Method for extracting phycocyanin from biogas residues

Abstract

The invention discloses a method for extracting phycocyanin from biogas residues, 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 residues, mixing precipitates with acetone, and stirring and centrifuging the mixed solution to obtain cell precipitates; (2) dissolving the net cell precipitate of the biogas residue 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 residues, a promising application is explored for resource utilization of the biogas residues, and a new thought is provided for improving the resource utilization efficiency and economic benefits of the biogas residues.

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 residues. Background Art

[0002] Biogas residue refers to the solid residue generated during anaerobic digestion, which is rich in organic matter and nutrients and is widely used in fields such as soil improvement, feed production, and fertilizer making. It can effectively improve soil fertility and promote crop growth; moreover, biogas residue can also be used for the production of biofuels or as a feed additive, with high utilization value. Through the reasonable treatment and utilization of biogas residues, it is possible to promote the sustainable development of agriculture while reducing environmental pollution. However, agricultural production has obvious seasonal characteristics, and the mismatch in the time dimension leads to prominent contradictions between the supply and demand of biogas residues. Secondly, the resource conversion efficiency in the process of biogas residue treatment is relatively low: in the process of converting organic matter into fertilizer, some nutrients may not be fully converted into forms that can be utilized by crops. To address the above problems, in order to improve the resource utilization efficiency of biogas residues and expand their application scenarios, it is necessary to explore new ways of resource utilization of biogas residues, extract their high-value components, and develop their high-value utilization pathways.

[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. Its main chain is based on aspartic acid, and the side chain contains arginine groups. From the perspective of the biosynthesis mechanism, CGP can be naturally produced by most cyanobacteria, and at the same time, 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, the heterologous expression of the cphA gene in engineering strains through genetic engineering technology has become the main way to mass-produce 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 it is difficult to apply to complex mixed systems such as biogas residues. At the same time, these traditional processes have limitations such as complex operation procedures, harsh environmental conditions, and high economic costs. In addition, in the prior art, Chinese patents CN115746292 A (a method for extracting cyanophycin from activated sludge) and CN 119320668 A (a method and device for double recovery of carbon and nitrogen by collecting oil and cyanophycin) respectively disclose methods for extracting cyanophycin from activated sludge and algae-bacteria symbiotic sewage treatment systems. However, there is still room for improvement in the extraction amount of cyanophycin in the above methods.

[0005] So far, there is still a blank for an efficient extraction scheme for the biogas residue mixed system. Therefore, developing a new method for extracting CGP applicable to this system has become an important problem that needs to be solved urgently. Summary of the Invention

[0006] Technical Problem

[0007] Some of the existing methods for extracting cyanophycin are only applicable to pure strains or genetically engineered strains, and there are problems such as complex operation procedures and harsh environmental conditions, and they cannot be directly applied to the biogas residue system. In addition, the extraction amount of cyanophycin in some methods for extracting cyanophycin is not high, and there is still room for improvement.

[0008] Technical Solution

[0009] To solve the above technical problems, the object of the present invention is to provide a method for extracting cyanophycin from biogas residues, which can extract cyanophycin from the biogas residue mixed system and realize the high-value utilization of biogas residues. The method is to take biogas residues and centrifuge them, discard the supernatant to obtain a precipitate, and after acetone pretreatment, centrifuge at high speed to separate the cell precipitate; then resuspend the cell precipitate in a buffer solution, repeat washing multiple times and then centrifuge to separate the net cell precipitate. Resuspend the net cell precipitate in ultrapure water and adjust the pH to obtain an acidic cell suspension and stir strongly to make the cells in the acidic suspension release cyanophycin, thereby obtaining an acidic cell-cyanophycin mixture. Centrifuge the mixture, collect and adjust the supernatant to weakly alkaline to precipitate cyanophycin, and obtain a cyanophycin suspension. Centrifuge the alkaline cyanophycin suspension at high speed to separate the crude cyanophycin extract; then redissolve the crude cyanophycin extract under acidic conditions and dialyze the solution to obtain a pure cyanophycin solution. Adjust the pH of the pure cyanophycin solution to weakly alkaline, let it stand and then centrifuge to separate to obtain a cyanophycin particle precipitate; dry the cyanophycin particle precipitate to obtain a cyanophycin powder. Through the method of the present invention, it is possible to extract cyanophycin from biogas residues, thereby enhancing the economic value of the resource utilization of biogas residues.

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

[0011] (1) Take the biogas residues for centrifugation, collect the precipitate, and mix the precipitate with acetone to obtain a mixed solution. Stir the mixed solution, and 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 residue system;

[0012] (2) Dissolve the net cell precipitate of the biogas residue 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 heated and stirred suspension. 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 residues in step (1) are the solid residues generated during the anaerobic digestion process.

[0015] Further, the raw material used for the anaerobic digestion is kitchen waste; the time of the anaerobic digestion is 20 - 30 days, and the temperature is 20 - 40 °C.

[0016] Further, the composition of the kitchen waste includes 10 - 20 wt% of protein, 20 - 40 wt% of carbohydrates, and 5 - 15 wt% of fat.

[0017] Further, the concentration of the precipitate in the mixed solution in step (1) is 0.6 - 0.7 g / mL.

[0018] Further, the conditions for the centrifugation in step (1) are centrifugation for 10 - 30 min at 20 - 30 °C and 5000 - 20000 × g.

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

[0020] Further, 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] Further, the pH of the acidic net cell suspension in step (2) is 1 - 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 centrifugation 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 dialysis for 12 - 48 h using a dialysis bag with a molecular weight cut-off of 3000 - 4000 Da.

[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 centrifugation 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 yield, and the extraction method includes the following steps:

[0031] (1) Centrifuge the biogas residue, collect the precipitate, mix the precipitate 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 residue system;

[0032] (2) Dissolve the net cell precipitate of the biogas residue 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 obtained in step (2) in an aqueous hydrochloric acid solution and perform 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 residue described in step (1) is the solid residue generated during the anaerobic digestion process.

[0035] In one embodiment of the present invention, the raw material used for anaerobic digestion is food waste; the time for anaerobic digestion is 20 - 30 days, and the temperature is 20 - 40 °C.

[0036] In one embodiment of the present invention, the composition of the food waste includes 10 - 20 wt% of protein, 20 - 40 wt% of carbohydrates, and 5 - 15 wt% of fat.

[0037] In one embodiment of the present invention, the concentration of the precipitate in the mixed solution in step (1) is 0.6 - 0.7 g / mL.

[0038] In one embodiment of the present invention, the conditions for centrifugation in step (1) are centrifugation at 20 - 30 °C and 5000 - 20000 × g for 10 - 30 min; the conditions for stirring are stirring at 20 - 30 °C and 1000 - 2000 rpm for 30 - 60 min.

[0039] In one embodiment of the present invention, 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.

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

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

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

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

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

[0045] In one embodiment of the present invention, the pH of the alkaline phycocyanin suspension in step (3) is 7.5 - 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 120 mg / g of dry sludge.

[0048] Beneficial effects

[0049] (1) Simple and efficient: The present invention extracts phycocyanin from biogas residues, achieving a yield of 129 ± 6.8 mg / g of dry sludge. The whole process is simple to operate, with mild conditions and good reproducibility, without involving harsh conditions or expensive raw materials.

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

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

[0052] Figure 2 It is an appearance figure of phycocyanin after freeze-drying.

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

[0054] Figure 4 It is a microscope figure of phycocyanin precipitate. Specific embodiments

[0055] Source of raw materials

[0056] The biogas residues are taken from the solid residues generated during anaerobic digestion. The raw material for anaerobic digestion is food waste (the main components are 10 - 20 wt% protein, 20 - 40 wt% carbohydrates, and 5 - 15 wt% fat. The protein mainly comes from meat and fish, the carbohydrates mainly come from rice, pasta, and fruits and vegetables, and the fat mainly comes from cooking oil. The remaining components of food waste include bones, soup, and other garbage generated during food processing production). The temperature of anaerobic digestion is 35 °C and the time is 25 days.

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

[0058] Take a certain volume of biogas residue in a 50mL centrifuge tube and centrifuge it at room temperature and 6000×g for 15min using a high-speed centrifuge. After centrifugation, collect the precipitate and take 4g of the precipitate in a 150mL conical flask, add 60mL of acetone and stir to obtain a mixed solution (concentration of 0.067g / mL). Use a magnetic stirrer to stir the above-mentioned mixed solution at high speed, and the stirring conditions are: 1600rpm, 50min. 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: room temperature, 18000×g centrifugation for 20min. The obtained cell precipitate is then resuspended in a Tris-HCl buffer with a concentration of 1mol / L and pH=8 for washing, and centrifuged at 18000×g for 20min using a high-speed centrifuge at room temperature. Repeat the washing and centrifugation 3 times to obtain a clean cell precipitate of the biogas residue system.

[0059] The clean cell precipitate of the sludge system was resuspended in 50 mL of ultrapure water, and then 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 placed in a magnetic stirring constant temperature water bath, and stirred continuously at 65°C and 1400rpm for 1 hour 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 minutes, the supernatant was collected, and the pH value was adjusted to 7.5 with an aqueous NaOH solution to precipitate the phycocyanin in the supernatant to obtain an alkaline phycocyanin suspension. It was observed that the precipitated phycocyanin was light green, as shown in FIG. Figure 1 Finally, the alkaline phycocyanin suspension was allowed to stand for 20 minutes, and then centrifuged at room temperature and 18000×g for 20 minutes to separate and obtain a crude phycocyanin extract.

[0060] The above crude phycocyanin 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 pure phycocyanin solution was adjusted to 7.5 using an aqueous NaOH solution to allow the phycocyanin to reprecipitate 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 obtained phycocyanin precipitate was freeze-dried for 24 hours to finally obtain a gray-green powdery phycocyanin, such as Figure 2 shown.

[0061] Example 2: Calculation of Phycocyanin Extraction Amount

[0062] In this example, the extraction amount of phycocyanin extracted from the biogas residue system in Example 1 was calculated. The powdered phycocyanin obtained in Example 1 was weighed and repeated 3 times. It was found that the extraction amount of phycocyanin for the biogas residue system could reach 129 ± 6.8 mg / g dry sludge.

[0063] Example 3: Characterization of Phycocyanin

[0064] The phycocyanin obtained in Example 1 was characterized by infrared spectroscopy. Figure 3 The infrared spectrum of phycocyanin is shown in the figure. As can be seen from the figure, there is an absorption peak at 3239 cm -1 , which can be considered as the O–H stretching vibration after the formation of hydrogen bonds by the carboxyl dimers of phycocyanin. The absorption peak near 2924 cm -1 corresponds to the anti-stretching vibration and stretching vibration of the methylene (–CH2–) in the phycocyanin molecule. The absorption peak at 2397 cm -1 is related to the combination frequency of the stretching vibration of –OH and the bending vibration of C–H in the carboxyl (–COOH). The peak at 1730 cm -1 can be attributed to the characteristic absorption peak of the carbonyl group. The absorption peak at 1628 cm -1 can be attributed to the vibration absorption of –C=NH. In addition, the absorption peaks at 1375 cm -1 and 1019 cm -1 come from the stretching vibrations of C–N and C–O, respectively.

[0065] In summary, the phycocyanin extracted from the biogas residue has similar chemical groups and structural compositions to the phycocyanin that has been reported so far, indicating that the phycocyanin in the biogas residue has been successfully extracted by this method.

[0066] Comparative Example 1: Influence of Extraction Conditions on the Extraction of Phycocyanin from Biogas Residue

[0067] Take a certain volume of biogas residue in a 50 mL centrifuge tube and centrifuge it at room temperature and 6000×g for 15 min using a high-speed centrifuge. After centrifugal separation, collect the precipitate and take 3 g of the precipitate in a 150 mL conical flask, add 30 mL of acetone and stir to mix to obtain a mixed solution (concentration: 0.01 g / mL). Use a magnetic stirrer to stir the above mixed solution at high speed, and the stirring conditions are: 1600 rpm, 30 min. After the stirring is completed, place the mixed solution in a centrifuge tube and centrifuge it using a high-speed centrifuge to obtain cell precipitates. 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 Tris-HCl buffer solution with pH = 8 for washing, and centrifuge at 18000×g for 20 min at room temperature using a high-speed centrifuge, and repeat the washing and centrifugation 3 times to obtain the net cell precipitates of the biogas residue system.

[0068] Resuspend the net cell precipitate of the biogas residue system in 50 mL of ultrapure water, and then 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 it at 65 °C and 1400 rpm for 30 min to promote cell lysis 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 the phycocyanin in the supernatant, obtaining an alkaline phycocyanin suspension. The phycocyanin in the solution precipitates, and the precipitated phycocyanin is observed to be light green. Finally, let the alkaline phycocyanin suspension stand for 20 min, and then 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 with a concentration 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 the 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 phycocyanin precipitate for 24 h to finally obtain gray-green powdered phycocyanin.

[0070] Comparative Example 2: Effect of extraction agents on the extraction of phycocyanin from biogas residue

[0071] Take a certain volume of biogas residue in a 50 mL centrifuge tube, and centrifuge it at room temperature and 6000×g for 15 min using a high-speed centrifuge. After centrifugal separation, collect the precipitate and take 4 g of the precipitate in a 150 mL conical flask, add 60 mL of ethanol and stir to mix to obtain a mixed solution (concentration: 0.067 g / mL). 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: centrifuge at 18000×g for 20 min at room temperature. Subsequently, resuspend the obtained cell precipitate in a Tris-HCl buffer solution with a concentration of 1 mol / L and pH = 8 for washing, and centrifuge it at 18000×g for 20 min at room temperature using a high-speed centrifuge, and repeat the washing and centrifugation 3 times to obtain the net cell precipitate of the biogas residue system.

[0072] The clean cell precipitate of the sludge system is resuspended in 50 mL of ultrapure water, and then the pH value is adjusted to 1 with an aqueous hydrochloric acid solution to obtain an acidic clean cell suspension. The above-mentioned acidic clean cell suspension is placed in a magnetic stirring constant temperature water bath, and stirred continuously for 50 minutes at 65°C and 1400rpm to cause the cells in the system to rupture and release phycocyanin. The heated and stirred suspension is then centrifuged at room temperature and 18000×g for 20 minutes, the supernatant is collected and the pH value is adjusted to 7.5 with an aqueous NaOH solution to precipitate the phycocyanin in the supernatant to obtain an alkaline phycocyanin suspension, and the precipitated phycocyanin is observed to be light green. Finally, the alkaline phycocyanin suspension is allowed to stand for 20 minutes, and then centrifuged at room temperature and 18000×g for 20 minutes to separate and obtain a crude phycocyanin extract.

[0073] The above-mentioned crude phycocyanin extract was dissolved in a 0.1 mol / L hydrochloric acid aqueous solution at a ratio of 1:500. After complete dissolution, it was placed in a 3500Da dialysis bag for 24 hours to obtain a relatively pure pure phycocyanin solution. The pH value of the pure phycocyanin 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 phycocyanin precipitate was freeze-dried for 24 hours to finally obtain gray-green powdered phycocyanin.

[0074] Comparative Example 3: Extraction of Phycocyanin from Aerobic Granular Sludge

[0075] Take a certain volume of aerobic granular sludge and water mixture in a 50mL centrifuge tube and centrifuge it at room temperature and 6000×g for 15min using a high-speed centrifuge. After centrifugation, collect the precipitate and take 4g of the precipitate in a 150mL conical flask, add 60mL of acetone and stir to obtain a mixed solution (concentration of 0.067g / mL). Use a magnetic stirrer to stir the above mixed solution at high speed for 50min at 1600rpm. After stirring, place the mixed solution in a centrifuge tube and centrifuge it in a high-speed centrifuge to obtain a cell precipitate. The separation conditions are: room temperature, 18000×g centrifugation for 20min. Subsequently, the obtained cell precipitate is resuspended in a Tris-HCl buffer with a concentration of 1mol / L and pH=8 for washing, and centrifuged at 18000×g for 20min at room temperature using a high-speed centrifuge. Repeat the washing and centrifugation 3 times to obtain a clean cell precipitate of the sludge system.

[0076] Resuspend the net cell precipitate of the sludge 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. Then 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 the phycocyanin in the solution, obtaining an alkaline phycocyanin suspension. It is observed that the precipitated phycocyanin appears grayish-white. Finally, let the alkaline phycocyanin suspension stand for 20 min, and then centrifuge it at room temperature and 18000×g for 20 min to separate and obtain the crude phycocyanin extract.

[0077] 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. Use an aqueous NaOH solution to readjust the pH value of this solution to 7.5, causing the phycocyanin to precipitate again, obtaining 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 phycocyanin precipitate for 24 h to finally obtain grayish-white powdered phycocyanin.

[0078] Weigh the powdered phycocyanin obtained in Comparative Example 1, Comparative Example 2, and Comparative Example 3, and calculate the phycocyanin extraction amount for each pair of groups. Each comparative example is repeated 3 times, and 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 preparation of the initial mixture has an impact on the phycocyanin extraction amount from biogas residue. The preparation of an overly concentrated mixture will reduce the final phycocyanin extraction amount. In addition, from the comparison between Comparative Example 2 and Example 1, it can be seen that the extraction reagent is also crucial for the phycocyanin extraction amount from biogas residue. Selecting 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 a large amount of phycocyanin cannot be extracted from the aerobic granular sludge mixed liquor of mud and water.

[0082] The examples provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit their execution order. Obvious improvements made by those skilled in the art to the present invention in combination with the existing common general knowledge also fall within the protection scope 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) Centrifuge the biogas residue, collect the precipitate, mix the precipitate with acetone to obtain a mixed solution, stir the mixed solution, and 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 residue system; the concentration of the precipitate in the mixed solution is 0.6-0.7 g / mL; the biogas residue is the solid residue generated during the anaerobic digestion process; the raw material used for the anaerobic digestion is food waste; (2) Dissolve the net cell precipitate of the biogas residue 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 heated and stirred suspension. 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; (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, and then centrifuge the alkaline phycocyanin suspension. Collect the precipitate and freeze-dry it to obtain phycocyanin.

2. The extraction method according to claim 1, wherein The components of the food waste described in step (1) include 10-20 wt% of protein, 20-40 wt% of carbohydrates, and 5-15 wt% of fat.

3. The extraction method according to claim 1, wherein The conditions of the centrifugation in step (1) are centrifugation for 10-30 min at 20-30 °C and 5000-20000×g; the conditions of the stirring are stirring for 30-60 min at 20-30 °C and 1000-2000 rpm. The concentration of the Tris-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 net cell suspension described in step (2) is 1-2.

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

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

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

10. The extraction method according to claim 1, wherein The high extraction amount means that the extraction amount of phycocyanin reaches 120 mg / g of dry sludge.

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