CN116239674A - Method for refining phycobiliprotein from dilute solution of phycobiliprotein and flocculation formula - Google Patents
Method for refining phycobiliprotein from dilute solution of phycobiliprotein and flocculation formula Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
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Abstract
The invention discloses a method for refining phycobiliprotein from a dilute solution of phycobiliprotein and a flocculation formula, belonging to the field of high-valued utilization of marine biological resources. The method comprises the steps of mixing and flocculating a pH regulator, chitosan, a dilute solution of phycobiliprotein and a pH stabilizer to obtain a precipitate phycobiliprotein-chitosan flocculate, mixing the phycobiliprotein-chitosan flocculate with an ion buffer solution, taking supernatant, and filtering to obtain phycobiliprotein. The method is suitable for refining and extracting phycobiliprotein from low-abundance phycobiliprotein solution with concentration less than 0.1g/L, and the phycobiliprotein is further extractedThe flocculation rate of white is above 90%, the recovery rate is above 70%, the purity (A 545 /A 280 )>3.0, the refining method is simple and the cost is low.
Description
Technical Field
The invention relates to the field of high-value utilization of marine organism resources, in particular to a method for refining phycobiliprotein from a dilute solution of phycobiliprotein and a flocculation formula.
Background
The phycobiliprotein is released from the phycobiliprotein body by physical destruction, freeze thawing and other means, and the concentration of the residual phycobiliprotein in the wastewater is low in the process, and is basically below 0.1g/L. Common methods for extracting phycobiliprotein from phycobilisomes mainly comprise an ammonium sulfate precipitation method, ultrafiltration, a two-aqueous-phase system and the like, and when the concentration of the phycobiliprotein is less than 0.1g/L, the chemical precipitation method and the ultrafiltration method which mainly use ammonium sulfate are not cost-effective in terms of energy and economic cost. Thus, obtaining phycobilipigments from wastewater remains a significant challenge.
Chitosan (CS) is a natural positively charged basic polysaccharide, which is of great interest in the biomedical industry due to its inherent safety, recyclability and biocompatibility. Since chitosan has positive charges, it is easy to form a flocculant with proteins, solid-liquid separation can be achieved, and is often used for removing proteins in food processing, but most of the above researches are directed to deproteinization problems in high protein concentration solutions, but rarely involve recovery or purification of proteins, especially refining of phycobiliprotein. Although the prior patent literature discloses a method for preparing high-purity phycoerythrin by using a chitosan adsorption method, the patent does not disclose the concentration of phycobiliprotein in a solution, and the concentration of phycoerythrin is estimated to be more than hundred grams per liter according to the content of the patent. By this means, it is difficult to achieve refining of phycobiliprotein from dilute solutions. Therefore, the research and development of the method for refining phycobiliprotein from the dilute solution of the phycobiliprotein has important economic significance for realizing synergy and emission reduction and improving the added value of byproducts.
Disclosure of Invention
The invention mainly aims to provide a method for refining phycobiliprotein from a dilute solution of phycobiliprotein and a flocculation formula, and aims to solve the technical problem that the prior art is difficult to enrich, recycle and purify the phycobiliprotein in the dilute solution of low-abundance phycobiliprotein.
To achieve the above object, the present invention provides a method for refining phycobiliprotein from a dilute solution of phycobiliprotein, which provides a flocculation formulation comprising chitosan, an ionic buffer, a pH adjustor and a pH stabilizer, and refining according to the following steps:
s10, mixing the chitosan, the pH regulator and the pH stabilizer with a dilute solution of phycobiliprotein to obtain phycobiliprotein-chitosan flocculate;
s20, mixing the phycobiliprotein-chitosan flocculate and the ion buffer solution, and then collecting supernatant;
s30, filtering the supernatant, and refining to obtain phycobiliprotein.
Optionally, the concentration of phycobiliprotein in the dilute solution of phycobiliprotein is below 0.1g/L.
Optionally, the molecular weight of the chitosan is 10kDa-80kDa.
Optionally, the pH of the mixture of the chitosan, the pH regulator and the pH stabilizer with the dilute solution of the phycobiliprotein is 4.5-6.0.
Optionally, the mass ratio of phycobiliprotein in the dilute solution of the phycobiliprotein to the chitosan is 1 (3-25).
Optionally, the pH adjuster comprises at least one of glacial acetic acid, hydrochloric acid, phosphoric acid, oxalic acid, and malic acid; and/or the pH stabilizer comprises at least one of phosphate buffer, citrate buffer and acetate buffer.
Optionally, the volume ratio of phycobiliprotein-chitosan floc to the ionic buffer is 1 (20-60).
Optionally, the ionic buffer comprises at least one of phosphate buffer, citrate buffer, acetate buffer.
Optionally, the ionic buffer has a pH of 6-8.
Optionally, the ionic strength of the ionic buffer is 6.0-7.5.
Optionally, the supernatant is filtered with an ultrafiltration membrane.
Optionally, the flocculation rate of phycobiliprotein in the dilute solution of the phycobiliprotein is more than 90%.
Optionally, purity A of phycobiliprotein obtained by refining 545 /A 280 ＞3.0。
Optionally, the recovery rate of phycobiliprotein in the dilute solution of the phycobiliprotein is more than 70%.
In addition, in order to achieve the above object, the present invention also provides a flocculation formulation for refining phycobiliprotein from a dilute solution of phycobiliprotein, the flocculation formulation comprising chitosan, an ionic buffer, a pH regulator and a pH stabilizer as described in the above method of the invention.
The invention has the beneficial effects that:
the invention provides a method for refining phycobiliprotein from a dilute solution of phycobiliprotein and a flocculation formula thereof, which can refine, recycle and purify the phycobiliprotein from the dilute solution of phycobiliprotein with the concentration of less than 0.1g/L, the method is simple, the cost is lower, the flocculation rate of the phycobiliprotein in the dilute solution of the phycobiliprotein is more than 90%, and the purity A of the phycobiliprotein obtained by refining 545 /A 280 More than 3.0, the recovery rate of phycobiliprotein in the dilute solution of the phycobiliprotein is more than 70%.
Drawings
For a clearer description of embodiments of the invention or of solutions in the prior art, the following brief description of the drawings is given for the purpose of illustrating the embodiments or the solutions in the prior art, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained from the structures shown in these drawings without the need for inventive effort for a person skilled in the art.
Fig. 1 is an SEM image and a TEM image of chitosan, phycobiliprotein and phycobiliprotein-chitosan floc according to example 1 of the present invention, wherein a, b, c are SEM images of chitosan, phycobiliprotein and phycobiliprotein-chitosan floc, and d, e, f are TEM images of chitosan, phycobiliprotein and phycobiliprotein-chitosan floc, respectively.
FIG. 2 is a diagram showing, from left to right, the appearance of a crude extract solution of Chlorella, phycobiliprotein-chitosan flocculate formed in example 1, and phycobiliprotein-containing precipitate formed in comparative example 12, respectively.
FIG. 3 is a SDS-PAGE pattern of B-Phycoerythrin (PE) in the crude extract solution of Synechococcus, 1, and refined B-Phycoerythrin (PE) of example 1 and comparative example 12, wherein the crude extract solution of Synechococcus was separated by polyacrylamide gel electrophoresis; 2. comparative example 10; 3. example 1.
FIGS. 4 to 5 are fluorescence spectra of B-phycoerythrin in the crude extract solution of Chlorella, and B-phycoerythrin refined in comparative example 12 and example 1.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The description as it relates to "first", "second", etc. in the present invention is for descriptive purposes only and is not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be considered to be absent and not within the scope of protection claimed in the present invention.
The invention provides a method for refining phycobiliprotein from a dilute solution of phycobiliprotein.
The method comprises a flocculation formula, wherein the flocculation formula comprises chitosan, an ion buffer solution, a pH regulator and a pH stabilizer, and is refined through the following steps:
s10, mixing the chitosan, the pH regulator and the pH stabilizer with a dilute solution of phycobiliprotein to obtain phycobiliprotein-chitosan flocculate;
s20, mixing the phycobiliprotein-chitosan flocculate and the ion buffer solution, and then collecting supernatant;
s30, filtering the supernatant, and refining to obtain phycobiliprotein.
According to the method, chitosan and phycobiliprotein are combined to form phycobiliprotein-chitosan flocculate through a coagulation-flocculation polymer bridging principle, coagulation treatment is completed, and then an ion buffer solution is added to separate chitosan from phycobiliprotein, so that phycobiliprotein with high purity and high recovery rate is obtained through refining.
By the method, phycobiliprotein can be recovered from low-abundance water containing algae pigment protein, high-purity phycobiliprotein is enriched, flocculation rate of the phycobiliprotein is more than 90%, and purity A of the phycobiliprotein is obtained by refining 545 /A 280 The recovery rate of phycobiliprotein in the dilute solution of the phycobiliprotein is more than 70 percent, which can meet the application requirements of food and even spectral grade, and the concentration of the phycobiliprotein in the water body is very highTo may be less than 0.1g/L.
It is understood that the method of the present invention can refine phycobiliprotein with high purity and high yield from phycobiliprotein concentration below 0.1g/L or from phycobiliprotein concentration above 0.1g/L. The dilute phycobiliprotein solution can be water bodies containing phycobiliprotein, such as microalgae solution, seaweed processing wastewater, algae crude extraction solution and the like, and the phycobiliprotein mainly contains R-phycoerythrin, B-phycoerythrin, phycocyanin and allophycocyanin.
In step S10, the pH adjuster includes at least one of glacial acetic acid, hydrochloric acid, phosphoric acid, oxalic acid and malic acid, which not only can play a role in dissolving chitosan, but also can adjust the pH value of the whole system to promote flocculation of phycobiliprotein in the dilute solution of phycobiliprotein. .
In step S10, the pH stabilizer includes at least one of phosphate buffer, citrate buffer, acetate buffer, and may stabilize the pH of the whole system to promote flocculation of phycobiliprotein in the dilute phycobiliprotein solution.
In some embodiments, the chitosan working solution can be obtained by mixing the pH regulator with chitosan, specifically, after the chitosan is dissolved by the pH regulator, the chitosan is refrigerated for 12 hours at the temperature of 4 ℃ to completely hydrate the chitosan to obtain the chitosan working solution. Then mixing the chitosan working solution with the dilute solution of the phycobiliprotein and the pH stabilizer to obtain phycobiliprotein-chitosan flocculate.
In some embodiments, the precipitate phycobiliprotein-chitosan flocculate can be better separated by mixing the chitosan working solution with the dilute solution of the phycobiliprotein and the pH stabilizer, standing for 1-2h and centrifuging at the rotating speed of 2500-3500rpm/min for 8-15 min.
In the method, the chitosan is used as a high molecular biological flocculant, has better safety, recoverability and biocompatibility, has natural positive charges, is easy to form a flocculant with protein so as to realize solid-liquid separation, is applied to recovery and refining of phycobiliprotein, and can form flocculate with the phycobiliprotein in water body to generate precipitation.
The viscosity of the chitosan solution can be increased along with the increase of the molecular weight, the molecular weight is too small, the viscosity of the chitosan is too small, flocculation with phycobiliprotein is not facilitated, the molecular weight is too large, the viscosity of the chitosan is too large, the mixing and collision of the chitosan and the phycobiliprotein are easy to be blocked, and the flocculation efficiency is also reduced. Thus, considering collectively the molecular weight of chitosan as 10kDa-80kDa, in some embodiments, the molecular weight of chitosan may be any one of the 10kDa-80kDa ranges of 10kDa, 20kDa, 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, etc.
The chitosan has positive charges, the phycobiliprotein has negative charges, the phycobiliprotein and the phycobiliprotein can be combined into phycobiliprotein-chitosan flocculate, the pH value can influence the quantity of the negative charges of the phycobiliprotein and the quantity of the positive charges of the chitosan, and under the acidic condition, the chitosan and the phycobiliprotein can carry almost equal and opposite charges especially at the pH value of 4.5-6.0. In some embodiments, the pH of the entire system after mixing the chitosan working solution with the dilute solution of phycobiliprotein and the pH stabilizer is any one of the values in the range of 4.5-6.0, such as 4.5, 4.8, 5.0, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, etc.
The mass ratio of phycobiliprotein to chitosan in the dilute phycobiliprotein solution can influence the active site combined with the phycobiliprotein in the solution, thereby influencing the adsorption rate of the phycobiliprotein. With the improvement of the mass ratio of phycobiliprotein to chitosan, the positive charge of the phycobiliprotein is excessive and covers the surface of the flocculating body, so that the flocculating efficiency is reduced, and therefore, the mass ratio of chitosan to the dilute solution of phycobiliprotein is 1 (3-25) in order to improve the adsorption rate of the phycobiliprotein. In some embodiments, the mass ratio of phycobiliprotein to chitosan in the dilute phycobiliprotein solution is 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, etc. 1: any one of the values in the range of (3-25). Under the condition of the mass ratio, the adsorption rate of chitosan to phycobiliprotein in the phycobiliprotein solution can be improved, so that the flocculation rate, recovery rate and purity of the phycobiliprotein are improved.
The purpose of step S20 of the present invention is to separate phycobiliprotein from chitosan in phycobiliprotein-chitosan flocs.
Because of the electrostatic attraction between phycobiliprotein and chitosan, the ion buffer added in the step has the main function of shielding the electrostatic attraction between phycobiliprotein and chitosan. When the ionic strength of the ion buffer solution is smaller, a better shielding effect cannot be exerted, and when the ionic strength of the ion buffer solution is larger, the high ionic strength has a stronger salting-out effect on phycobiliprotein, so that the phycobiliprotein is difficult to release from floccules. Thus, to increase phycobiliprotein recovery, the ionic strength of the ionic buffer used in step S20 is from 6.0 to 7.5, and in some embodiments, the ionic strength of the ionic buffer may be any one of values in the range of 6.00, 6.10, 6.20, 6.30, 6.40, 6.45, 6.50, 6.55, 6.60, 6.65, 6.70, 6.73, 6.75, 6.77, 6.80, 6.85, 6.88, 6.89, 6.90, 6.92, 6.95, 6.96, 6.99, 7.00, 7.1, 7.2, 7.5, etc. from 6.0 to 7.5.
In some embodiments, the ionic buffer of step S20 comprises a phosphate buffer, a citrate buffer, an acetate buffer.
In some embodiments, the pH range of the ion buffer solution in step S20 is 6-8, and the ion buffer solution in the above pH range has a good shielding effect on electrostatic attraction between phycobiliprotein and chitosan, so that separation of phycobiliprotein and chitosan can be promoted, and the recovery rate of phycobiliprotein is improved.
In some embodiments, the volume ratio of phycobiliprotein-chitosan floc to added ionic buffer of step S20 is 1: (20-60), specifically, may be 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, etc. 1: any one of the ratios in the range of (20-60).
In some embodiments, after mixing the phycobiliprotein-chitosan flocculate with the ion buffer, the mixture may be centrifuged at 2500-3500rpm/min for 8-15min until the precipitate is colorless, and the precipitate is removed to obtain a supernatant, wherein the supernatant is rich in the separated phycobiliprotein, and the precipitate is chitosan.
In step S30, phycobiliprotein may be extracted from the supernatant by filtration, specifically, an ultrafiltration membrane may be selected for filtration, where the ultrafiltration membrane includes any one of cellulose acetate, aromatic polyamide, polyethersulfone, and polyvinylidene fluoride, and the molecular weight cutoff of the ultrafiltration membrane is 150K, so that the recovery rate and purity of phycobiliprotein may be improved.
In addition, the invention also provides a flocculation formula for refining phycobiliprotein from the dilute solution of phycobiliprotein, and the formula comprises chitosan, an ion buffer solution, a pH regulator and a pH stabilizer in the refining method.
The flocculation formula of the invention can enrich and purify phycobiliprotein from the dilute solution of phycobiliprotein less than 0.1g/L, and can enrich and purify phycobiliprotein from the dilute solution of phycobiliprotein more than 0.1g/L, the flocculation rate of the phycobiliprotein is more than 90%, and the purity A of the phycobiliprotein obtained by refining 545 /A 280 The recovery rate is more than 70 percent and is more than 3.0.
The flocculation formula of the invention can also be prepared into a kit for refining phycobiliprotein, which is more beneficial to the popularization and use of the method and the formula.
The technical scheme of the present invention will be further described in detail with reference to the following specific examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure.
Example 1
Reagent preparation:
the dilute solution of phycobiliprotein to be refined is a rough extraction solution of Synechococcus, and the content of phycobiliprotein in the solution is below 0.1 g/L;
chitosan: the molecular weight is 30kDa;
ion buffer: phosphate buffer with ionic strength of 6.77 and pH of 7;
pH regulator: hydrochloric acid solution with the mass concentration of 0.2% -1%;
pH stabilizer: phosphate buffer.
The method for refining phycobiliprotein from the dilute solution of phycobiliprotein in the embodiment comprises the following steps:
s10, weighing chitosan, regulating and dissolving the chitosan by using a pH regulator, refrigerating at 4 ℃ for 12 hours to ensure that the chitosan is completely hydrated to obtain a chitosan working solution, mixing the chitosan working solution and the crude extraction solution of the rhodococcus, namely phycobiliprotein, according to the mass ratio of 1:10 of the phycobiliprotein to the chitosan in the crude extraction solution of the rhodococcus, adding a pH stabilizer to obtain a mixed solution with the pH of 5.4, standing for 1 hour, centrifuging at the rotating speed of 2500-3500rpm/min for 8-15 minutes to obtain precipitate phycobiliprotein-chitosan flocculate, and collecting the phycobiliprotein-chitosan flocculate;
s20, adding an ion buffer solution into phycobiliprotein-chitosan floccules in the step S10 according to the volume ratio of 1:40, then oscillating, and centrifugally collecting supernatant at the speed of 2500-3500 rpm/min;
s30, filtering the supernatant in the step S20 by using an ultrafiltration membrane polyvinylidene fluoride with a molecular cut-off of 150kDa to obtain phycobiliprotein.
Performance test 1:
the chitosan, phycobiliprotein and phycobiliprotein-chitosan flocs obtained in step S10 before refining in example 1 were characterized by a scanning electron microscope and a transmission electron microscope, and the results are shown in FIG. 1, wherein a, b and c are SEM images of chitosan, phycobiliprotein and phycobiliprotein-chitosan flocs, d, e and f are TEM images of chitosan, phycobiliprotein and phycobiliprotein-chitosan flocs, respectively, and as compared with chitosan and phycobiliprotein, phycobiliprotein-chitosan flocs show cross-linking and compact network structures, which indicate that bridging plays a main role in flocculation.
Examples 2 to 3
Examples 2-3 were identical to the refining process of example 1, except that the chitosan had molecular weights of 10kDa and 80kDa, respectively.
Comparative examples 1 to 3
Comparative example 1 was identical to the refining process of example 1, except that the molecular weight of chitosan was 100kDa, 150kDa and 200kDa, respectively.
Examples 4 to 5
Examples 4 to 5 are the same as the refining method of example 1, except that the addition amount of the reagent is different in that the mass ratio of phycobiliprotein to chitosan in the crude extract solution of Synechococcus is 1:25, 1:3, respectively.
Comparative examples 4 to 5
Comparative examples 4 to 5 are identical to the refining method of example 1, except that the mass ratio of phycobiliprotein to chitosan is 1:1 and 1:2, respectively.
Examples 6 to 7
Examples 6 to 7 are basically the same as the refining method of example 1 except that in step S10, the pH is 4.5 and 6.0, respectively, after the chitosan working solution is mixed with the dilute solution of phycobiliprotein and the pH stabilizer.
Comparative examples 6 to 7
Comparative examples 5 to 6 are basically the same as the refining method of example 1 except that in step S10, the pH is 3.5 and 6.5, respectively, after the chitosan working solution is mixed with the dilute solution of phycobiliprotein and the pH stabilizer.
Examples 8 to 9
Examples 8 to 9 were substantially identical to the refining method of example 1, except that the ionic strength of the ionic buffer was 6.0 and 7.5, respectively.
Comparative examples 8 to 9
Comparative examples 8 to 9 were substantially the same as the refining method of example 1 except that the ionic strength of the ionic buffer was 5.5 and 8, respectively.
Examples 10 to 11
Examples 10 to 11 were substantially identical to the refining method of example 1, except that the reagents used were such that the pH of the ionic buffer was 6 and 8, respectively.
Performance test 2:
1) The flocculation rate of B-Phycoerythrin (PE) in examples 1 to 7 and comparative examples 1 to 7 was calculated by the following calculation formula of flocculation rate, and the result is shown as 1.
2) The recovery rate of B-Phycoerythrin (PE) of example 1 and examples 8 to 11 and comparative examples 8 to 9 was calculated by the following calculation formula for recovery rate, and the results are shown in Table 1.
TABLE 1 flocculation Rate vs. recovery of examples 1-11 and comparative examples 1-9
Table 1 analysis of results:
examples 1 to 3 and comparative examples 1 to 3: when the molecular weight of the chitosan is in the range of 10kDa to 80kDa, the flocculation rate of the B-Phycoerythrin (PE) is higher and reaches more than 90 percent. In comparative examples 1 to 3, however, the molecular weight of chitosan was large, the viscosity of the solution was increased, the mixing and collision of particles were hindered, and the flocculation efficiency was lowered.
Examples 4 to 5 and comparative examples 4 to 5: when the mass ratio of phycobiliprotein to chitosan is in the range of 1:25 to 1:3, the flocculation rate of B-Phycoerythrin (PE) is higher, especially when the mass ratio of phycobiliprotein to chitosan is 1:10, almost all B-Phycoerythrin (PE) is adsorbed, but when the mass ratio of phycobiliprotein to chitosan is further improved to 1:1 and 1:2, the positive charge of phycobiliprotein is excessive, the formed floccule surface is covered, and the flocculation efficiency is reduced.
Examples 6 to 7 and comparative examples 6 to 7: when the pH of the solution is extremely low and is 3.5, the number of negative charges of phycobiliprotein is very low, and conversely, when the pH of the solution is higher and is 6.5, the number of positive charges of chitosan is smaller due to deprotonation. Only when the pH of the solution is in the proper range of 4.5-6.0, the chitosan and phycobiliprotein can carry equal but opposite charges, so that the system is completely neutralized, and the flocculation rate is improved, and particularly when the pH of example 1 is 5.4, the flocculation rate reaches 97%.
Examples 8 to 9 and comparative examples 8 to 9: the ionic strength of the ionic buffer is low at 5.5, which is insufficient to shield electrostatic attraction between B-Phycoerythrin (PE) and chitosan, resulting in low recovery rate. On the other hand, when the ionic strength of the ionic salt buffer is 8, the high ionic strength has a strong salting-out effect on B-Phycoerythrin (PE) and makes it difficult to release from the floccule. And the ionic strength was in the range of 6.0 to 7.5, with a higher recovery rate, which reached the peak at 6.77 of example 1.
Examples 10 to 11: when the pH value of the phosphate buffer solution is 6-8, the recovery rate is not obviously different and reaches more than 70%.
Comparative example 12
Comparative example 12 phycobiliprotein in the same crude extract solution of Synechococcus as in example 1 was purified by ammonium sulfate salting-out method using 80% by mass of saturated ammonium sulfate.
Performance test 3:
the properties of phycobiliprotein refined in example 1 and comparative example 12 were calculated, and the fluorescence intensity and purity of phycobiliprotein were measured by using the crude extract of rhodococcus as a control group, and the flocculation state was observed. The results are shown in Table 2, FIGS. 2 to 5.
TABLE 2 comparison of Performance of phycobiliproteins extracted from example 1 and comparative example 12
As shown in Table 2, phycobiliprotein obtained by the method of example 1 has higher purity, higher recovery rate and higher fluorescence intensity than phycobiliprotein obtained by the ammonium sulfate salting-out method of comparative example 12, but has much lower cost than the ammonium sulfate precipitation method, which indicates that the method of the invention is more suitable for simultaneously enriching and purifying phycobiliprotein from a dilute solution system of phycobiliprotein.
From left to right in fig. 2, the phycobiliprotein-chitosan flocculent formed in example 1 and the phycobiliprotein-containing precipitate formed in comparative example 12 are respectively a rough extraction solution of rhodococcus, and it can be seen that the phycobiliprotein-chitosan flocculent formed in example 1 has a fibrous structure, a high density, a stable structure and is easier to perform subsequent operations such as centrifugation.
FIG. 3 shows SDS-PAGE patterns of B-Phycoerythrin (PE) in the crude extract solution of Synechococcus, and refined B-Phycoerythrin (PE) of example 1 and comparative example 12, wherein 1 is the crude extract solution of Synechococcus, 2 is comparative example 12, and 3 is example 1. Among them, the specific band of Phycoerythrin (PE) of example 1B is particularly apparent.
FIGS. 4 to 5 are fluorescence spectra of B-Phycoerythrin (PE) in the crude Chlorella extract solution and B-phycoerythrin refined in comparative example 12 and example 1, and as can be seen from FIG. 4, both example 1 and comparative example 12 have the same absorption peak as B-Phycoerythrin (PE) in the crude Chlorella extract solution, and as can be seen from FIG. 5, the purity (A 545 /A 280 )>3.0。
Examples 12 to 17
Examples 12 to 17 are substantially identical to the refining method of example 1, except that the reagents used are as follows:
the ionic buffers used in examples 12 to 13 were respectively: citrate buffer with an ionic strength of 6.77, ph 7 and acetate buffer with an ionic strength of 6.77, ph 7;
the pH adjusters used in examples 14 to 15 were respectively: phosphoric acid with mass concentration of 0.2% -1% and glacial acetic acid with mass concentration of 0.2% -1%;
the pH stabilizers used in examples 16 to 17 were respectively: acetate buffer and citrate buffer.
Performance test 4
Flocculation rate and recovery rate of B-Phycoerythrin (PE) of examples 12 to 17 were calculated by the following calculation formulas for flocculation rate and recovery rate, and phycobiliprotein purity thereof was measured, and the results are shown in Table 3.
TABLE 3 flocculation Rate, recovery Rate and phycobiliprotein purity results for examples 12-17
| Experimental group | Flocculation rate (%) | Recovery (%) | Phycobiliprotein purity (A) 545 /A 280 ) |
| Example 12 | 96 | 75 | 3.5 |
| Example 13 | 95 | 75 | 3.4 |
| Example 14 | 97 | 78 | 3.2 |
| Example 15 | 97 | 77 | 3.4 |
| Example 16 | 95 | 76 | 3.5 |
| Example 17 | 96 | 76 | 3.5 |
As shown in Table 3, flocculation rate of phycobiliprotein in examples 12 to 17The recovery rate is more than 70% and the purity A of refined phycobiliprotein is more than 90% 545 /A 280 ＞3.0。
The purity, flocculation rate and recovery rate of phycobiliprotein refined in the above examples and comparative examples were calculated as follows:
1. the flocculation efficiency of phycobiliprotein was measured, and since rhodococcus rhodochrous is mainly B-Phycoerythrin (PE), the purity of B-Phycoerythrin (PE) was calculated and represented as the purity of phycobiliprotein in the crude extract solution of rhodochrous.
The calculation formula is as follows:
b-phycoerythrin purity CPE:
wherein a562 represents the absorbance of the sample at 562 nm.
2. The rhodococcus rhodochrous is mainly B-Phycoerythrin (PE), so that the flocculation rate and the recovery rate of the B-phycoerythrin are measured, and the flocculation rate and the recovery rate of phycobiliprotein in the rhodochrous crude extract solution can be respectively represented.
1) Flocculation rate YF (%) of phycobiliprotein:
wherein, C1 and V1 are respectively the concentration of B-phycoerythrin in the dilute solution of phycobiliprotein before refining and the total volume of the dilute solution of phycobiliprotein; c2 and V2 are the concentration of B-phycoerythrin in the solution obtained by centrifugation and the total volume of the solution after the treatment of step S10 is completed.
2) Recovery of phycobiliprotein YR (%):
wherein, C1 and V1 are respectively the concentration of B-phycoerythrin in the dilute solution of phycobiliprotein before refining and the total volume of the dilute solution of phycobiliprotein; c3 and V3 are the concentration of B-phycoerythrin in the supernatant and the total volume of the supernatant, respectively, after the completion of the treatment of step S20.
The foregoing description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the invention, but rather is intended to cover any equivalents of the structures or equivalent processes disclosed herein or in the alternative, which may be employed directly or indirectly in other related arts.
Claims (10)
1. A method for refining phycobiliprotein from a dilute solution of phycobiliprotein, characterized in that a flocculation formula is provided, the flocculation formula comprises chitosan, an ion buffer, a pH regulator and a pH stabilizer, and the refining is carried out according to the following steps:
mixing the chitosan, the pH regulator, the pH stabilizer and the dilute solution of the phycobiliprotein to obtain phycobiliprotein-chitosan flocculate;
mixing the phycobiliprotein-chitosan floc with the ion buffer, and then collecting the supernatant;
filtering the supernatant, and refining to obtain phycobiliprotein.
2. The method for refining phycobiliprotein from a dilute solution of phycobiliprotein according to claim 1, wherein the concentration of phycobiliprotein in the dilute solution of phycobiliprotein is less than 0.1g/L.
3. The method for refining phycobiliprotein from a dilute solution of phycobiliprotein as recited in claim 1, wherein the chitosan has a molecular weight of 10KDa-80KDa.
4. The method for refining phycobiliprotein from a dilute solution of phycobiliprotein according to claim 1, wherein the pH of the mixture of chitosan, pH adjustor, pH stabilizer and dilute solution of phycobiliprotein is 4.5-6.0;
and/or the mass ratio of phycobiliprotein to chitosan in the dilute solution of the phycobiliprotein is 1: (3-25).
5. The method of refining phycobiliprotein from a dilute phycobiliprotein solution of claim 1, wherein the pH adjustor comprises at least one of glacial acetic acid, hydrochloric acid, phosphoric acid, oxalic acid, and malic acid;
and/or the pH stabilizer comprises at least one of phosphate buffer, citrate buffer and acetate buffer.
6. The method for refining phycobiliprotein from a dilute solution of phycobiliprotein according to claim 1, wherein the volume ratio of phycobiliprotein-chitosan floc to the ionic buffer is 1 (20-60).
7. The method of refining phycobiliprotein from a dilute phycobiliprotein solution of claim 1, wherein the ionic buffer comprises at least one of a phosphate buffer, a citrate buffer, and an acetate buffer;
and/or the pH of the ion buffer is 6-8;
and/or the ionic strength of the ionic buffer is 6.0-7.5.
8. The method for refining phycobiliprotein from a dilute solution of phycobiliprotein according to claim 1, wherein the supernatant is filtered with an ultrafiltration membrane.
9. The method for refining phycobiliprotein from a dilute solution of phycobiliprotein according to any of claims 1-8,
the flocculation rate of phycobiliprotein in the dilute solution of the phycobiliprotein is more than 90%;
and/or purity A of phycobiliprotein obtained by refining 545 /A 280 ＞3.0；
And/or the recovery rate of phycobiliprotein in the dilute solution of the phycobiliprotein is more than 70%.
10. A flocculation formulation for refining phycobiliprotein from a dilute solution of phycobiliprotein, comprising the chitosan of any one of claims 1-9, an ionic buffer, a pH adjustor, and a pH stabilizer.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101734746A (en) * | 2009-12-31 | 2010-06-16 | 中国科学院等离子体物理研究所 | Method for adsorbing and removing phycobiliprotein and microcystin-LR by modified attapulgite |
| CN101899102A (en) * | 2010-07-12 | 2010-12-01 | 华南理工大学 | Method for separating high purity phycocyanin from spirulina |
| CN106496312A (en) * | 2016-09-30 | 2017-03-15 | 烟台大学 | A kind of method that employing shitosan absorption method prepares high-purity phycoerythrin |
| WO2019193614A2 (en) * | 2018-04-01 | 2019-10-10 | Hoseini Fahime | The process of achieving the highest degree of drug's purity of c-phycocyanin from spirulina alga |
| CN112812176A (en) * | 2021-01-13 | 2021-05-18 | 江南大学 | Method for extracting phycocyanin from spirulina by low-salt flocculation method |
| CN113292648A (en) * | 2021-07-02 | 2021-08-24 | 华东理工大学 | Method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae and phycobiliprotein |
-
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101734746A (en) * | 2009-12-31 | 2010-06-16 | 中国科学院等离子体物理研究所 | Method for adsorbing and removing phycobiliprotein and microcystin-LR by modified attapulgite |
| CN101899102A (en) * | 2010-07-12 | 2010-12-01 | 华南理工大学 | Method for separating high purity phycocyanin from spirulina |
| CN106496312A (en) * | 2016-09-30 | 2017-03-15 | 烟台大学 | A kind of method that employing shitosan absorption method prepares high-purity phycoerythrin |
| WO2019193614A2 (en) * | 2018-04-01 | 2019-10-10 | Hoseini Fahime | The process of achieving the highest degree of drug's purity of c-phycocyanin from spirulina alga |
| CN112812176A (en) * | 2021-01-13 | 2021-05-18 | 江南大学 | Method for extracting phycocyanin from spirulina by low-salt flocculation method |
| CN113292648A (en) * | 2021-07-02 | 2021-08-24 | 华东理工大学 | Method for separating and purifying high-purity phycobiliprotein from nitrogen-fixing blue algae and phycobiliprotein |
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