phycocyanin extract.

[0069] The crude phycocyanin extract was dissolved in 0.1 mol / L hydrochloric acid solution at a ratio of 1:500. After complete dissolution, the solution was dialyzed through a 3500 Da dialysis bag for 24 hours to obtain a relatively pure phycocyanin solution. The pH of the pure phycocyanin solution was adjusted to 7.5 using NaOH aqueous solution, causing the phycocyanin to redefine and form 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, and the phycocyanin precipitate was collected. The obtained precipitate was freeze-dried for 24 hours to finally obtain a light yellow powdered phycocyanin.

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

[0071] Take 30 mL of biogas slurry and place it in a 150 mL Erlenmeyer flask. Add 60 mL of ethanol solution and stir to obtain a mixture (concentration 33.3 v / v%). Stir the mixture at high speed using a magnetic stirrer at 1600 rpm for 50 min. After stirring, transfer the mixture to a centrifuge tube and centrifuge at 18000 × g for 20 min at room temperature to obtain cell pellet. Resuspend the cell pellet in PBS buffer (pH 8) and wash. Centrifuge at 18000 × g for 20 min at room temperature. Repeat the washing and centrifugation process three times to obtain a net cell pellet from the biogas slurry system.

[0072] The cell precipitate from the biogas slurry system was resuspended in 50 mL of ultrapure water, and the pH was adjusted to 1 using hydrochloric acid to obtain an acidic cell suspension. This acidic cell suspension was placed in a magnetically stirred constant-temperature water bath and stirred continuously at 65°C and 1400 rpm for 50 min to induce cell 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 was adjusted to 7.5 using NaOH solution to precipitate the phycocyanin, resulting in an alkaline phycocyanin suspension. The precipitated phycocyanin was observed to be yellow-green. 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 obtain a crude phycocyanin extract.

[0073] The crude phycocyanin extract was dissolved in 0.1 mol / L hydrochloric acid solution at a ratio of 1:500, and dialyzed in a 3500 Da dialysis bag for 24 h to obtain a relatively pure phycocyanin solution. The pH of the solution was adjusted to 7.5 using NaOH aqueous solution, causing the phycocyanin to redefine and form an alkaline phycocyanin suspension. Subsequently, the alkaline phycocyanin suspension was centrifuged at room temperature and 18000 × g for 20 min using a high-speed centrifuge, and the phycocyanin precipitate was collected. The precipitate was freeze-dried for 24 h to finally obtain a light yellow powdered phycocyanin.

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

[0075] Take 30 mL of the sludge-water mixture from the aerobic tank of a wastewater treatment plant into a 150 mL Erlenmeyer flask, add 60 mL of acetone solution, and stir to obtain a mixture (concentration 33.3 v / v%). Stir the mixture at 1600 rpm for 50 min using a magnetic stirrer. After stirring, transfer the mixture to a centrifuge tube and centrifuge at 18000 × g for 20 min at room temperature to obtain cell precipitate. The cell precipitate was then resuspended in Tris-HCl buffer (pH 8) and washed, followed by centrifugation at 18000 × g for 20 min at room temperature. This washing process was repeated three times to obtain a clean cell precipitate from the sludge system.

[0076] The purified cell precipitate was resuspended in 50 mL of ultrapure water, and the pH was adjusted to 1 using hydrochloric acid solution to obtain an acidic purified cell suspension. This acidic suspension was then placed in a magnetically stirred constant-temperature water bath and stirred continuously at 65°C and 1400 rpm for 50 min to induce cell 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 was adjusted to 7.5 using NaOH solution to precipitate the phycocyanin, resulting in an alkaline phycocyanin suspension. The obtained phycocyanin was light brown in color. 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 obtain the crude phycocyanin extract.

[0077] The crude phycocyanin extract was dissolved in 0.1 mol / L hydrochloric acid solution at a ratio of 1:500. After complete dissolution, the solution was dialyzed through a 3500 Da dialysis bag for 24 h to obtain a relatively pure phycocyanin solution. The pH of the solution was adjusted to 7.5 using NaOH aqueous solution, causing the phycocyanin to redefine and form an alkaline phycocyanin suspension. Subsequently, the alkaline phycocyanin suspension was centrifuged at 18000 × g for 20 min at room temperature, and the phycocyanin precipitate was collected. The obtained precipitate was freeze-dried for 24 h to finally obtain a light brown powdery phycocyanin.

[0078] The powdered phycocyanin obtained from Comparative Examples 1, 2 and 3 were weighed and the phycocyanin extraction amount of each comparative 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] The comparison between Comparative Example 1 and Example 1 in Table 1 shows that the initial concentration of the mixed solution affects the extraction yield of phycocyanin from the biogas slurry; an overly concentrated mixed solution will reduce the final extraction yield of phycocyanin. Furthermore, the comparison between Comparative Example 2 and Example 1 shows that the extraction agent is also crucial for the extraction yield of phycocyanin from the biogas slurry; using ethanol does not yield good results. Finally, the comparison between Comparative Example 3 and Example 1 shows that different extraction raw materials have different extraction effects; phycocyanin cannot be extracted in large quantities from the aerobic pond sludge-water mixture.

[0082] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for extracting phycocyanin with high extraction yield, characterized in that, The extraction method includes the following steps: (1) Mix biogas slurry and acetone to obtain a mixture, stir the mixture, and then centrifuge to obtain cell precipitate. Then, use Tris... The mixture was washed with HCl buffer and then centrifuged to obtain the cell-free precipitate of the biogas slurry system; the volume concentration of biogas slurry in the mixture was 30-40%; the biogas slurry was the liquid formed by anaerobic fermentation of the wastewater treatment plant's residual sludge; (2) Dissolve the cell precipitate of the biogas slurry system obtained in step (1) in water, then adjust the pH to obtain an acidic cell suspension. Heat and stir the acidic cell suspension, then centrifuge the heated and stirred suspension, collect the supernatant and adjust the pH of the supernatant to alkaline to obtain an alkaline phycocyanin suspension. Finally, centrifuge the alkaline phycocyanin suspension to obtain crude phycocyanin extract. (3) Dissolve the crude phycocyanin extract obtained in step (2) in hydrochloric acid aqueous solution and dialyze to obtain pure phycocyanin solution. Then adjust the pH value of the pure phycocyanin solution to alkaline to obtain alkaline phycocyanin suspension. Then centrifuge the alkaline phycocyanin suspension, collect the precipitate and freeze dry to obtain phycocyanin. The biogas slurry is a liquid formed after anaerobic fermentation; the raw material used for anaerobic fermentation is the residual sludge from the sewage treatment plant; the anaerobic fermentation time is 20-40 days and the temperature is 30-40℃; the residual sludge refers to the activated sludge discharged from the secondary sedimentation tank or sedimentation zone of the activated sludge system.

2. The extraction method according to claim 1, characterized in that, The centrifugation conditions in step (1) are centrifugation at 20~30℃ and 15000~20000×g for 10~30 min; the stirring conditions are stirring at 20~30℃ and 1000~2000rpm for 30~60 min.

3. The extraction method according to claim 1, characterized in that, The Tris mentioned in step (1) The concentration of the HCl buffer solution 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 cell suspension mentioned in step (2) is 1~2.

5. The extraction method according to claim 1, characterized in that, The heating and stirring conditions in step (2) are stirring at 60~70℃ and 1000~2000 rpm for 0.5~2h; the centrifugation conditions are centrifugation at 20~30℃ and 15000~20000×g for 10~30 min.

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

7. The extraction method according to 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, characterized in that, The centrifugation conditions described in step (3) are centrifugation at 20~30℃ and 15000~20000×g for 10~30 min.

10. The extraction method according to claim 1, characterized in that, The high extraction rate refers to the extraction rate of phycocyanin reaching 130 mg / g dry sludge.

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