阅读说明：本技术 一种在提取甘油葡萄糖苷过程中回收藻蓝蛋白的方法 (Method for recovering phycocyanin in process of extracting glycerol glucoside ) 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,特别涉及一种在提取甘油葡萄糖苷过程中回收藻蓝蛋白的方法,取微藻藻泥,经过加水回收、膜过滤等步骤,在提取甘油葡萄糖苷的过程中回收和纯化藻蓝蛋白。本发明能在不影响甘油葡萄糖苷的提取效率下,充分对藻蓝蛋白进行回收,最终获得食品级的藻蓝蛋白产品,应用范围更广。(The invention belongs to the technical field of biological extraction, and particularly relates to a method for recovering phycocyanin in a process of extracting glycerol glucoside. The method can fully recover the phycocyanin without influencing the extraction efficiency of the glycerol glucoside, finally obtains food-grade phycocyanin products, and has wider application range.)

1. A method for recovering phycocyanin in the process of extracting glycerol glucoside is characterized by comprising the following steps:

obtaining salt stressed algae mud; and comprises:

step one, obtaining a hypotonic extract: mixing and extracting the algae mud and the hypotonic solution to obtain hypotonic extract liquor and algae mud solids which are rich in glycerol glucoside, phycocyanin and salt;

and a second step: finely filtering the low-permeability extraction liquid obtained in the first step to remove impurities, and performing ultrafiltration to obtain an extraction liquid containing phycocyanin and a permeate containing glycerol glucoside;

and a third step of: mixing the solid algae mud obtained in the first step with CaCl with the concentration of 1g/l to 10g/l₂Mixing the solution, mixing the solid of algae mud with CaCl₂Mixing the solutions according to the volume ratio of 1: 10-100, performing swelling wall breaking in an environment of 4-40 ℃, and shading for 6-72 hours to obtain a mixed solution of phycocyanin and algae residue;

step four: performing solid-liquid separation on the mixed solution obtained in the third 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;

and a fifth step: fine filtering the phycocyanin coarse extract obtained in the step four and/or the phycocyanin-containing extract obtained in the step two to remove impurities, and performing ultrafiltration to obtain a phycocyanin concentrated solution;

and a sixth step of drying: adding trehalose and sodium citrate into the phycocyanin concentrated solution obtained in the fifth step, and drying to obtain phycocyanin dry powder;

wherein, according to different obtained products, the method sequentially comprises a first procedure and a second procedure; or, sequentially performing the first step, the third step and the fourth step; or the first step, the second step, the third step and the fourth step are sequentially carried out; or the first step, the second step, the third step, the fourth step and the fifth step are sequentially carried out; or, sequentially performing the first step, the third step, the fourth step and the fifth step; or sequentially performing the first step, the third step, the fourth step, the fifth step and the sixth step; or, the operation is performed according to the first step, the second step, the third step, the fourth step, the fifth step and the sixth step in sequence.

2. The method of claim 1, wherein in step one, the hypotonic extract is prepared by: mixing the algae mud and the hypotonic solution according to the volume ratio of 1:1, extracting for 1-8 times, and filtering out microalgae to obtain hypotonic extract.

3. The method of claim 2, wherein in step one, the hypotonic solution is a solution having a lower osmotic pressure than the microalgae cells, and comprises a clean salt-free aqueous solution, sterile deionized water, and an aqueous solution of potassium or magnesium salts for reducing the cell osmotic pressure.

4. The method according to claim 1, wherein the fine filtration in the second step and the fifth step is specifically carried out by filtering the low-permeability extract obtained in the second step or the crude extract obtained in the fifth step with a filter having a pore size of 0.1 to 5 μm to remove impurities and macromolecular substances, thereby obtaining a permeate containing phycocyanin.

5. The method as claimed in claim 4, wherein the fine filtration is performed by using a filtration device comprising at least two stages of filter membranes with different pore sizes, wherein the pore size of the first stage of filter membrane is 0.1 μm to 5 μm, and the pore size of the last stage of filter membrane is 1500D to 0.1 μm.

6. The method as claimed in claim 5, wherein the ultrafiltration in the second and fifth steps comprises concentrating and desalting with a filter membrane with pore size of 1500D-0.1 μm, and leaving phycocyanin in the feed liquid tank.

Technical Field

The invention belongs to the technical field of biological extraction, and particularly relates to a method for recovering phycocyanin in a glycerol glucoside extraction process.

Background

Glycerol glucoside (GG for short) is a compatible substance generated by microalgae in a high-salt environment to resist damage of external high osmotic pressure to cells, is a substance formed by combining glycerol and glucose molecules by glycosidic bonds, has the functions of moisture preservation and oxidation resistance, and can be applied to various fields of food, pesticides, medicines, energy sources and the like.

Phycocyanin is a deep blue component separated from microalgae, is an excellent natural edible pigment, has the effects of resisting cancer, promoting blood cell regeneration, maintaining ovary, promoting elastin synthesis in human body and the like, and can be applied to the fields of food, cosmetics, agriculture and the like.

At present, the extraction process related to the glycerol glucoside is mature, but phycocyanin is lost in the extraction process, so that the application value of microalgae cannot be fully utilized, and the economic benefit cannot be maximized.

Disclosure of Invention

Aiming at the defects of the prior art, the inventor researches and designs a method for recovering phycocyanin in the process of extracting glycerol glucoside in long-term practice, under the condition of not influencing the extraction efficiency of the glycerol glucoside, the phycocyanin in the glycerol glucoside is recovered, the recovery efficiency is greatly improved, and the purity of the recovered phycocyanin can reach the food-grade standard.

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

a method for recovering phycocyanin in the process of extracting glycerol glucoside comprises the following steps:

obtaining salt stressed algae mud; and comprises:

step one, obtaining a hypotonic extract: mixing and extracting the algae mud and a hypotonic solution to obtain hypotonic extract liquor and algae mud solids which are rich in GG, phycocyanin and salt;

and a second step: finely filtering the hypotonic extract obtained in the first step to remove impurities, and performing ultrafiltration to obtain an extract containing phycocyanin and a permeate containing GG;

and a third step of: mixing the solid algae mud obtained in the first step with CaCl with the concentration of 1g/l to 10g/l₂Mixing the solution, mixing the solid of algae mud with CaCl₂Mixing the solutions according to the volume ratio of 1: 10-100, placing the mixture in an environment of 4-40 ℃ for swelling and wall breaking,and shading, swelling for 6-72 hours to obtain a mixed solution of phycocyanin and algae residue;

step four: performing solid-liquid separation on the mixed solution obtained in the third 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;

and a fifth step: fine filtering the phycocyanin coarse extract obtained in the step four and/or the phycocyanin-containing extract obtained in the step two to remove impurities, and performing ultrafiltration to obtain a phycocyanin concentrated solution;

and a sixth step of drying: adding trehalose and sodium citrate into the phycocyanin concentrated solution obtained in the fifth step, and drying to obtain phycocyanin dry powder;

wherein, according to different obtained products, the method sequentially comprises a first procedure and a second procedure; or, sequentially performing the first step, the third step and the fourth step; or the first step, the second step, the third step and the fourth step are sequentially carried out; or the first step, the second step, the third step, the fourth step and the fifth step are sequentially carried out; or, sequentially performing the first step, the third step, the fourth step and the fifth step; or sequentially performing the first step, the third step, the fourth step, the fifth step and the sixth step; or, the operation is performed according to the first step, the second step, the third step, the fourth step, the fifth step and the sixth step in sequence.

Further, in the first step, the hypotonic extract is prepared by the following method: mixing the algae mud and the hypotonic solution according to the volume ratio of 1:1, extracting for 1-8 times, and filtering out microalgae to obtain hypotonic extract.

Further, in the first step, the hypotonic solution is a solution having a lower osmotic pressure than that in the microalgae cells, and includes a clean salt-free aqueous solution, sterile deionized water, and a potassium salt or magnesium salt aqueous solution for reducing the cellular osmotic pressure.

Further, the fine filtration in the second step and the fifth step is specifically to filter the low-permeability extract obtained in the second step or the crude extract obtained in the fifth step by using a filter membrane with the pore size of 0.1-5 μm, so as to remove impurities and macromolecular substances, thereby obtaining a permeate containing phycocyanin.

Furthermore, the filtering device adopted by the fine filtration comprises at least two stages of filter membranes with different pore diameters, the pore diameters are gradually reduced, the pore diameter of the first stage filter membrane is 0.1-5 μm, and the pore diameter of the last stage filter membrane is 1500D-0.1 μm.

Further, the ultrafiltration in the second and fifth working procedures is specifically that a filter membrane with the aperture of 1500D-0.1 μm is adopted for concentration and desalination treatment, and phycocyanin is left in a feed liquid barrel.

Further, ultrafiltration is carried out at a temperature of 4 ℃ to 40 ℃ and a pressure of 50Kpa to 2 MPa.

Further, in the fourth 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.

In the sixth step, the addition amounts of trehalose and sodium citrate are, in terms of mass ratio, 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 sixth 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) the method is characterized in that a solution containing GG and phycocyanin is subjected to membrane filtration, and the phycocyanin remained in the phycoresidues after GG is extracted is obtained by swelling and breaking the walls, so that the phycocyanin is fully recovered on the premise of not influencing the extraction of GG, the recovery rate of the phycocyanin can reach 85%, and the purity of the phycocyanin meets the standard of food-grade phycocyanin.

(2) By CaCl₂The swelling method is used for swelling and breaking walls of the algae residues after GG extraction, so that the cell walls of the microalgae can be rapidly swelled and broken, the effective components in the algae residues can be fully recycled, resource waste is avoided, industrialized mass recovery of phycocyanin is facilitated, and the nutritional value and the medicinal value of the microalgae are increased.

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 prior art causes phycocyanin loss in the process of extracting glycerol glucoside, cannot fully utilize the application value of microalgae, and cannot maximize economic benefit. The invention provides a method for recovering phycocyanin in the process of extracting glycerol glucoside, which can fully recover phycocyanin without influencing the extraction efficiency of the glycerol glucoside, and finally produce food-grade phycocyanin products for sale.

The method comprises the following steps:

obtaining salt stress algae mud; and comprises:

step one, obtaining a hypotonic extract: mixing and extracting the algae mud and the hypotonic solution to obtain a hypotonic extract mixture rich in GG, phycocyanin and salt and algae mud solids.

And a second step: and (2) finely filtering the hypotonic extraction liquid obtained in the first step to remove impurities such as macromolecular substances, colloids and the like in the hypotonic extraction liquid, performing ultrafiltration to keep phycocyanin in a feed liquid barrel, enabling GG to flow out along with the permeate liquid to obtain the extraction liquid containing phycocyanin and the permeate liquid containing GG, and performing GG extraction on the permeate liquid.

And a third step of: mixing the solid algae mud obtained in the first step with CaCl with the concentration of 1g/l to 10g/l₂Mixing the solution, mixing the solid of algae mud with 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 phycocyanin and algae residues.

The procedure is to recover the residual phycocyanin in the phycobiont, because only a small part of phycocyanin can be extracted by adding hypotonic solution, and a large amount of phycocyanin in the phycobiont is not broken and dissolved out, CaCl is utilized₂The swelling method recovers the residual phycocyanin.

Step four: and (4) performing solid-liquid separation on the mixed solution obtained in the third step at the temperature of 4-40 ℃ by using a centrifugal machine or a filtering device to obtain a mixture of the phycocyanin crude extract and the phycoresidues.

And a fifth step: mixing the crude phycocyanin extracting solution obtained in the step four and/or the phycocyanin-containing extracting solution obtained in the step two, and repeating the step two to obtain the phycocyanin concentrated solution.

And a sixth step of drying: and adding trehalose and sodium citrate into the concentrated solution obtained in the fifth step, and drying to obtain the food-grade phycocyanin.

Wherein, according to different obtained products, the method sequentially comprises a first procedure and a second procedure; or, sequentially performing the first step, the third step and the fourth step; or sequentially performing the first process, the second process and the sixth process; or the first step, the second step, the third step and the fourth step are sequentially carried out; or the first step, the second step, the third step, the fourth step and the fifth step are sequentially carried out; or the first step, the second step, the third step, the fourth step and the sixth step are sequentially carried out; or, sequentially performing the first step, the third step, the fourth step and the fifth step; or, sequentially performing the first step, the third step, the fourth step and the sixth step; or sequentially performing the first step, the third step, the fourth step, the fifth step and the sixth step; alternatively, the operations are performed in the first step, the second step, the third step, the fourth step, the fifth step, the sixth step, and the like in this order.

Further, the hypotonic extraction solution in the first step is prepared by the following method: mixing the collected microalgae rich in GG with a hypotonic solution according to the volume ratio of 1:1, extracting for 1-8 times, filtering out the microalgae to obtain a hypotonic extraction liquid, wherein the concentration of GG in the obtained extraction liquid is reduced along with the reduction of the extraction times.

Furthermore, in the first step, the hypotonic extraction solution is prepared by the following method: dehydrating the collected microalgae mud rich in GG, mixing the microalgae mud with a hypotonic solution according to the volume ratio of 1:1, extracting, separating the microalgae mud with water to obtain microalgae mud solid and GG water solution, extracting for 1-8 times in the way, and collecting the extraction liquid after each extraction or combining the extraction liquids after multiple extractions to obtain the hypotonic extraction liquid.

In one embodiment of the present invention, in the first step, the hypotonic extraction liquid is an extraction liquid obtained by extracting the microalgae enriched with GG with a hypotonic solution, and the extraction liquid comprises phycocyanin, salts, (a small amount of) saccharides, and GG.

In one embodiment of the present invention, in the first step, the hypotonic solution is a solution having a lower osmotic pressure than the algal cells, and includes a clean salt-free aqueous solution, a low-salt aqueous solution, and an aqueous solution of potassium salt or magnesium salt for reducing the cellular osmotic pressure. The concentration of the saline solution may be prepared as required, but the present invention is not limited thereto.

Preferably, the hypotonic solution is a clean salt-free aqueous solution; further preferably sterile deionized water.

If a salt solution with high osmotic pressure and a high-concentration ethanol solution are used as extraction liquid, the extraction liquid has good capacity of penetrating cell walls or dissolving cell membranes, so that the internal tissue structure of the microalgae cells can be damaged during extraction, and the microalgae cells can die.

Further, the fine filtration in the second step is specifically to remove impurities from the obtained low-permeability extract by a membrane filtration method, wherein the selected membrane aperture is 0.1-5 μm, to remove impurities such as macromolecular substances, colloids and the like in the crude extract, and phycocyanin and GG flow out along with the permeate.

Furthermore, in the second working procedure, the filtering device adopted by the fine filtration comprises at least two stages of filter membranes with different pore diameters, the pore diameters are gradually reduced, the pore diameter of the first stage filter membrane is 0.1-5 μm, and the pore diameter of the last stage filter 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.

Further, in the second step, the ultrafiltration is specifically to perform concentration and desalination treatment on the permeate obtained after the fine filtration by adopting a membrane filtration method, wherein the selected membrane aperture is 1500D-0.1 μm, so as to ensure that salt ions and GG permeate the filter membrane and the concentrated solution of phycocyanin in the feed liquid barrel.

Furthermore, in the second working procedure, in the ultrafiltration process, 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 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.

Further, in the second step, the permeate containing GG and small molecular substances is subjected to GG extraction, in which the obtained permeate is concentrated by a nanofiltration device to remove small molecular substances, and the obtained GG concentrate is dehydrated to obtain purified glycerol glucoside.

The nanofiltration device adopts membrane filtration equipment, the pore diameter of the membrane is 100-250Da, small molecules in the permeate can pass through the membrane in the pore diameter range, but GG cannot pass through the membrane, in the process, the extraction rate of GG can be more than 90%, and the loss rate of GG is less than 10%.

It should be noted that, in the present invention, it is impossible to perform desalination treatment by using ion exchange resin, because this desalination method will lose more than 5% of GG, which will make the purity of the final GG less than 90%, and it is impossible to obtain high purity GG.

As an alternative embodiment, the desalination device is GG adsorption resin column, including but not limited to styrene type macroporous adsorption resin, such as D101 resin, XAD-1 resin, etc. The resin has specific adsorption on GG, and does not adsorb substances such as salt, glycerol and the like. After the adsorption saturation of GG, pure water with a volume being a plurality of times of that of the column is used for eluting impurities participating in the column, and then ethanol with a volume being a plurality of times of 10-60 (v/v)% is used for eluting the GG.

And the dehydrating device further removes redundant moisture from the desalted and concentrated extract liquor, and finally obtains a GG pure product with the purity of more than 90%.

Preferably, the dehydration device comprises a vacuum reaction kettle, a rotary evaporator and the like.

Further, in the third step, the solid 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 fourth 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.

Furthermore, in the sixth step, trehalose and sodium citrate need to be added to the concentrated solution of phycocyanin to ensure that phycocyanin is not oxidized, and the addition ratio 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 after drying treatment.

Further, the sixth step is a drying process 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 is specifically 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 ℃, 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.

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.

At present, the salt-stressed 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 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 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 conditions of the medium in the first stage of GG pulling-up are basically the same as those in the stage of GG production, and the medium is used for cell cultureThe GG is synthesized in the algae cells while ensuring the growth of 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, culture is performed in the same way as the first step of GG elevationNutrient conditions, with the difference that the concentration of the substance capable of altering the osmotic pressure of the cells in the culture medium is 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 algae cells enriched with GG from the culture medium in the first GG elevation stage or the second GG elevation stage to obtain microalgae 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 microalgae mud, and removing surface attachments to obtain clean microalgae 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.