CN110903384A - A kind of extraction and purification method of phycocyanin - Google Patents
A kind of extraction and purification method of phycocyanin 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
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- Life Sciences & Earth Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
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- Biomedical Technology (AREA)
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Abstract
本发明属于生物提取技术领域，特别涉及一种藻蓝蛋白的提取和纯化方法，首先用CaCl2溶液对藻泥进行溶胀破壁，再经过固液分离和两次膜过滤得到高纯度藻蓝蛋白。本发明可在盐胁迫的微藻细胞中提取藻蓝蛋白，提取出的藻蓝蛋白纯度可达到食品级标准，并且操作步骤简单，可实现快速连续生产，应用范围更广。The invention belongs to the technical field of biological extraction, and in particular relates to a method for extracting and purifying phycocyanin. First, algal slime is swelled and broken by a CaCl solution, and then high - purity phycocyanin is obtained through solid-liquid separation and two membrane filtrations. . The invention can extract phycocyanin from salt-stressed microalgae cells, the purity of the extracted phycocyanin can reach food-grade standards, and the operation steps are simple, rapid and continuous production can be realized, and the application range is wider.
Description
Technical Field
The invention belongs to the technical field of biological extraction, and particularly relates to a method for extracting and purifying phycocyanin.
Background
The microalgae contains various bioactive substances, wherein the phycobiliprotein is rich in the microalgae, and has important application values in the aspects of medicine, health care, bioengineering and the like. At present, after microalgae cultured in a high-salt stress environment is subjected to high-salt stress, phycocyanin contained in the microalgae is difficult to extract, and a large amount of salt ions cannot be effectively removed in a later purification process, so that phycocyanin in the microalgae is lost, and the nutritional value of the microalgae cannot be fully utilized.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor researches and designs a phycocyanin extracting and purifying method in long-term practice, and phycocyanin with high recovery rate and high purity can be extracted from salt-stressed microalgae mud.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for extracting and purifying phycocyanin comprises the following steps:
obtaining salt stress algae mud; and comprises:
step one, swelling and wall breaking: mixing the obtained salt stress algae mud with CaCl with the concentration of 1-10 g/l2Mixing the solution, salt stress algae mud and CaCl2Mixing the solutions according to the volume ratio of 1: 10-100 to obtain a mixed solution of phycocyanin and algae residue;
and (2) solid-liquid separation: carrying out solid-liquid separation on the mixed solution obtained in the first step at the temperature of 4-40 ℃ by using a centrifugal machine or a filtering device to obtain a crude phycocyanin extracting solution;
fine filtering in the third step: treating the crude extract obtained in the second step by adopting a membrane filtration method, wherein the selected membrane aperture is 0.1-5 microns, removing impurities, and obtaining a permeate as a phycocyanin solution;
ultrafiltration in the fourth step: concentrating and desalting the crude extract obtained in the second step or the phycocyanin solution obtained in the third step by adopting a membrane filtration method, wherein the selected membrane aperture is 1500D-0.1 μm, and the phycocyanin concentrated solution is in a feed solution barrel;
and a fifth step of drying: adding trehalose and sodium citrate into the phycocyanin concentrated solution, and drying to obtain phycocyanin dry powder;
according to different obtained products, the method comprises a first process and a second process or any combination of the first process, the second process and other subsequent processes, and specifically comprises the first process, the second process and the third process in sequence; or, sequentially performing the first step, the second step and the fourth step; or, sequentially performing the first step, the second step and the fifth step; or the first step, the second step, the third step and the fourth step are sequentially carried out; or, sequentially performing the first step, the second step, the third step and the fifth step; or the first step, the second step, the fourth step and the fifth step are sequentially carried out; or, the operation is performed according to the first step, the second step, the third step, the fourth step and the fifth step in sequence.
Further, in the first process, the salt-stressed algae mud is mixed with CaCl2And placing the mixed solution of the solution in an environment of 4-40 ℃ for swelling and wall breaking, wherein the swelling time is 6-72 hours.
Further, in the first process, the salt-stressed algae mud is mixed with CaCl2The mixed solution of the solution is subjected to swelling wall breaking in a lightproof environment.
Further, in the second step, the centrifuge is a tubular centrifuge, a disk centrifuge or a three-leg centrifuge; the filter device is a plate filter, and the size of filter cloth of the plate filter is 600-2000 meshes.
Further, in the third step, a filtering device is adopted for fine filtering; the filter device comprises at least two stages of filter membranes with different apertures, the apertures are gradually reduced, the aperture of the first stage filter membrane is 0.1-5 μm, and the aperture of the last stage filter membrane is 1500D-0.1 μm.
Further, in the fourth step, ultrafiltration is carried out at a temperature of 4 to 40 ℃ and a pressure of 50Kpa to 2 MPa.
Further, in the fifth step, the addition amount of the trehalose and the sodium citrate in the phycocyanin concentrated solution is as follows according to the mass ratio of phycocyanin: sodium citrate: trehalose is 40% -90%: 10% -40%: 10 to 40 percent of the additive, and the sum of the proportions of the three is 1.
Further, in the fifth step, the drying treatment is performed by vacuum freeze drying, spray drying or oven drying.
Further, the vacuum freeze drying is divided into a pre-freezing stage, a sublimation drying stage and an analysis drying stage, and the phycocyanin concentrated solution is subjected to three stages to obtain phycocyanin dry powder.
Further, the spray drying is specifically to spray dry the phycocyanin concentrated solution added with the trehalose and the sodium citrate at the air inlet temperature of 110-130 ℃ and the air outlet temperature of 70-90 ℃ to obtain the phycocyanin dry powder.
Further, the drying specifically comprises the step of putting the phycocyanin concentrated solution added with the trehalose and the sodium citrate into a drying machine, and drying at the temperature of 50 ℃ to obtain phycocyanin dry powder.
The invention has the beneficial effects that:
(1) in the process of extracting phycocyanin, CaCl is added2The swelling method enables the salt concentration difference value to be generated inside and outside the cell, and the drop value is utilized to enable the cell to fully absorb water and break the cell wall, so that the phycocyanin is dissolved out, and the wall breaking rate can be greatly improved; CaCl2The swelling method can effectively ensure the stability of phycocyanin, has high yield, low cost and simple operation, and overcomes the defects of high cost of repeated freeze thawing, difficult realization of glass bead oscillation method equipment and easy denaturation of phycocyanin caused by ultrasonic waves.
(2) Through two times of membrane filtration, firstly 0.1-5 mu m is taken as the membrane aperture, macromolecular impurities in the crude extract are effectively removed, then 1500D-0.1 mu m is taken as the membrane aperture, salt ions are removed, the operation is simple, the recovery rate of phycocyanin can reach 85%, the purity can be more than 1, and food-grade phycocyanin is obtained.
(3) By adopting a vacuum freeze-drying method, the phycocyanin is ensured not to be denatured, the volume and the shape of the dried phycocyanin are well preserved, and the shelf life of the product is prolonged; the freeze-drying method can control the water content of the phycocyanin to be between 0.5 and 5 percent, greatly controls the water content of a finished product, and can effectively protect the phycocyanin from being oxidized because little oxygen is generated during freeze-drying.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present invention, the present invention will be further described with reference to the following preferred embodiments.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting.
Interpretation of terms:
microalgae: generally refers to algae whose morphology is microscopic.
As introduced in the background art, the extraction method of phycocyanin in microalgae after high salt stress in the prior art is difficult, and a large amount of salt ions cannot be effectively removed in the later purification process, so that the phycocyanin is lost. The invention provides an extraction and purification method of phycocyanin, which can extract phycocyanin with high purity and high yield under the condition of salt stress, and the recovery rate can reach 85%.
The method comprises the following steps:
obtaining salt stress algae mud; and comprises:
step one, swelling and wall breaking: mixing the obtained salt stress algae mud with CaCl with the concentration of 1-10 g/l2Mixing the solution, salt stress algae mud and CaCl2Mixing the solutions according to the volume ratio of 1: 10-100, and placing the mixture in an environment with the temperature of 4-40 ℃ for swelling for 6-72 hours to obtain a mixed solution of the algae residue and the phycocyanin.
And (2) solid-liquid separation: and (3) carrying out solid-liquid separation on the mixed solution obtained in the first step at the temperature of 4-40 ℃ by using a centrifugal machine or a filtering device to obtain the crude phycocyanin extracting solution.
Fine filtering in the third step: and (4) treating the crude extract obtained in the second step by adopting a membrane filtration method, wherein the selected membrane aperture is 0.1-5 microns, and removing impurities to obtain a permeate which is a phycocyanin solution.
Ultrafiltration in the fourth step: concentrating and desalting the crude extract obtained in the second step or the phycocyanin solution obtained in the third step by adopting a membrane filtration method, wherein the selected membrane aperture is 1500D-0.1 μm, and the phycocyanin is left in a feed liquid barrel to obtain a phycocyanin concentrated solution.
And a fifth step of drying: adding trehalose and sodium citrate into the phycocyanin concentrated solution, and drying to obtain food-grade phycocyanin with purity higher than 1.
According to different obtained products, the method comprises a first process and a second process or any combination of the first process, the second process and other subsequent processes, and specifically comprises the first process, the second process and the third process in sequence; or, sequentially performing the first step, the second step and the fourth step; or, sequentially performing the first step, the second step and the fifth step; or the first step, the second step, the third step and the fourth step are sequentially carried out; or, sequentially performing the first step, the second step, the third step and the fifth step; or the first step, the second step, the fourth step and the fifth step are sequentially carried out; alternatively, the operations are performed in the first step, the second step, the third step, the fourth step, the fifth step, and the like in this order.
Further, in the first process, the salt-stressed algae mud is mixed with CaCl2The mixed solution of the solution is subjected to swelling wall breaking in a lightproof environment.
The purity of the phycocyanin is gradually reduced along with time at normal temperature, and the purity of the phycocyanin solution in the illumination environment is reduced rapidly. It is shown that light has an influence on the stability of phycocyanin, so that the phycocyanin needs to be stored in a dark place.
Furthermore, the first step utilizes the condition that the algae cells are in the environment of low salt solution to generate salt concentration difference values inside and outside the cells, and the difference values are utilized to ensure that the cells fully absorb water and break cell walls, so that the algae protein substances are dissolved out, and the wall breaking rate can be greatly improved. The extraction rate can reach more than 90 percent and is not lower than that of a glass bead oscillation method, so that CaCl is extracted2The extraction method can be used for extracting phycocyanin.
Further, in the second process, the centrifugal machine is a tubular centrifugal machine, a disc centrifugal machine or a three-leg centrifugal machine; the filtering device is a plate and frame filter, and the size of filter cloth of the plate and frame filter is 600-2000 meshes.
The recovery rate of phycocyanin is 45-95% after solid-liquid separation.
Further, in the third step, fine filtration is performed by using a filtration device, wherein the filtration device comprises at least two stages of filtration membranes with different pore diameters, the pore diameter is gradually reduced, the pore diameter of the first stage filtration membrane is 0.1-5 μm, and the pore diameter of the last stage filtration membrane is 1500D-0.1 μm.
The membrane element is the key component of the system, the device intercepts substances with corresponding molecular weight by distributing filter membranes with certain apertures on the membrane element, plays a role in separating and purifying target substances, and aims to remove substances such as colloid in feed liquid so as to prevent the influence on the filtration efficiency of subsequent experiments.
The invention has the advantages that the recovery rate of the phycocyanin is 20 to 90 percent after fine filtration.
Furthermore, in the ultrafiltration process of the step four, the denaturation of phycocyanin is influenced by the overhigh temperature and pressure in the feed liquid barrel, and the purity is reduced. In order to ensure the invariance of phycocyanin, the material liquid barrel needs to be kept in a low-temperature and low-pressure state.
In one embodiment of the present invention, condensed water is continuously injected into the jacket of the feed liquid barrel, and the temperature in the barrel is kept constant at a low temperature (i.e., 4 to 40 ℃) by the cooling and heating machine. In addition, the pressure is set to 50Kpa to 2MPa to sufficiently ensure the phycocyanin activity.
The recovery rate of phycocyanin is 10-85% by ultrafiltration.
Further, in the fifth step, the addition amount of trehalose and sodium citrate in the phycocyanin concentrated solution is as follows according to the mass ratio of phycocyanin: sodium citrate: trehalose is 40% -80%: 10% -40%: 10 to 40 percent of the phycocyanin is added, the sum of the proportion of the phycocyanin, the phycocyanin and the phycocyanin is ensured to be 1, and the phycocyanin meeting the food grade standard is finally obtained through drying treatment, wherein the recovery rate of the working procedure is 5 to 85 percent.
Further, in the fifth step, the drying treatment is performed by vacuum freeze drying, spray drying or oven drying. The drying method is not limited, and vacuum drying is further preferable.
Vacuum freeze-drying, freeze-drying for short, is a method of freezing a water-containing substance into a solid state, and then sublimating water in the solid state into a gas state, thereby drying the substance.
Compared with drying, spray drying and vacuum drying, the freeze drying is carried out at low temperature, so that the phycocyanin is not denatured, and simultaneously, the microorganisms can lose activity, and the freeze drying method is particularly suitable for storing some bioactive products, biochemical products and the like with poor thermal stability. The volume and the shape of the dried phycocyanin are well preserved, no drying shrinkage exists, the rehydration speed is high, and the original shape of the material can be quickly recovered.
Drying at low temperature greatly inhibits the growth of microorganisms and the action of enzyme, and prolongs the shelf life of the product; meanwhile, the loss of volatile components, aromatic components, heat-sensitive nutritional components and the like contained in the substances is reduced, so that the drying method is the best drying method for some chemicals, medicines and foods.
The freeze-drying can control the moisture of the dried substance to be between 0.5 and 5 percent, the moisture content of the finished product is greatly controlled, the freeze-drying is carried out under the vacuum condition, and the substances which are easy to oxidize are well protected to a certain extent due to little oxygen.
The freeze-dried product has lighter weight, the transportation cost is reduced, the product can be stored for a long time, and the economic loss caused by the deterioration of the product can be reduced. Therefore, a distinct advantage of lyophilization itself over other drying means is the optimal choice for processing the dried product.
The vacuum freeze drying is divided into 3 stages, namely a pre-freezing stage, a sublimation drying stage and an analysis drying stage. Specifically, the phycocyanin concentrated solution added with trehalose and sodium citrate is subpackaged, is placed into a vacuum pump for pre-freezing for 5-6 hours to reach-50 ℃, then the vacuum pump is opened to enter a sublimation drying stage, the time consumption of the stage is long, and when the temperature of a partition plate reaches the set maximum temperature of 40 ℃, the material enters an analysis drying stage and is dried at the temperature until the drying is finished.
The spray drying is specifically to spray dry the phycocyanin concentrated solution added with the trehalose and the sodium citrate at the air inlet temperature of 110-130 ℃ and the air outlet temperature of 70-90 ℃, and finally obtain the phycocyanin dry powder in the main material.
The drying specifically comprises the step of putting the phycocyanin concentrated solution added with the trehalose and the sodium citrate into a drying machine, and drying at the temperature of 50 ℃ to obtain phycocyanin dry powder.
After the phycocyanin is extracted, the ultrafiltration permeate can be subjected to nanofiltration again by selecting a proper membrane aperture, and glycerol glucoside (GG for short) is recovered. Under high salt stress, glycerol glucoside is synthesized in vivo as a compatible substance to resist damage of cells caused by external high osmotic pressure. The glycerol glucoside is widely applied to the aspects of medicine, health care and the like, so the glycerol glucoside can be recovered, the algae cell raw material in the salt stress environment is fully utilized, the waste of resources is greatly avoided, and the economic benefit is improved.
In a specific embodiment of the present invention, the species of the salt-stressed algae mud is multicladium, nostoc, synechococcus, cryptophyceae, spirulina, etc., the species is not limited, and spirulina is further preferred.
Currently, salt-stressed microalgae algal mud can be obtained by various culture methods, and is not particularly limited. However, in order to be able to produce large quantities of algal cells quickly and efficiently, the present invention proposes a preferred method for algal species cultivation, which the applicant has protected as a further patent application, which comprises: inoculating algae cells into low-salt fresh water culture medium for culture, wherein the salt concentration is less than 100 mmol/L.
Preferably, the incubation time at this stage is 3-20 days.
Preferably, the culture temperature at this stage is: 15-40 deg.C, and preferably, the culture temperature at this stage is 20-40 deg.C.
Preferably, the low-salt fresh water culture medium contains nitrogen, phosphorus, iron, magnesium, sodium, potassium and trace elements required for the growth of microalgae.
Preferably, the formula of the low-salt fresh water culture medium is Zarrouk culture medium, and the detailed components are shown in tables 1 and 2. The low-salt fresh water culture medium can enable a large amount of algae cells to be propagated, and is used as a basis for synthesizing and accumulating a large amount of GG.
TABLE 1Z's Medium formulation
TABLE 2 mother liquor formula
In this stage, light and carbon source with proper wavelength are selected for photosynthesis.
Selecting carbon source from carbon dioxide-containing mixed air under autotrophic conditions, wherein the concentration of carbon dioxide is within 10% (v/v), preferably, the concentration of carbon dioxide is 1-5% (v/v), or selecting inorganic carbonate, or selecting the carbon dioxide-containing mixed air and the inorganic carbonate at the same time; under heterotrophic conditions, glucose, maltose, glycerol, acetic acid, etc. are additionally added to the low-salt fresh water medium.
In a preferred embodiment of the present invention, the step of culturing the algal cells further comprises a harvesting step, which comprises: and harvesting algae cells from the culture medium to obtain algae mud.
In a preferred embodiment of the present invention, the harvesting step is followed by a washing step comprising: and cleaning the surface of the algae mud, and removing surface attachments to obtain the clean algae mud.
In a preferred embodiment of the present invention, the stage of culturing the algal cells further comprises a GG production stage.
In the GG generation phase:
the algal cells are selected from those grown after the culture in the stage of obtaining algal cells after the culture, and normally, the cells do not contain GG component or contain little if any GG component before being inoculated into the culture medium in the GG production stage.
The culture medium used for cell culture is not only required to ensure the growth of the algal cells, but also to allow the algal cells to synthesize GG, and in addition to obtaining nutrient elements required for the growth of the microalgae in the stage of the cultured algal cells, substances which can stress the cells and induce GG synthesis reaction are required to be added, and usually, substances which can change the cell osmotic pressure, such as substances which can change the cell osmotic pressure and are added at a concentration of 100-300 mmol/L, and the substances can be sodium chloride, potassium chloride and the like, and are not limited to the above substances, as long as GG synthesis reaction can be induced.
Although continuous light irradiation can promote accumulation of GG in addition to the light energy in the wavelength range required for the above-described photosynthesis, intermittent light irradiation can promote accumulation of GG more than continuous light irradiation, and temperature difference can promote accumulation of GG more. During the dark reaction process of intermittent illumination, the accumulation of GG can be promoted by reducing the oxygen concentration in the introduced carbon dioxide gas mixture.
Based on the above, the culture conditions in the stage are optimized to obtain algal cells containing GG, so that a good basis is provided for the stage of pulling up.
Preferably, the culturing time in this stage is 3 to 20 days, and more preferably, the culturing time in this stage is 5 to 10 days.
Preferably, the culture temperature is: 15-40 deg.C, preferably 20-40 deg.C.
Preferably, the stage selects intermittent illumination with a light-to-dark ratio of 1:1, light-to-dark time periods of 6-18h and 6-18h, respectively, light intensity of 500--2·s-2。
Further preferably, in the process of the dark reaction of the intermittent illumination, the oxygen concentration is reduced to 1-2% (v/v), and a large number of experiments prove that the accumulation of GG can be further promoted by reducing the oxygen concentration in the introduced carbon dioxide gas mixture in the process of the dark reaction of the intermittent illumination.
In a preferred embodiment of the present invention, after the GG generation phase, a GG pull-up phase is further included.
In the first stage of GG pulling-up:
in a most preferred embodiment, the algal cell feature is that it has been cultured to contain an amount of GG after the GG generation stage. When the GG is continuously cultured for a long time in the GG production stage, the cells secrete substances which can inhibit the growth of the cells, so that the metabolic activity of the cells is reduced, and the synthesis capability of the GG in the cells is reduced. After the cells containing a certain amount of GG after being cultured are inoculated into a culture medium of the first stage of GG elevation, the cells can recover activity and continue to grow, a driving force is provided for GG synthesis reaction, and the accumulation efficiency of GG is improved.
The culture medium conditions in the first GG pulling-up stage are basically the same as those in the GG generation stage, and the culture medium used for cell culture is used for ensuring the growth of algae cells and synthesizing GG in the algae cells. In addition to the nutrient elements required for the growth of microalgae, it is necessary to add a substance which can induce GG synthesis reaction under the condition of stress on cells, wherein the addition amount of the GG-inducing substance is at least as large as the GG production stage, and the increase of the addition amount of the GG-inducing substance can promote the GG synthesis reaction more effectively, for example, 300 < C1Sodium chloride at a concentration of 800mmol/L or less, potassium chloride or the like, and the like are not limited to the above, as long as the GG synthesis reaction can be induced.
Although continuous light irradiation can promote accumulation of GG in addition to the light energy in the wavelength range required for the above-described photosynthesis, intermittent light irradiation can promote accumulation of GG more than continuous light irradiation, and temperature difference can promote accumulation of GG more. During the dark reaction process of intermittent illumination, the accumulation of GG can be promoted by reducing the oxygen concentration in the introduced carbon dioxide gas mixture.
Based on the above, the culture conditions in the first stage of GG elevation are optimized so as to obtain algal cells with higher content of GG.
Preferably, the cultivation time is more than 2 days, preferably 3-5 days.
Preferably, the culture temperature is: 15-40 ℃; further preferably, the temperature during the dark period is set to 15-25 ℃ and the temperature during the light period is set to 25-40 ℃.
Preferably, the stage selects intermittent illumination with a light-to-dark ratio of 1:1, light-to-dark time periods of 6-18h and 6-18h, respectively, light intensity of 500--2·s-2。
Further preferably, in the process of the dark reaction of the intermittent illumination, the oxygen concentration is reduced to 1-2% (v/v), and a large number of experiments prove that in the process of the dark reaction of the intermittent illumination, the introduced mixed gas can reduce the oxygen concentration and promote the accumulation of GG.
In the most preferred embodiment, algal cells having a GG content of 10% (w/w) or more can be obtained by GG pull-up stage-one culture.
In the GG pull-up stage two:
for autotrophic microalgae, the culture conditions are the same as those in the first step-up stage of GG, except that the concentration of substances capable of changing the osmotic pressure of cells in the culture medium is more than 800 < C2≤1500mmol/L。
For heterotrophic microalgae or microalgae cells with improved cell wall micromolecule permeability after genetic engineering technology modification, the concentration of substances capable of changing the osmotic pressure of the cells in the culture medium is more than 800 < C2In addition to less than or equal to 1500mmol/L, reaction substrates for synthesizing GG, such as glycerol or available sugars, such as glucose and maltose, but not limited to the two sugars, are added, so that the GG content of the microalgae cells can be further increased. Preferably, the glycerol or glucose is maintained at a concentration of 0.5-2g/L by feeding or the like, and the glucose is maintained at a concentration of 0.5-5 g/L.
Preferably, the cultivation time is more than 2 days, preferably 3-5 days.
The inventor finds that different culture methods and culture conditions have great influence on the content of GG in algae cells, and the invention can obtain the algae cells with high content of GG by a specific culture method (including day and night combination mode, high-salinity culture after high-salinity culture of algae seeds, high-light condition and the like) and a step-by-step regulation mode.
In a preferred embodiment of the present invention, after the step of GG pulling-up stage one or step of GG pulling-up stage two, a harvesting step is further included, which includes: and harvesting the alga cells enriched with GG from the culture medium in the first GG pulling-up stage or the second GG pulling-up stage to obtain alga mud.
In a preferred embodiment of the present invention, the harvesting step is followed by a washing step comprising: and cleaning the surface of the algae mud, and removing surface attachments to obtain the clean algae mud.
The reactor used in each step of the present invention is not limited, and may be a closed reactor or an open raceway pond. For heterotrophic microalgae cells, the closed reactor is used to more effectively prevent contamination by other bacteria.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1
Salt stress algae mud is obtained by the method, and the algae mud is processed by the first working procedure and the second working procedure to obtain crude extract containing phycocyanin with different concentrations. An example of the method is as follows:
1) swelling and breaking cell wall in the first step, mixing the salt stress algae mud with 1g/l CaCl2Mixing the solution, salt stress algae mud and CaCl2Mixing the solutions according to the volume ratio of 1:10, placing in an environment at 4 ℃, and shading for 6 hours to obtain a mixed solution of phycocyanin and algae residue.
2) And (2) carrying out solid-liquid separation on the mixed solution obtained in the step (1) through a second working procedure, and carrying out high-speed centrifugation at the temperature of 4 ℃ by adopting a tubular centrifuge to obtain the crude phycocyanin extracting solution.
Through the above steps, the recovery rate of phycocyanin was 53.3%, and the purity was 0.54.
Example 2
The salt stress algae mud is obtained by the method, and the algae mud is subjected to impurity removal treatment through the first process, the second process and the third process to obtain the phycocyanin with low impurities. An example of the method is as follows:
1) swelling and breaking cell wall in the first step, mixing the salt stress algae mud with 10g/l CaCl2Mixing the solution, salt stress algae mud and CaCl2The volume ratio of the solutionMixing at a ratio of 1:100, and swelling at 40 deg.C for 72 hr to obtain mixed solution of phycocyanin and algae residue.
2) And (2) carrying out solid-liquid separation on the mixed liquor obtained in the step (1) through a second working procedure, filtering by adopting a plate and frame filter at the temperature of 40 ℃, and obtaining the crude phycocyanin extract when the size of filter cloth is 600 meshes.
3) And (3) removing impurities such as macromolecular substances, colloids and the like in the crude extract obtained in the step (2) by a membrane filtration method in a third working procedure, wherein the selected membrane aperture is 0.1 mu m, so as to obtain the phycocyanin concentrated solution.
Through the above steps, the recovery rate of phycocyanin was 69.1%, and the purity was 0.72.
Example 3
Salt-stressed algae mud is obtained by the method, and the algae mud is desalted through the first step, the second step and the fourth step to obtain the phycocyanin with low salt concentration. An example of the method is as follows:
1) swelling and breaking cell wall in the first step, mixing the salt stress algae mud with 8g/l CaCl2Mixing the solution, salt stress algae mud and CaCl2Mixing the solutions according to the volume ratio of 1:40, placing the mixture in an environment at 15 ℃, and shading the mixture for 12 hours to obtain a mixed solution of the blue protein and the algae residue.
2) And (2) carrying out solid-liquid separation on the mixed liquor obtained in the step (1) through a second working procedure, filtering by adopting a plate and frame filter under the environment of 15 ℃, and obtaining the crude phycocyanin extract when the size of filter cloth is 2000 meshes.
3) Concentrating and desalting the crude extractive solution obtained in step 2) by membrane filtration method, selecting membrane with pore diameter of 2000D, filtering at 4 deg.C and pressure of 2Mpa, removing salt ions from the crude extractive solution, and recovering phycocyanin to obtain phycocyanin concentrated solution.
Through the above procedures, the recovery rate of phycocyanin is 46.3%, and the purity is 1.09
Example 4
The salt stress algae mud is obtained by the method, and the algae mud is subjected to impurity removal and desalination treatment through the first process, the second process, the third process and the fourth process to obtain the phycocyanin with low impurity and low salt concentration. An example of the method is as follows:
1) swelling and breaking cell wall in the first step, mixing the salt stress algae mud with 8g/l CaCl2Mixing the solution, salt stress algae mud and CaCl2Mixing the solutions according to the volume ratio of 1:40, placing in an environment at 15 ℃, and carrying out shading treatment for 30 hours to obtain a mixed solution of phycocyanin and algae residue.
2) And (2) carrying out solid-liquid separation on the mixed liquor obtained in the step (1) through a second working procedure, filtering by adopting a plate and frame filter under the environment of 15 ℃, and obtaining the crude phycocyanin extract when the size of filter cloth is 1200 meshes.
3) And (3) removing impurities such as macromolecular substances, colloids and the like in the crude extract obtained in the step (2) by a membrane filtration method in a third working procedure, wherein the selected membrane aperture is 5 mu m, and obtaining a permeate containing phycocyanin.
4) Concentrating and desalting the permeate obtained in step 3) by membrane filtration method, wherein the selected membrane has a pore size of 0.1 μm, filtering at 15 deg.C and 50Kpa, removing salt ions from the permeate, and recovering phycocyanin to obtain phycocyanin concentrate.
Through the above procedures, the recovery rate of phycocyanin was 67.8%, and the purity was 1.35.
Example 5
The salt stress algae mud is obtained by the method, and the food-grade phycocyanin is obtained by carrying out impurity removal, desalination and freeze drying on the algae mud through the first process, the second process, the third process, the fourth process and the fifth process. An example of the method is as follows:
1) swelling and breaking cell wall in the first step, mixing the salt stress algae mud with 8g/l CaCl2Mixing the solution, salt stress algae mud and CaCl2Mixing the solutions according to the volume ratio of 1:40, and placing the mixture in an environment at 4 ℃ for swelling for 60 hours to obtain a mixed solution of phycocyanin and algae residue.
2) And (2) carrying out solid-liquid separation on the mixed liquor obtained in the step (1) through a second working procedure, filtering by adopting a plate and frame filter under the environment of 4 ℃, and obtaining the crude phycocyanin extract when the size of filter cloth is 600 meshes.
3) And (3) removing impurities such as macromolecular substances, colloids and the like in the crude extract obtained in the step (2) by a membrane filtration method in a third working procedure, wherein the selected membrane aperture is 0.2 mu m, and obtaining the permeation solution containing phycocyanin.
4) Concentrating and desalting the permeate obtained in step 3) by membrane filtration method, selecting membrane with pore diameter of 2500D, filtering at 10 deg.C and pressure of 200Kpa, removing salt ions from the crude extractive solution, and recovering phycocyanin to obtain phycocyanin concentrated solution.
5) Adding trehalose and sodium citrate into the concentrated solution obtained in the step 4), wherein the addition mass of the trehalose and the sodium citrate is as follows: sodium citrate: trehalose is 40%: 40%: adding 20% of the above components, pre-freezing the phycocyanin concentrated solution added with trehalose and sodium citrate in a vacuum pump for 5 hours to-50 ℃, then opening the vacuum pump to enter a sublimation drying stage, and when the temperature of the partition board reaches the set maximum temperature of 40 ℃, entering an analysis drying stage for the materials at the temperature until the drying is finished.
Through the above procedures, the recovery rate of phycocyanin was 71.2%, and the purity was 1.65.
Example 6
The crude phycocyanin extract obtained in the first step and the second step or phycocyanin with low impurity concentration obtained by impurity removal can be dehydrated to obtain phycocyanin with different phycocyanin concentrations in the fifth step.
An example of the method is as follows:
1) swelling and breaking cell wall in the first step, mixing the salt stress algae mud with 8g/l CaCl2Mixing the solution, salt stress algae mud and CaCl2Mixing the solutions according to the volume ratio of 1:40, placing the mixture in an environment at 4 ℃, and shading the mixture for 60 hours to obtain a mixed solution of phycocyanin and algae residue.
2) And (2) performing solid-liquid separation on the mixed solution obtained in the step (1) through a second working procedure, and performing high-speed centrifugation at the temperature of 4 ℃ by using a disc centrifuge to obtain the crude phycocyanin extracting solution.
3) Adding trehalose and sodium citrate into the crude extract obtained in the step 2), wherein the addition mass of the trehalose and the sodium citrate is as follows: sodium citrate: trehalose is 60%: 30%: adding 10% of the crude phycocyanin extract, placing the crude phycocyanin extract added with trehalose and sodium citrate into a vacuum pump for pre-freezing for 6 hours to reach-50 ℃, then opening the vacuum pump to enter a sublimation drying stage, when the temperature of a partition board reaches the set maximum temperature of 40 ℃, entering an analysis drying stage, and at the temperature, drying until the drying is finished.
Through the above steps, the recovery rate of phycocyanin was 81.65%, and the purity was 0.54.
Example 7
The phycocyanin with different phycocyanin concentrations can be obtained by dehydrating phycocyanin with low salt concentration or low impurity and low salt phycocyanin obtained by impurity removal and desalination in the first step, the second step and the fourth step. An example of the method is as follows:
1) swelling and breaking cell wall in the first step, mixing the salt stress algae mud with 8g/l CaCl2Mixing the solution, salt stress algae mud and CaCl2Mixing the solutions according to the volume ratio of 1:40, placing in an environment at 15 ℃, and carrying out shading treatment for 12 hours to obtain a mixed solution of phycocyanin and algae residue.
2) And (2) carrying out solid-liquid separation on the mixed solution obtained in the step (1) through a second working procedure, and carrying out high-speed centrifugation at 15 ℃ by adopting a three-leg centrifuge to obtain a crude phycocyanin extracting solution.
3) Concentrating and desalting the crude extractive solution obtained in step 2) by membrane filtration method, selecting membrane with pore diameter of 2500D, filtering at 15 deg.C and pressure of 200Kpa, removing salt ions from the crude extractive solution, and recovering phycocyanin to obtain phycocyanin concentrated solution.
4) Adding trehalose and sodium citrate into the concentrated solution obtained in the step 3), wherein the addition mass of the trehalose and the sodium citrate is as follows: sodium citrate: trehalose is 70%: 25%: adding 5% of the above extract, and spray drying at air inlet temperature of 130 deg.C and air outlet temperature of 70 deg.C to obtain phycocyanin dry powder.
Through the above steps, the recovery rate of phycocyanin was 53.95%, and the purity was 0.64.
Comparative experiment 1: CaCl2Experimental background for the swelling method.
The purpose of the experiment; comparative CaCl2Influence of swelling method, repeated freezing and thawing method, glass bead oscillation method and ultrasonic crushing method on recovery rate of phycocyanin
The experimental method comprises the following steps: selecting salt-stressed algae mud, and sequentially performing repeated freeze thawing method, glass bead shaking method, ultrasonic crushing method and CaCl2The recovery rate and purity of the swelling method were compared.
The experimental results are as follows:
TABLE 3 CaCl2Phycocyanin recovery rate under swelling method and other methods
And (3) knotting: assuming that all phycocyanin can be extracted as 1 by a glass bead oscillation method, CaCl2Compared with other methods, the swelling method can extract phycocyanin with high purity and high content, so CaCl is selected2The swelling method is used for extracting phycocyanin.
The above detailed description is only for the preferred embodiment of the present invention, and the present invention should not be limited to the embodiment, i.e. all equivalent changes and modifications should be made within the scope of the present invention.
Claims (10)
1. A method for extracting and purifying phycocyanin is characterized by comprising the following steps:
obtaining salt stress algae mud; and comprises:
step one, swelling and wall breaking: mixing the obtained salt stress algae mud with CaCl with the concentration of 1-10 g/l2Mixing the solution, salt stress algae mud and CaCl2Mixing the solutions according to the volume ratio of 1: 10-100 to obtain a mixed solution of phycocyanin and algae residue;
and (2) solid-liquid separation: carrying out solid-liquid separation on the mixed solution obtained in the first step by a centrifugal machine or a filtering device to obtain a crude phycocyanin extracting solution;
fine filtering in the third step: treating the crude extract obtained in the second step by adopting a membrane filtration method, wherein the selected membrane aperture is 0.1-5 microns, removing impurities, and obtaining a permeate as a phycocyanin solution;
ultrafiltration in the fourth step: desalting the crude extract obtained in the second step or the phycocyanin solution obtained in the third step by adopting a membrane filtration method, wherein the selected membrane aperture is 1500D-0.1 μm, and the phycocyanin is left in a feed solution barrel to obtain a phycocyanin concentrated solution;
and a fifth step of drying: adding trehalose and sodium citrate into the phycocyanin concentrated solution obtained in the step four, and drying to obtain phycocyanin dry powder;
according to different obtained products, the method comprises a first process and a second process or any combination of the first process, the second process and other subsequent processes, and specifically comprises the first process, the second process and the third process in sequence; or, sequentially performing the first step, the second step and the fourth step; or, sequentially performing the first step, the second step and the fifth step; or the first step, the second step, the third step and the fourth step are sequentially carried out; or, sequentially performing the first step, the second step, the third step and the fifth step; or the first step, the second step, the fourth step and the fifth step are sequentially carried out; or, the operation is performed according to the first step, the second step, the third step, the fourth step and the fifth step in sequence.
2. The method according to claim 1, wherein in the second step, the mixed solution obtained in the first step is subjected to solid-liquid separation in an environment of 4 to 40 ℃.
3. The method of claim 2, wherein in step one, the salt-stressed algal mash is mixed with CaCl2And placing the mixed solution of the solution in an environment of 4-40 ℃ for swelling and wall breaking, wherein the swelling time is 6-72 hours.
4. The method of claim 3, wherein in step one, the salt-stressed algal mash is mixed with CaCl2The mixed solution of the solution is coveredSwelling and wall breaking are carried out in a light environment.
5. The method according to claim 2, wherein in the second step, the centrifuge is a tube centrifuge, a disk centrifuge or a three-leg centrifuge; the filter device is a plate filter, and the size of filter cloth of the plate filter is 600-2000 meshes.
6. The method according to claim 2, wherein in the third step, fine filtration is performed by using a filtration device; the filter device comprises at least two stages of filter membranes with different apertures, the apertures are gradually reduced, the aperture of the first stage filter membrane is 0.1-5 μm, and the aperture of the last stage filter membrane is 1500D-0.1 μm.
7. The method according to claim 2, wherein in the fourth step, the ultrafiltration is carried out at a temperature of 4 ℃ to 40 ℃ and a pressure of 50Kpa to 2 MPa.
8. The method according to claim 2, wherein in step five, the trehalose and sodium citrate are added in an amount of at least one of phycocyanin: sodium citrate: trehalose is 40% -90%: 10% -40%: 10 to 40 percent of the additive, and the sum of the proportions of the three is 1.
9. The method according to claim 8, wherein in the fifth step, the drying treatment is performed by vacuum freeze drying, spray drying or oven drying.
10. The method as claimed in claim 9, wherein the vacuum freeze drying is divided into a pre-freezing stage, a sublimation drying stage and an analysis drying stage, and the phycocyanin concentrated solution is subjected to three stages to obtain phycocyanin dry powder;
specifically, the spray drying is to spray dry the phycocyanin concentrated solution added with the trehalose and the sodium citrate at the air inlet temperature of 110-130 ℃ and the air outlet temperature of 70-90 ℃ to obtain phycocyanin dry powder;
the drying specifically comprises the step of putting the phycocyanin concentrated solution added with the trehalose and the sodium citrate into a drying machine, and drying at the temperature of 50 ℃ to obtain phycocyanin dry powder.
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| CN114478813A (en) * | 2022-01-24 | 2022-05-13 | 徐州工程学院 | Method for extracting polysaccharide from hericium erinaceus spore powder |
| WO2023085941A1 (en) | 2021-11-15 | 2023-05-19 | Ful Foods B.V. | A method of producing a composition, an water soluble extract composition and a non-water soluble extract composition, each composition being a phycobiliprotein containing micro-organism-based composition |
| WO2023115326A1 (en) * | 2021-12-21 | 2023-06-29 | 佛山蓝强生物科技有限公司 | Composition for enhancing photothermal stability of phycobiliproteins, and preparation method therefor and use thereof |
| WO2024206398A1 (en) * | 2023-03-28 | 2024-10-03 | Arizona Board Of Regents On Behalf Of Arizona State University | Methods for extracting and encapsulating phycocyanin |
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| WO2023085941A1 (en) | 2021-11-15 | 2023-05-19 | Ful Foods B.V. | A method of producing a composition, an water soluble extract composition and a non-water soluble extract composition, each composition being a phycobiliprotein containing micro-organism-based composition |
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| WO2024206398A1 (en) * | 2023-03-28 | 2024-10-03 | Arizona Board Of Regents On Behalf Of Arizona State University | Methods for extracting and encapsulating phycocyanin |
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