阅读说明：本技术 一种可节约用水量的藻蓝蛋白提取方法 (Phycocyanin extraction method capable of saving water consumption ) 是由 吕雪峰 李新 段仰凯 张凯 吴怀之 刘祥 于 2019-12-17 设计创作，主要内容包括：本发明属于生物提取技术领域,特别涉及一种可节约用水量的藻蓝蛋白提取方法,首先取微藻藻泥与高浓度CaCl<Sub>2</Sub>溶液以不小于1:10的体积比混合进行溶胀破壁,再经过过滤脱水,得到食品级藻蓝蛋白产品。本发明操作简单,既能保证藻蓝蛋白的稳定性和产率,又能节约用水、降低能耗,有利于工业化快速连续生产,应用范围更广。(The invention belongs to the technical field of biological extraction, and particularly relates to a phycocyanin extraction method capable of saving water consumption 2 Mixing the solutions at a volume ratio of not less than 1:10 for swelling and wall breaking, and filtering and dehydrating to obtain food-grade phycocyanin product. The method is simple to operate, can ensure the stability and yield of the phycocyanin, can save water and reduce energy consumption, is beneficial to industrial rapid continuous production, and has wider application range.)

1. A phycocyanin extraction method capable of saving water consumption is characterized by comprising the following steps:

obtaining salt stress algae mud; and comprises:

step one, swelling and wall breaking: mixing the obtained algae mud with CaCl with the concentration of 10-50 g/l₂Mixing the solution, mixing the algae mud with CaCl₂Mixing the solutions according to the volume ratio of 1: 1-10 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 between 4 and 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: 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 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 the algal 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.

4. The method according to claim 3, wherein in the first step, the algal mud is mixed with CaCl₂The mixed solution of the solution is subjected to swelling wall breaking in a lightproof 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 the fine filtration in the third step is performed by 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 the trehalose and the sodium citrate in the fifth step are added with phycocyanin in a mass ratio of: sodium citrate: trehalose is 40% -90%: 10% -40%: 10 to 40 percent, and the sum of the proportion 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 added with the trehalose and the sodium citrate 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 conditions of 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.

Technical Field

The invention belongs to the technical field of biological extraction, and particularly relates to a phycocyanin extraction method capable of saving water consumption.

Background

Phycocyanin is one of the rare pigment proteins in nature, not only has bright color, but also is a protein with rich nutrition, has complete amino acid composition and high essential amino acid content, has the functions of resisting cancer and stimulating the generation of erythrocyte colony, and has similar action with Erythropoietin (EPO); phycocyanin also has fluorescence effect, and can be used as a research reagent of some photodynamics in biology, cytology and the like.

At present, all extraction methods utilize microalgae dry powder as a raw material to extract phycocyanin, the water consumption is large in the extraction process, the yield of phycocyanin obtained by taking water as a buffer solution is low, the purity is not high, the energy consumption is large, and the continuous production requirements of enterprises are not facilitated.

Disclosure of Invention

Aiming at the defects of the prior art, the inventor researches and designs a phycocyanin extracting method capable of saving water consumption in long-term practice, so that the extraction rate and purity of phycocyanin can be ensured, and the water consumption can be greatly saved.

In order to achieve the purpose, the invention provides the following technical scheme:

a phycocyanin extraction method capable of saving water consumption comprises the following steps:

obtaining salt stress algae mud; and comprises:

step one, swelling and wall breaking: mixing the obtained algae mud with CaCl with the concentration of 10-50 g/l₂Mixing the solution, mixing the algae mud with CaCl₂Mixing the solutions according to the volume ratio of 1: 1-10 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: 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 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.

Further, mixing the algae mud 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 step, the 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, the fine filtration is carried out by adopting a filtering 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.

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 trehalose and the sodium citrate are added with phycocyanin according to the mass ratio: sodium citrate: trehalose is 40% -90%: 10% -40%: 10 to 40 percent, and the sum of the proportion 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 added with the trehalose and the sodium citrate is subjected to three stages to obtain the 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) by using high concentrations of CaCl₂Solutions, i.e. CaCl₂The solution is not less than 10g/l, and simultaneously the algae mud and CaCl are added₂The mixing proportion of the solution is improved, namely the volume ratio is not less than 1:10, so that the water consumption in the phycocyanin extraction process can be greatly saved, the energy consumption is reduced, the effect of saving water is achieved, and the production cost is further saved.

(2) The membrane filtration technology is adopted to remove impurities, salt and flora, the product quality is improved, the quality guarantee period is prolonged, the operation is simple, the extraction time is short, the stability of phycocyanin can be effectively ensured, and the industrial rapid continuous production is facilitated.

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, all the existing industrial extraction methods have large water consumption in the extraction process, the invention provides a phycocyanin extraction method capable of saving water consumption, and the phycocyanin with high purity and high yield is extracted under the condition of saving water consumption, and the recovery rate is between 5 and 80 percent.

The method comprises the following steps:

obtaining microalgae mud; and comprises:

step one, swelling and wall breaking: mixing the obtained algae mud with CaCl with the concentration of 10-50 g/l₂Mixing the solution, mixing the algae mud with CaCl₂Mixing the solutions according to the volume ratio of 1: 1-10, 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 phycocyanin and algae residues.

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 removing impurities from the obtained crude extract by adopting a membrane filtration method, wherein the selected membrane aperture is 0.1-5 mu m, removing impurities such as macromolecular substances, colloids and the like in the crude extract, and obtaining a permeate as a phycocyanin solution.

Ultrafiltration in the fourth step: and (3) 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 mu m, removing salt ions in the crude extract, and leaving a phycocyanin concentrated solution in a material liquid barrel.

Step five, freeze drying: and adding trehalose and sodium citrate into the concentrated solution obtained in the step four, and drying to obtain the phycocyanin meeting the food-grade standard.

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 step, the 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.

Further, in the second step, a tubular centrifuge, a disc centrifuge or a three-leg centrifuge is used for high-speed centrifugation to separate solid from liquid, or a plate filter is used for membrane filtration to separate solid from liquid. Wherein the size of the filter cloth of the plate and frame filter is 600-2000 meshes.

The recovery rate of phycocyanin is 40-90% through solid-liquid separation.

And further, in the third step, fine filtration is carried out by adopting 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 20-85 percent after fine filtration.

Furthermore, in the ultrafiltration process of the fourth step, the denaturation of phycocyanin is affected 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 temperature in the feed 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 10-80% by ultrafiltration.

Furthermore, in the fifth step, trehalose and sodium citrate are added into the concentrated solution of phycocyanin to ensure that phycocyanin is not oxidized, and the adding quality is as follows: 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 with the purity meeting the requirement of food grade is finally obtained through drying treatment, and the recovery rate can reach 5 to 80 percent.

Further, the fifth step is drying treatment 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 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, when the temperature of a partition board reaches the set maximum temperature of 40 ℃, the material enters an analysis drying stage, and the drying is completed at the temperature.

The spray drying specifically comprises the step of carrying out spray drying on the phycocyanin concentrated solution added with the trehalose and the sodium citrate under the conditions that the air inlet temperature is 110-130 ℃ and the air outlet temperature is 70-90 ℃, and finally obtaining phycocyanin dry powder from 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.

In one embodiment of the present invention, the microalgae mud is multicladium, nostoc, synechococcus, cryptophyceae, spirulina, etc., and the species is not limited, and further preferably spirulina.

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 fed-batch-fed or the like to maintain the concentration of the glycerol at 0.5-2g/L,the concentration of glucose is 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.